Search Results (387)

Background/Objectives: Thymic epithelial tumors (TETs) are rare, histologically heterogeneous neoplasms lacking robust molecular biomarkers. Hippo pathway dysregulation—driving YAP/TEAD-dependent transcription—has been implicated across cancers, but transcript-level data in TETs are limited. Methods: We profiled 26 (23 TETs and three normal thymus) formalin-fixed and paraffin-embedded (FFPE) specimens by SYBR real-time quantitative polymerase chain reaction (RT-qPCR) across World Health Organization (WHO) subtypes, focusing on core Hippo components YAP1, TEAD4, MST1, SAV1, LATS1, and MOB1A. Expression was normalized to the geometric mean of HPRT1 and TBP and reported as log₂ fold change (log₂FC) using the 2^−ΔΔCq method relative to the pooled normal. Group differences were compared using non-parametric tests. Results: Median log₂FC values showed subtype-dependent upregulation of YAP1/TEAD4, notably in type A (YAP1 ≈ +3.43) and B3 (YAP1 ≈ +2.78) thymomas, with TEAD4 strongly increased in thymic carcinoma (TC; ≈ +3.49) and elevated in type A/B3. Upstream kinases tended to be subtype-specifically reduced, particularly in TC (MST1 ≈ −1.38; LATS1 ≈ −1.34), and modestly in B1. SAV1 was elevated in type A (≈+2.25) and B3 (≈+2.01), while MOB1A remained near baseline. Differential expression among WHO subtypes (Kruskal–Wallis) was significant for YAP1 (p = 0.003), TEAD4 (p = 0.015), SAV1 (p = 0.004), MST1 (p = 0.012), and LATS1 (p = 0.036), but not for MOB1A (p = 0.09). Conclusions: TETs seem to exhibit subtype-dependent expression patterns of core Hippo pathway components, characterized by enhanced YAP1–TEAD4 transcriptional output in selected subtypes and marked reduction of the MST1/LATS1 kinase module, most pronounced in TC. These exploratory patterns nominate candidate markers for subtype stratification and clinical validation. Full article

Splenic nodules in dogs that were historically classified under the broad term “fibrohistiocytic nodules” are now recognised as distinct entities within likely a biological continuum. These include lymphoid hyperplasia extending to indolent lymphoma and complex hyperplasia to stromal sarcoma. However, the molecular mechanisms underpinning these proposed progressions remain largely unexplored, particularly at the genomic and transcriptomic levels. This study aimed to delineate and compare the transcriptomic landscapes of four distinct canine splenic nodules through differential gene expression profiling. RNA sequencing was performed on twelve formalin-fixed, paraffin-embedded (FFPE) splenic tissue samples obtained from dogs diagnosed with lymphoid hyperplasia, complex hyperplasia, histiocytic sarcoma, and stromal sarcoma, with normal canine spleen serving as a control tissue. Comparative transcriptomic analysis identified 47 differentially expressed genes (DEGs) between splenic nodules and normal spleen, including CSRP1, SLC40A1, C1QA, C1QC, DLA-12, FTL, FXYD6, MPEG1, OAS3, CSF1, and JMJD6. Furthermore, 39 DEGs were significantly altered among the four splenic lesion types, such as MLC1, ERAS, MOV10L1, LOC102152143, COL4A1, COL4A2, COL12A1, NOTCH3, PLOD2, CPXM2, MRC1, GALNT5, TIMP1, and TFPI2. Many of these genes have previously been implicated in tumorigenesis and metastasis in other malignancies. These findings suggest that dysregulated gene expression may contribute to the activation of stromal cells and macrophages within the spleen, facilitating malignant transformation. Overall, these findings deliver novel transcriptomic insights into canine splenic tumorigenesis that may improve diagnostic precision, inform prognostic assessment, and support the development of targeted therapeutic strategies in veterinary oncology. Full article

