Search Results (477)

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

Mammary gland tumors are among the most common neoplasms in female dogs. Macrophages are believed to play an important role in tumor progression and metastasis. The aim of this study was to determine whether hemosiderin-laden macrophages (HLMs) may be involved in the development of metastatic lesions in regional lymph nodes. Forty-two cases of mammary gland cancers in female dogs and their regional lymph nodes were included in the analysis. The samples were divided into two groups based on the presence or absence of metastases. The sections were stained with hematoxylin and eosin (HE) and Prussian blue and were additionally subjected to immunohistochemical labeling using antibodies against pan-cytokeratin (Pan-CK) and Ki-67. In 20 cases, no metastatic changes were detected in the regional lymph node, whereas metastases were identified in 22 cases. A positive correlation was observed between the number of HLMs in the tumor stroma and the number of HLMs in the regional lymph node. Furthermore, a positive correlation was found between Ki-67 nuclear immunoreactivity in the mammary tumor and the number of HLMs present within its stroma. HLMs may represent a component of the tumor microenvironment that promotes cancer cell proliferation and potentially contributes to the propensity for metastasis formation in mammary tumors of female dogs. Full article

Aflatoxins (AFs) are potent hepatotropic mycotoxins—AFB₁ being the best-characterized—yet their ability to induce intrahepatic cholangiocarcinoma (iCCA) remains underexplored. Male Wistar rats received vehicle (controls; n = 5) or an AFB₁-dominant AF mixture (AFB₁ 39.46 μg/mL; AFB₂ 1.13 μg/mL; AFG₁ 17.44 μg/mL; AFG₂ 0.59 μg/mL— n = 10) by daily gavage for 90 days, at a dose equivalent to 400 μg AFB₁ per kg of diet. After 12 months, twelve iCCA tumors were resected and analyzed by histology (H&E) and tissue-microarray-based immunohistochemistry (Cytokeratin-19, Hep Par-1, p53, Cyclin D1, Rb, β-catenin, and PCNA). Lesions predominantly showed glandular/tubular architecture consistent with iCCA and were cytokeratin-19-positive and Hep Par-1-negative. Cell proliferation was high (PCNA ≈ 69%). p53 displayed nuclear accumulation in 83% of tumors. G1/S control was perturbed, with cyclin D1 overexpression (67%), and Rb was positive in 58% of iCCA. Aberrant Wnt activation was rare (nuclear β-catenin in 8%). Subchronic exposure to an AFB₁-dominant AF mixture in rats was associated with iCCA characterized by high proliferative activity, p53 accumulation, and disruption of G1/S checkpoint components. These findings broaden the oncogenic spectrum of AFs and warrant genomic confirmation of AF mutational signatures. Full article

Background/Objectives: Intrauterine growth restriction (IUGR) is a condition in which a fetus does not reach its normal growth potential and is associated with increased neonatal morbidity. Surveillance relies on cardiotocography, a biophysical ultrasound, and a Doppler assessment, but placental pathology remains insufficiently integrated into clinical evaluations. This study aimed to compare placentas from IUGR and normal pregnancies. Methods: This cohort included 34 pregnancies (16 IUGR, 18 controls) managed at Hunedoara County Hospital (Romania). The ultrasound and Doppler parameters were documented. The placentas were collected after delivery, fixed in formalin, and processed using standard histopathological protocols. The villous morphology and maternal vascular malperfusion features were assessed on H&E sections, focusing on syncytial knots, villous caliber reduction, stromal fibrosis, fibrin deposition, and infarctions. Immunohistochemistry for CD34, cytokeratin 7 (CK7), CD68, vascular endothelial growth factor (VEGF), and Hypoxian inducible factor 1 (HIF-1α)was performed using a semi-quantitative 0–3 scoring system. A statistical analysis was performed using chi-squared testing for categorical variables and t-tests for continuous variables. Results: The ultrasound evaluation showed an estimated fetal weight below the 10th percentile and abnormal Doppler indices in the IUGR group. The histopathology demonstrated a strong association between IUGR and villous abnormalities, including an increased number of syncytial knots, stromal fibrosis, a reduced villous caliber, and placental infarctions. The immunohistochemistry showed a marked overexpression of VEGF and HIF-1α and increased CD68-positive Hofbauer cells in IUGR placentas (p < 0.0001), while CD34 and CK7 displayed preserved strong staining in both groups. Conclusions: Placentas from IUGR pregnancies exhibited advanced maternal vascular malperfusion with consistent hypoxic and inflammatory changes, correlating with Doppler alterations. These findings highlight the diagnostic relevance of placental pathology in pregnancies with IUGR. Full article