Background: Scabies, caused by Sarcoptes scabiei var. hominis, remains difficult to confirm histologically when parasites are sparse or fragmented. Conventional microscopy is particular but limited by small sample size, tissue destruction, and observer dependence. Objective: To evaluate visible–near-infrared hyperspectral imaging (VIS–NIR HSI) as a label-free optical method for detecting S. scabiei in human skin sections and to assess its compatibility with routine HE staining. Methods: Formalin-fixed, paraffin-embedded (FFPE) skin tissue from six patients with histologically verified scabies was analysed using VIS–NIR HSI (500–1000 nm). Unstained sections mounted on CaF₂ substrates and parallel HE-stained slides were imaged. Spectral datasets were processed by principal component analysis and segmentation to distinguish mite structures from epidermal and dermal compartments. Results: The chitin-rich mite exoskeleton exhibited a reproducible reflectance slope in the near-infrared range (R₈₅₀/R₅₅₀ > 1.5), clearly separating parasite from host tissue (R₈₅₀/R₅₅₀ < 1.0). PCA confirmed consistent cluster separation across all cases (ΔPC ≈ 3.7 ± 0.2). These contrasts remained detectable in HE-stained sections, validating applicability to conventional slides. Conclusions: VIS–NIR HSI enables reliable, label-free detection of S. scabiei mites in both unstained and HE-stained human skin tissue. By combining morphological and biochemical information in a single modality, HSI represents a promising adjunct to digital dermatopathology and may improve diagnostic sensitivity in challenging or atypical cases. Full article

The reasons for the aggressive clinical phenotype of signet ring cell carcinoma (SRCC) have not been fully elucidated. Previous studies suggest similarities in the genotype of colorectal and gastric SRCC and a clear distinction from non-SRCC. The immune microenvironments of gastric and colorectal SRCC have not been comprehensively examined. We isolated RNA from formalin-fixed, paraffin-embedded (FFPE) sections of 34 tumor specimens, 10 colorectal SRCC, 24 gastric SRCC, 4 non-SRCC colorectal (CCC), and 3 gastric adenocarcinoma (GCC) samples. The PanCancer Immune Profiling Panel was used to evaluate the expression of 770 immune-related genes. We compared the expression profiles of colorectal and gastric SRCC and non-SRCC adenocarcinoma. We found that the immune-related gene expression profiles (GEPs) of colorectal SRCC (CR-SRCC) and gastric SRCC (G-SRCC) were distinct from the non-SRCC. A total of 127 genes were upregulated and 32 downregulated in CR-SRCC compared to CCC. Only two genes (CCL27 and LAIR2 reached statistical significance (p-adj < 0.05)) among the differentially expressed genes in G-SRCC compared to GCC. None of the clinically relevant immune checkpoints were significantly differentially expressed in SRCC vs. non-SRCC. Overall, we noted a relative abundance of CD8+ cells in CR-SRCC and G-SRCC and relative overexpression of genes involved in innate immune response including the complement pathway. Finally, we identified IL13RA2 as a potential biomarker and therapeutic target candidate for CR-SRCC. The immune microenvironments of CR-SRCC and G-SRCC are distinct from non-SRCC. Broadly, both CR-SRCC and G-SRCC are characterized by a complex immune microenvironment that features cytotoxic cells and innate immune activity that may facilitate immune evasion. Full article

Background/Objectives: The 2021 WHO Classification of Central Nervous System (CNS) tumors emphasizes the integration of molecular data with histopathological features. Lower-grade gliomas (LGGs) represent a heterogeneous group of neoplasms with variable clinical behavior. This study aimed to explore the molecular landscape of a single-institution series of LGGs using targeted next-generation sequencing (NGS). Methods: Eleven adult patients diagnosed with LGG between 2015 and 2024 at Cattinara University Hospital (Trieste, Italy) were retrospectively analyzed. DNA and RNA were extracted from formalin-fixed, paraffin-embedded (FFPE) tissue and analyzed using the TruSight Oncology 500 panel (Illumina). Mutational, amplification, and transcriptomic profiles were evaluated. Results: IDH1 mutations were the most frequent alteration (75%), commonly co-occurring with TP53 and ATRX mutations, consistent with the canonical IDH-mutant astrocytoma profile. CDK4 amplification was found in four cases, while MYCN amplification and MET amplification were each identified in isolated cases. Two diffuse IDH-wild-type gliomas displayed aggressive clinical courses and shorter survival, and one was reclassified as glioblastoma (grade 4) based on EGFR amplification. The transcriptome analysis revealed heterogeneous expression signatures and distinct clustering of IDH1/ATRX-mutant tumors. Conclusions: Targeted NGS confirmed the key molecular features of diffuse gliomas and enabled precise WHO 2021 classification even in archival FFPE samples. Despite the exploratory nature of the analysis on a small population, the study underscores the biological and transcriptional heterogeneity of LGGs and highlights the limitations of tumor-only sequencing approaches. Broader genomic profiling and matched normal controls are warranted to refine the interpretation of rare or non-canonical variants. Full article

Background: MicroRNAs (miRNAs) such as miR-155-5p and miR-221-3p are key regulators of gene expression in cancer. Although both have been implicated in colorectal cancer (CRC) and papillary thyroid carcinoma (PTC), data on their regional expression profiles and clinical associations remain scarce, particularly in the Middle East. This study assessed the expression patterns and clinical relevance of miR-155-5p and miR-221-3p in CRC and PTC patients from Sulaymaniyah Province, Iraq. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor and adjacent normal tissue samples were collected from 60 CRC patients and 50 PTC patients. miRNA expression levels were quantified using real-time quantitative PCR (RT-qPCR) and analyzed by the ΔΔCt method, adjusted for tumor cellularity. Statistical analyses were conducted to evaluate associations between miRNA expression and clinicopathological parameters. Results: miR-155-5p and miR-221-3p were frequently overexpressed in both CRC (65%) and PTC (72% and 68%, respectively). In CRC, miR-155-5p expression correlated significantly with histological grade, tumor location, and TNM stage (p < 0.05), while miR-221-3p did not show significant associations with clinicopathological features. In PTC, miR-155-5p exhibited a trend toward association with TNM stage (p = 0.02). No significant differences in expression levels of these miRNAs were observed between CRC and PTC samples. Conclusions: Overall, miR-155-5p and miR-221-3p are consistently overexpressed in CRC and PTC, indicating their potential as diagnostic biomarkers. miR-155-5p, in particular, shows promise as a marker of disease progression in CRC. These findings underscore the importance of region-specific studies in advancing our understanding of the molecular landscape of cancer. Full article

Background: Colorectal cancer (CRC) remains a major global health problem, with rising incidence among younger individuals. The implementation of next-generation sequencing (NGS) has enabled comprehensive multigene analysis to identify cancer-predisposing variants and molecular alterations in tumors. However, data on age-related genetic differences in CRC from Central and Eastern European populations, including Poland, remain limited. Methods: This study aimed to explore molecular differences in CRC between patients aged ≤50 and >50 years in a Polish cohort. Tumor DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from 54 treatment-naive patients. Targeted sequencing of hot spot regions of 50 genes with known association to cancer was performed using an AmpliSeq for Illumina Cancer Hotspot Panel v2. Results: Variant frequencies in younger vs. older patients were: TP53 (71.4% vs. 57.6%), APC (57.1% vs. 45.5%), KRAS (28.1% vs. 72.7%), NRAS (28.6% vs. 0%), SMAD4 (9.5% vs. 12.1%), PIK3CA (14.3% vs. 24.2%), and FBXW7 (4.8% vs. 14.7%). Co-occurrence of APC/KRAS/TP53 variants was observed in 20% of cases. KRAS mutations were significantly more frequent in older patients (p-value = 0.001), while NRAS mutations occurred exclusively in younger patients (29% vs. 0%, p = 0.021). Overall, 46% of patients exhibited multiple gene alterations (≥3 mutations). Notably, IDH1 and CTNNB1 variants were found only in patients with better prognosis, whereas TP53 variants were nearly five times more frequent in patients with worse outcomes. Conclusions: Multigene panel sequencing revealed distinct age-related molecular patterns in CRC. Younger patients were more likely to harbor NRAS variants, whereas KRAS alterations predominated in older individuals. These findings underscore the relevance of NGS-based multigene profiling for risk stratification and personalized therapy in colorectal cancer. Full article