Background: Frostbite injury is a thermal injury where ice crystals form in skin tissues and subsequently lead to damage due to prolonged exposure to cold temperatures below 0 °C. The extremities are mostly affected, leading to potential amputation. As there is no pharmacological treatment of frostbite injury, we recently reported that non-clinically viable hydrogen sulfide (H₂S) donors promote frostbite wound healing in mice. In this study, we investigated whether commonly used cosmetic creams supplemented with sodium thiosulfate (STS), a clinically viable H₂S donor drug, also promote healing of frostbite wounds. Methods: Frozen magnets (−80 °C) were placed on the dorsal skin of 40 C57BL/6 mice for 3 min to induce frostbite injury. Next, commercially available cosmetic creams (Aveeno, Dove, Neutrogena, and Nivea) were topically applied on frostbite wounds daily for 14 days with or without 150 µM of STS supplementation. The mice were sacrificed on day 15 after induction of frostbite injury, and samples of the injured dorsal skin tissue were collected for analysis. Results: Addition of STS enhanced frostbite wound healing, as evidenced by progressive and significantly reduced wound area by about 50% and inflammation (p < 0.05), and markedly increased granulation tissue formation by >45%, fibroblast maturation by >28%, and re-epithelialization by >63% compared to control groups (p < 0.05), with Nivea producing a superior wound-healing effect. Also, STS supplementation significantly upregulated the expression of CD31 (by >25%), KI-67 (by >25%), CD163 (by >20%), fibronectin (by >14%), and cytokeratin (by >50%) in frostbite wounds compared to control groups, with Aveeno and Nivea producing a better wound-healing effect than Dove and Neutrogena creams. Conclusions: In conclusion, STS accelerated healing of frostbite wounds. Therefore, it could be considered as a novel pharmacological treatment of clinical frostbite. Full article

Single-cell sequencing (scRNA-seq) has emerged as a pivotal technology for deciphering the complex cellular heterogeneity and tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC), positioning it as a critical tool for informing novel therapeutic strategies. This review explores how scRNA-seq reveals diverse cellular subpopulations and their functional roles within the PDAC TME, including malignant epithelial cells with transitional phenotypes, heterogeneous cancer-associated fibroblasts (CAFs), functionally distinct immune cells such as tumor-associated neutrophils (TANs) and macrophages (TAMs), and actively participating neural components like Schwann cells. These cellular constituents form specialized functional units that drive tumor progression, immune evasion, neural invasion, and therapy resistance through metabolic reprogramming, immunosuppressive signaling, and cellular plasticity. The review further examines technological advances in single-cell sequencing from 2023 to 2025, focusing on sample preprocessing innovations, multi-omics integration (combining transcriptomics with epigenomics and proteomics), spatial resolution enhancements, and customized computational tools that address PDAC-specific challenges. Clinically, single-cell sequencing enables precise cellular subtyping, identification of novel biomarkers, and development of personalized therapeutic approaches, including combination therapies targeting specific cellular subpopulations and their interactions. Despite these advances, significant challenges remain in standardizing clinical applications such as liquid biopsy for early detection and tumor microenvironment assessment for diagnostic staging, validating biomarkers like CLIC4, GAS2L1, Cytokeratins, Vimentin and N-cadherin in circulating tumor cells, and comprehensively integrating multi-omics data. Future research focusing on both technology refinement and biological validation will be essential for translating single-cell insights into improved diagnostic and therapeutic outcomes for pancreatic cancer. Full article