Three-dimensional cell model systems such as tumour organoids allow for in vitro modelling of self-organized tissue with functional and histologic similarity to in vivo tissue. However, there is a need for standard protocols and techniques to confirm the presence of cancer within organoids derived from tumour tissue. The aim of this study was to assess the utility of a Nanostring gene expression-based machine learning classifier to determine the presence of cancer or normal organoids in cultures developed from both benign and cancerous stomach biopsies. A prospective cohort of normal and cancer stomach biopsies were collected from 2019 to 2022. Tissue specimens were processed for formalin-fixed paraffin-embedding (FFPE) and a subset of specimens were established in organoid cultures. Specimens were labelled as normal or cancer according to analysis of the FFPE tissue by two pathologists. The gene expression in FFPE and organoid tissue was measured using a 107 gene Nanostring codeset and normalized using the Removal of Unwanted Variation III algorithm. Our machine learning model was developed using five-fold nested cross-validation to classify normal or cancer gastric tissue from publicly available Asian Cancer Research Group (ACRG) gene expression data. The models were externally validated using the Cancer Genome Atlas (TCGA), as well as our own FFPE and organoid gene expression data. A total of 60 samples were collected, including 38 cancer FFPE specimens, 5 normal FFPE specimens, 12 cancer organoids, and 5 normal organoids. The optimal model design used a Least Absolute Shrinkage and Selection Operator model for feature selection and an ElasticNet model for classification, yielding area under the curve (AUC) values of 0.99 [95% CI: 0.99–1], 0.90 [95% CI: 0.87–0.93], and 0.79 [95% CI: 0.74–0.84] for ACRG (internal test), FFPE, and organoid (external test) data, respectively. The performance of our final model on external data achieved AUC values of 0.99 [95% CI: 0.98–1], 0.94 [95% CI: 0.86–1], and 0.85 [95% CI: 0.63–1] for TCGA, FFPE, and organoid specimens, respectively. Using a public database to create a machine learning model in combination with a Nanostring gene expression assay allows us to allocate organoids and their paired whole tissue samples. This platform yielded reasonable accuracy for FFPE and organoid specimens, with the former being more accurate. This study re-affirms that although organoids are a high-fidelity model, there are still limitations in validating the recapitulation of cancer in vitro. Full article

Viral infections in humans and animals are increasing, and retrospective studies using formalin-fixed, paraffin-embedded (FFPE) samples reveal recurring outbreaks over past decades. However, the impact of pre-analytical factors like fixation and autolysis on immunohistochemistry (IHC) remains insufficiently understood. To examine how autolysis, fixation duration (6–72 h) and formalin concentration (2.5–25%) influence histology and IHC of canine distemper virus (CDV, Morbillivirus canis), interferon-β (IFN-β), and selected IFN-stimulated genes (ISGs), the study was conducted using an in vitro model based on persistently CDV-infected and non-infected DH82 cells (canine histiocytic sarcoma cell line). Autolysis led to a progressive loss of cell morphology, whereas formalin fixation had minimal impact. CDV nucleoprotein, ISG15, and myxovirus resistance protein (Mx) showed stable immunohistochemical signals across all fixation conditions and remained detectable after prolonged autolysis. CDV infection upregulated ISG15 and Mx. In contrast, IFN-β and phosphorylated protein kinase R (pPKR) exhibited variable staining and did not distinguish infected from non-infected samples. Overall, autolysis had a stronger negative impact on IHC signal quality than fixation parameters. Despite the limitations of the in vitro model, the robustness of CDV, ISG15, and Mx under suboptimal conditions highlights their potential utility as virus-sensing markers in FFPE material. Full article

Scabies, caused by Sarcoptes scabiei var. hominis, remains difficult to diagnose in histological routine when mite fragments are sparse or degraded. We explored whether Fourier-transform infrared (FTIR) microscopy can detect chitin-associated spectral signatures of scabies mites in formalin-fixed paraffin-embedded (FFPE) human skin sections and distinguish them from surrounding host tissue. FFPE sections from six patients with histologically confirmed crusted scabies were analysed by FTIR imaging, univariate mapping of selected bands, and multivariate image analysis within the 1000–1200 cm⁻¹ carbohydrate region. Spectra from mite exoskeleton, stratum corneum, and dermis were compared, and absorbance at 1072 cm⁻¹ was quantified across all samples. Mite regions showed consistently higher 1072 cm⁻¹ absorbance than adjacent epidermal and dermal compartments, and unsupervised clustering reproducibly delineated mite-associated domains that co-localised with structures identified on haematoxylin-and-eosin-stained sections. Within this small, preliminary proof-of-concept cohort and in the absence of healthy or disease controls, the data do not allow estimation of clinical diagnostic performance at the patient level, but demonstrate the technical feasibility and analytical robustness of FTIR microscopy for intra-lesional detection of chitin-rich parasite structures in scabies lesions and provide a framework for future comparative studies in larger, prospectively collected cohorts. Full article

Background/Objectives: Formalin-fixed paraffin-embedded (FFPE) tissues are sometimes the only DNA source for forensic applications. The quantity and integrity of the DNA extracted from these samples depend on multiple factors. In this work, we analyzed, for the first time, whether Masson’s trichrome (MT) staining alters the results of genetic profiles obtained from DNA extracted from FFPE tissue sections. Methods: Three pairs of sections from the year 2024 and three pairs from the year 2001 were analyzed. Each pair consisted of serial sections, one stained with hematoxylin and eosin and the other with MT. DNA was extracted using the PrepFiler Express BTA™ Forensic DNA Extraction Kit and quantified by real-time PCR using the Quantifiler™ HP DNA Quantification Kit. DNA samples were processed for short tandem repeat (STR) profiling using the GlobalFiler™ PCR Amplification Kit. The amplified alleles were separated and analyzed using an ABI PRISM^® 3500 genetic analyzer. Results: All MT-stained samples showed deficiency in most or all of the parameters assessed: DNA yield, degradation index, number of alleles detected, random match probability value, and intensity of the electropherogram peaks. In fact, DNA could not even be quantified in the samples processed in 2001. Conclusions: These results could be due to the large number of acids used in MT staining, which cause chemical modification and hydrolysis of DNA, affecting the success of PCR-based methods used subsequently. In conclusion, DNA obtained from MT-stained FFPE tissue sections may be highly degraded and should therefore be used with great caution in forensic settings. Full article

Fusobacterium nucleatum (Fn) is increasingly recognized as a cancer-associated bacterium, yet reliable quantification in human specimens is challenging due to low bacterial burden and abundant host DNA. We analyzed 145 Fusobacterium genomes to design primers targeting conserved regions of the fadA adhesin gene and developed a duplex quantitative real-time PCR (qPCR) assay for simultaneous detection of fadA and a human PGT as an internal control. Analytical sensitivity, specificity, precision, and reproducibility were evaluated using serially diluted Fn DNA, spike-in experiments with human DNA, and cross-platform/operator validation. Clinical performance was assessed in colorectal cancer patient tissues, including fresh tissue (n = 24) and formalin-fixed paraffin-embedded (FFPE) samples (n = 22), using 16S rRNA-based methods as references. The assay successfully detected all four major Fn subspecies (nucleatum, animalis, polymorphum, and vincentii). The limit of detection was ≤0.1 pg, with no interference between duplex targets. Spike-in experiments demonstrated consistent target detection in human-DNA-rich samples, with strong linearity (R² = 0.998) across dilutions. High precision (coefficient of variations < 5%) was observed across intra-day, inter-day, inter-instrument, and inter-operator evaluations. In fresh tissues, the assay yielded 86% sensitivity, 94% specificity, and 92% accuracy. Using the FFPE samples, the assay achieved 91% sensitivity and 100% specificity, confirming robust classification in both clinical samples. This duplex qPCR assay enables broad detection of Fn with high analytical performance in both fresh and FFPE tissues. Its simplicity, reproducibility, and compatibility with pathology workflows support deployment in multi-center studies and downstream applications in diagnostic studies and prognostic modeling. Full article