Background/Objectives: The Vesical Imaging-Reporting and Data System (VI-RADS) provides high diagnostic accuracy for muscle-invasive bladder cancer; however, its prognostic value remains limited. We propose serum cytokeratin 19 fragment (CYFRA 21-1) and tumor contact length (TCL) as complementary prognostic factors. We aimed to construct a composite risk score integrating VI-RADS, CYFRA 21-1, and TCL for prognostic stratification. Methods: We retrospectively analyzed data from 101 patients with bladder cancer (BC) who underwent transurethral resection of bladder tumor (TURBT), magnetic resonance imaging, and postoperative serum CYFRA 21-1 measurement. For each factor, cut-off values were determined using receiver operating characteristic (ROC) analysis; meeting each threshold contributed one point (score range, 0–3). Overall survival (OS) was assessed using Kaplan–Meier and Cox regression analyses. Results: ROC analysis identified cut-offs of VI-RADS ≥ 3 (area under the curve [AUC] 0.779), TCL ≥ 40 mm (AUC 0.817), and CYFRA 21-1 ≥ 2.1 ng/mL (AUC 0.875). Based on these, patients were stratified into low- (0–1, n = 81), intermediate- (2, n = 12), and high-risk (3, n = 8) groups with 3-year OS rates of 95.1%, 75.0%, and 25.0%, respectively (p < 0.001). In univariate Cox regression, all factors significantly predicted poor OS: VI-RADS ≥ 3 (hazard ratio [HR], 6.51; p = 0.015), TCL ≥ 40 mm (HR, 8.36; p < 0.001), and CYFRA 21-1 ≥ 2.1 ng/mL (HR, 14.02; p < 0.001). In multivariate analysis, only CYFRA 21-1 remained independently significant (HR, 11.80; p < 0.001). Conclusions: A composite risk score combining VI-RADS, TCL, and CYFRA 21-1 effectively stratified patients with BC into distinct groups using minimally invasive, peri-TURBT assessments. Prospective multicenter validation is warranted. Full article

Salivary gland carcinomas (SGCs) are aggressive malignancies with limited treatment options, primarily due to the complexity of the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) remodel the extracellular matrix (ECM), enhance cancer cell stemness, and drive drug resistance. This study introduces a decellularized CAF-derived spheroid system as a biomimetic platform to study tumor–stromal interactions in SGC. Multicellular spheroids were generated by co-culturing Medical Research Council cell strain 5 (MRC-5) fibroblasts (fetal lung-derived) with A253 salivary gland cancer cells, producing distinct spatial architecture, with fibroblasts at the core and cancer cells at the periphery. Compared with A253-only spheroids, A253/MRC-5 spheroids exhibited enhanced proliferation and elevated expression of stemness markers (aldehyde dehydrogenase 1 [ALDH1], CD133, cytokeratin 19 [CK19]). MRC-5 spheroids displayed robust ECM and growth factor expression that persisted after decellularization. Decellularized spheroids retained biological activity, enabling A253 cells to develop invasive phenotypes, metabolic reprogramming, and stemness-associated signatures. Transcriptomic analysis revealed a transition from proliferative pathways to stress-adaptive survival programs, mirroring in vivo tumor behavior. Moreover, A253 cells cultured with decellularized fibroblast spheroids exhibited altered cisplatin sensitivity, highlighting the critical role of stromal ECM in therapeutic response. In conclusion, this study establishes decellularized CAF spheroids as a simplified yet biologically relevant TME-mimetic platform. By recapitulating tumor–stromal crosstalk without live co-culture, this system provides a powerful tool for mechanistic studies of salivary gland cancer, preclinical drug screening, and development of stroma-targeted therapies. Full article

The immune control of cancer growth is an area of active investigation. In this study, we demonstrated the feasibility of using standard immunohistochemistry methods in conjunction with a set of newly developed chromogens to demonstrate immune cell markers in a multiplex staining system. Immune infiltrating cells in breast cancer were identified using antibodies to CD20 (B-cells), CD3 (T-cells), and CD163 (macrophages). Additionally, the tumor compartment was identified using cytokeratin (AE1/AE3), and Ki67 was used to determine the proliferation index. These stains showed a significant immune cell infiltrate surrounding and within the tumors. B-cells, T-cells, and macrophages were abundant at the tumor periphery, particularly in areas where tertiary lymphoid structures were also present. In contrast, B-cells were significantly reduced within the tumor interior compared to an abundant infiltrate of T-cells and macrophages. Patterns of B-cell, T-cell, and macrophage infiltration were identified. Depending upon the particular set of markers chosen for analysis, a simple visual examination, without the aid of computer-assisted imaging systems, was sufficient to differentiate up to five different markers. Full article