Objectives: Anti-TNF-α therapies have transformed the management of Inflammatory Bowel Disease (IBD), yet a substantial proportion of patients fail to respond, highlighting the urgent need for predictive biomarkers. Mucosal TNF-α mRNA quantification in fresh biopsies has shown promise but showed several problems for routine clinical use. This study aimed to validate a clinically feasible method for TNF-α quantification in formalin-fixed paraffin-embedded (FFPE) tissues and to assess its correlation with established measures of disease activity. Methods: FFPE and matched fresh-frozen biopsies from 54 ulcerative colitis patients were analyzed. Total RNA was extracted from FFPE sections, and TNF-α RNA was quantified by RT-qPCR and compared with fresh tissue expression levels. Molecular data were correlated with clinical (pMayo), endoscopic (Mayo Endoscopic Score, MES), and histological (Geboes) indices. Results: TNF-α expression in FFPE samples strongly correlated with fresh tissue levels (r = 0.83, p < 0.0001). High TNF-α expression in FFPE tissue was significantly associated with active endoscopic mucosal disease (MES ≥ 1; OR 28, 95% CI 3.31–237; p < 0.0001) and with histological inflammation (Geboes ≥ 3.1; OR 0.12, 95% CI 0.02–0.59; p = 0.009). Fresh tissue TNF-α levels showed similar associations. Clinical parameters such as age, sex, and pMayo score did not significantly correlate with mucosal TNF-α expression. RT-qPCR quantification of TNF-α in FFPE tissue is a reliable, cost-effective surrogate for fresh biopsy analysis and correlates strongly with endoscopic and histological disease activity. Conclusions: This method offers a practical approach for integrating molecular biomarkers into routine pathology workflows, supporting the implementation of personalized treatment strategies in IBD. Full article

Background: Vulvar squamous cell carcinoma (VSCC) is an uncommon yet increasingly relevant malignancy characterized by two distinct etiopathogenetic pathways: HPV-associated and HPV-independent. Data from Eastern Europe remain scarce, where demographic and diagnostic variability may influence disease presentation and outcomes. Purpose: This study aimed to assess the epidemiologic characteristics, HPV status, surgical management, and postoperative morbidity of VSCC in a Romanian single-center cohort, providing real-world evidence from an underrepresented region. Methods: A retrospective analysis was conducted on all 35 consecutive patients with histologically confirmed vulvar squamous cell carcinoma (VSCC) diagnosed and treated between January 2017 and December 2024 at the Department of Obstetrics and Gynecology, County Emergency Clinical Hospital of Craiova, Romania. Demographic, histopathologic, and surgical data were reviewed. HPV genotyping was performed on formalin-fixed paraffin-embedded (FFPE) tissue using PCR-based methods. Results: HPV DNA was detected in 31.4% of cases, predominantly genotypes 16, 18, and 33. HPV-positive patients were significantly younger than HPV-negative ones (median 58 vs. 72.5 years, p < 0.001), supporting the dual-pathway model of carcinogenesis. Early postoperative complications occurred in 65.7% of patients and late morbidity in 71.4%, secondary lymphedema. Surgical radicality was not significantly associated with early complications or length of hospitalization. Conclusions: This study highlights the epidemiologic and surgical patterns of VSCC in an Eastern European population, showing that conservative surgical strategies can maintain oncologic safety while reducing morbidity. These findings emphasize the need for standardized HPV testing, optimized perioperative care, and improved surveillance programs to enhance outcomes and survivorship. Full article

Background: Molecular diagnostics has become a critical component of precision oncology in solid tumors, including colorectal cancer, yet the use of formalin-fixed, paraffin-embedded (FFPE) tissue often suffers from DNA degradation that compromises sequencing quality. This study aimed to evaluate the feasibility and effectiveness of using fresh, intraoperatively collected tumor tissue for next-generation sequencing-based molecular diagnostics in colorectal cancer. Methods: Tissue samples from 24 patients undergoing colorectal tumor resection were obtained based on macroscopic evaluation and tested with a custom gene panel. Sequencing metrics, mutation profiles, and correlations with clinical and pathological features were analyzed. Results: All samples yielded high-quality sequencing data. Oncogenic or likely oncogenic variants were detected in 21 out of 24 samples (87.5%), predominantly affecting genes frequently involved in colorectal cancer carcinogenesis, including APC, TP53, and KRAS. In three cases, no typical mutations were found despite visual confirmation of tumor tissue during surgery, which may be attributed to insufficient tumor cellularity or molecular alterations beyond the panel’s scope. Conclusions: The results support the use of fresh tissue as a high-quality source for molecular diagnostics, capable of reducing turnaround time and avoiding formalin-induced artifacts. However, the findings also highlight the diagnostic risk of relying solely on macroscopic tumor assessment without histological confirmation. Overall, fresh tissue-based testing represents a promising yet currently investigational approach that can enhance molecular diagnostics in colorectal cancer. Full article