Canine prostatic carcinoma is a highly aggressive neoplasm with a strong tendency to metastasize, most commonly to regional lymph nodes, lungs, and bones. However, skeletal muscle and myocardial involvement are rarely reported. This report describes an 11-year-old intact male Maltese dog with a two-month history of anorexia and lethargy, referred for further evaluation after failing to respond to piroxicam therapy. Diagnostic imaging revealed a prostatic mass and multiple rim-enhancing lesions in the epaxial musculature and left ventricular wall, without evidence of metastasis to the lymph nodes, lungs, or other visceral organs. Hemostatic analysis indicated a hypercoagulable state. Postmortem examination confirmed metastatic prostatic carcinoma involving the semispinalis, multifidus, and myocardium. Histologically, the neoplastic cells exhibited similar morphology at the primary and metastatic sites. Immunohistochemistry revealed strong cytokeratin expression and absence of uroplakin III, consistent with a non-urothelial epithelial origin. No evidence of lymphatic involvement was observed. To the best of our knowledge, this is the first documented case of canine prostatic carcinoma with exclusive myotropic and myocardial metastases. These findings suggest a possible hematogenous metastatic route and highlight the importance of including muscle and cardiac tissues in staging protocols for canine prostatic carcinoma, even when lymphadenopathy or pulmonary lesions are absent. Full article

Alcohol-associated liver disease is a global health burden with high morbidity and mortality, and the fungal microbiome is important for its progression. In particular, Candida albicans and C. albicans-reactive T helper 17 (Th17) cells contribute to alcohol-associated liver disease. Specific C. albicans antigens that activate Th17 cells during this disease are unknown. The C. albicans 65 kDa mannoprotein (CaMp65p) is one of the most abundant and immunodominant proteins of C. albicans, and is capable of eliciting robust T cell and interleukin (IL)-17A responses. The aim of this study was to measure levels of CaMp65p in serum of patients with alcohol use disorder and liver disease. Serum CaMp65p levels were measured in the serum of 60 patients with alcohol use disorder using an indirect competitive Enzyme-Linked Immunoabsorbent Assay (ELISA). Serum CaMp65p levels were correlated with liver disease severity. Serum CaMp65p levels positively correlated with several clinical and biochemical markers of liver injury and disease within the patient group with alcohol use disorder, including serum aspartate aminotransferase (AST; R = 0.33, p = 0.0092), alanine aminotransferase (ALT; R = 0.27, p = 0.037), gamma-glutamyl transferase (GGT; R = 0.35, p = 0.0055) and alkaline phosphatase (R = 0.36, p = 0.0052), and with the circulating M65 fragment of cytokeratin 18 (CK18-M65; R = 0.51, p = 0.0012), a marker of hepatocyte death. In addition, patients with alcohol use disorder in the upper quartile had significantly higher liver stiffness (p = 0.0022). Serum CaMp65p was significantly higher in patients with fibrosis stage F2–F4 as compared with patients with no or minimal fibrosis F0–F1 (p = 0.0082). The area under the curve (AUC) for detecting F2–F4 fibrosis was 0.70. Elevated serum CaMp65p levels are associated not only with more severe hepatic injury, but also with liver fibrosis in patients with alcohol use disorder. Therefore, CaMp65p may serve as a non-invasive biomarker for fibrosis assessment in patients with alcohol use disorder. Full article

Objectives: This study aimed to assess the dynamics of liver tests (LT) and detect signs of liver fibrosis and steatosis 2.5 years after the first COVID-19 episode in patients without pre-existing liver-related conditions. Methods: The study included 65 adult patients hospitalized with COVID-19 (including 18 with severe or critical illness) in 2020. After 2.5 years, in addition to regular LT, liver health status was assessed by the FIB-4 index, hyaluronic acid, cytokeratin 18 fragment M30 (serum, ELISA), cardiometabolic risk factors, and the multiparametric ultrasound examination. Results: LT abnormalities in the acute COVID-19 period were observed more frequently (p = 0.036) in patients with severe or critical COVID-19 (83%) than in patients with non-severe COVID-19 (55%). LT dynamics in 2.5 years showed an improvement of liver health status in most patients (p = 0.006). Persistent LT abnormalities were associated with LT abnormalities during hospitalization (p = 0.021). After 2.5 years, the presence of cardiometabolic risk factors and signs of liver fibrosis were associated with the severity of the first COVID-19 episode. However, regression analyses did not support disease severity as a predictor for LT abnormalities and liver stiffness. The latter was predicted by cardiovascular diseases in the anamnesis. Conclusions: In most patients, LT normalized despite potential risk factors. Simultaneously, in some patients, signs of liver fibrosis after COVID-19 might be stimulated by COVID-19-related metabolic dysfunction and the presence of cardiovascular diseases. Full article

Over the past decade, the interest in using 3D cell culture models for studying the mammary gland in biomedical and veterinary fields has increased, but a fully functional in vitro model for domestic species is still lacking. Multiple cellular components, including epithelial cells, vascular endothelial cells, and stromal/stem cells, sustain the secretory mammary gland tissue in a well-organized 3D architecture. Considering the Göttingen Minipigs widely used for translational lactation studies, this work aimed to establish a 3D culture protocol to generate mammary heterogeneous multicellular spheroids composed of three different Göttingen Minipigs primary cells: mammary epithelial cells (mpMECs), aortic endothelial cells (mpAECs), and vascular-wall mesenchymal stem cells (mpVW-MSCs). Cells were cultured with hanging-drop (HD) and ultra-low-adherence plate (ULA) methods, evaluating aggregate formation in both monocultures and co/triple co-cultures. Brightfield area, eccentricity, viability, and cell distribution were analyzed. Results showed mpMECs formed irregular aggregates in both HD and ULA, while more compact and viable spheroids were formed when co-cultured with mpVW-MSCs and mpAECs by ULA. A well-organized cellular distribution was demonstrated by cytokeratin-18, vimentin, and e-NOS immunofluorescence analysis. In conclusion, this study established a stable 3D mammary multicellular spheroid model, representing a promising tool for future studies on hormonal modulation and mammary gland physiology. Full article

The benefit of neoadjuvant chemotherapy (NAC) over upfront surgery (UFS) for resectable pancreatic ductal adenocarcinoma (PDAC) is increasingly recognized, yet prognostic biomarkers remain undefined. We evaluated tumor–stroma ratio (TSR), β-catenin (β-CTN) expression, and tumor budding (TB) in 84 resected PDACs (35 NAC, 49 UFS) using digital image analysis of multi-cytokeratin (m-CK) and β-CTN immunohistochemistry. TSR was defined as the proportion of malignant epithelial area within the tumor, and the β-CTN/m-CK index as the ratio of β-CTN to m-CK immunoreactivity in tumor tissue relative to intralobular ducts. TB was significantly less frequent in NAC than UFS (p = 0.003), suggesting that NAC may indirectly modulate epithelial–mesenchymal transition, with TB regarded as its morphological correlate. In the NAC cohort, low TSR was associated with more favorable histological response (Evans IIa/IIb, median 7%; Evans I, 16%; p = 0.009), likely reflecting NAC-induced tumor shrinkage with relative stromal predominance. In multivariable analysis, low β-CTN/m-CK index (<0.5) predicted shorter relapse-free survival in both NAC (HR = 2.516, p = 0.043) and UFS (HR = 2.230, p = 0.025) subgroups. High TSR (≥13%) was associated with shorter cancer-specific survival (HR = 2.414, p = 0.034) in the overall cohort, indicating prognostic value complementing its association with NAC response. These results identify the β-CTN/m-CK index and TSR as prognostic biomarkers in PDAC. Full article

Background/Objectives: Cervical Pap smears are routinely used to detect cellular abnormalities as a cervical cancer screening tool and to assess the presence of HPV for risk stratification of the disease. Here, we aimed to extend the applications of this sampling procedure by combining it with multicolor flow cytometry to characterize cell populations across cervical cancer disease stages. Methods: Cervical Pap smears from 30 patients with various disease stages ranging from normal to intraepithelial neoplasia up to treated cancers were analyzed as biofluids using multicolor flow cytometry. Individual samples were evaluated, and statistical analyses were performed over all sample stages. Cancer cell lines (CaSki, SiHa, HeLa, A549, U2OS) were examined as tumor cell controls. Results: Cervical biofluids were subdivided into cell populations according to their scattering properties and the expression of specific biomarkers: EpCAM and cytokeratin 8 for epithelial cells from tumors as well as healthy ectocervical and endocervical regions, and CD45 for immune cells. Discrimination of tumor cells was facilitated with cancer cell lines. Statistical analysis revealed that the composition of cell populations differs among disease stages, whereas treated cancer samples were consistently associated with a reduction in squamous epithelial cells and an increase in immune cells compared to normal samples. Conclusions: Herein, we identified the major cell populations in cervical biofluid samples and demonstrated that this method can detect changes in the cellular composition across different disease stages. This approach could be further exploited in cancer research and potentially serve as a companion diagnostic tool in tumor development, progression and during treatment. Full article