J. Mol. Pathol., Volume 7, Issue 1 (March 2026) – 14 articles

Background/Objectives: Chronic lymphocytic leukemia (CLL) is characterized by genetic and epigenetic alterations. This study aimed to assess the methylation status of E-Cadherin and MMP-9 gene promoters and to explore their relationships with disease pathogenesis and hematological parameters in CLL patients. Methods: A case–control study was conducted with 70 newly diagnosed CLL patients and 70 age- and sex-matched healthy controls. Promoter methylation of E-Cadherin and MMP-9 genes was evaluated using methylation-specific PCR (MSP) and methylation-sensitive restriction enzyme PCR (MSRE-PCR), respectively. Results: The median patient age was 62 years, and 68.5% were males. Binet stage A was the most common stage (57.3%). E-Cadherin promoter methylation was detected in 75.7% of CLL patients and 77.1% of controls (p = 0.91), showing no significant association with disease occurrence; however, it showed a significant correlation with higher lymphocyte counts (p = 0.01). In contrast, MMP-9 promoter methylation was significantly less frequent in CLL cases (70.0%) than in controls (100%, p = 0.001). Unmethylated MMP-9 correlated significantly with female gender (p = 0.02), lower hemoglobin (p = 0.031), reduced platelet counts (p = 0.001), and higher lymphocyte counts (p = 0.035). Conclusions: MMP-9 promoter hypomethylation may play a pathogenic role in CLL and is associated with female gender and cytopenia, whereas E-Cadherin methylation appears to be non-specific. MMP-9 methylation status could therefore serve as a potential biomarker for CLL biology and prognosis. Full article

Objective: This study examined the frequency of entosis in solid tumors of various origins (colorectal cancer, breast cancer, and lung cancer) and its association with clinical and pathological characteristics. It also examined survival and copy number alterations (CNAs) in genes associated with stem cells. The aim was to assess the potential prognostic value of entotic events in tumors. Methods: A total of 238 patients were included: 96 with colorectal cancer (CRC), 45 with lung cancer (LC), and 97 with breast cancer (BC). Entotic cell-in-cell (CIC) structures were evaluated on hematoxylin–eosin–stained slides using Mackay’s criteria. A CIC frequency >0.1 per 20 high-power fields was considered positive. Clinicopathological parameters, overall survival (CRC), metastasis-free survival (LC and BC), and CNA profiles of stemness-related genes were analyzed. Amplifications of MAP1LC3A and other chromosomal loci were assessed. Results: CRC demonstrated the highest entosis rate, more than two-fold higher compared with BC and LC (p < 0.05). Entosis correlated with high tumor grade (G3) in CRC (p = 0.03). In LC, CIC-positive tumors were more frequent in patients with lymph-node metastases (p = 0.02), whereas in BC, the opposite trend was observed (p = 0.02). It was noted that in patients with stage III–IV LC, the frequency of entosis was significantly higher than in patients with stage I–II cancer (p = 0.03). CIC-positive status was associated with poorer overall survival in CRC (p = 0.03) and reduced metastasis-free survival in LC (p = 0.011). In breast cancer, no statistically significant survival differences were observed. Tumors harboring two or more stemness-gene amplifications showed significantly higher entosis frequency regardless of tumor site. A strong association was identified between entosis and MAP1LC3A amplification. Conclusions: Enosis is not a random morphological phenomenon but a process associated with unfavorable tumor characteristics, high malignancy, reduced survival, and amplification of stem cell-related genes. The results of this study confirm the working hypothesis that entosis may contribute to the emergence of aneuploid clones of tumor cells, including those containing amplifications of stem cell-associated genes. This positions entosis as a potential factor in tumor genetic heterogeneity, which is particularly important in the context of therapeutic selection pressure. The observed association between high entosis frequency and the presence of ≥2 stem cell gene amplifications, as well as its association with poor prognosis in colorectal and lung cancer, highlights its potential value as a prognostic indicator. Furthermore, MAP1LC3A amplification data may serve as a molecular marker of entotic activity and a potential therapeutic target. Full article

Periodontitis is a dysbiosis-driven inflammatory disease in which a pathogenic subgingival biofilm disrupts the host–microbe equilibrium and promotes progressive loss of tooth-supporting tissues. While periodontal destruction has traditionally been explained mainly through the host immune response, increasing experimental and clinical evidence suggests that epithelial–mesenchymal transition (EMT)-like changes in the gingival epithelium may contribute to barrier failure and tissue remodeling during disease progression. EMT is characterized by reduced epithelial adhesion and polarity, alongside a shift toward a mesenchymal-like phenotype with enhanced motility and impaired epithelial barrier function. This narrative review focuses on how periodontal pathogens, particularly red complex organisms and keystone species, may trigger gingival EMT through virulence factors such as gingipains, fimbriae, lipopolysaccharide, and outer membrane vesicles. These microbial signals can hijack host pathways including TGF-β/Smad, Wnt/β-catenin, and Notch to drive EMT-associated transcriptional changes and downstream functional consequences. Collectively, pathogen-induced gingival EMT may facilitate deeper microbial invasion, perpetuate chronic inflammation, impair wound healing, and contribute to fibrotic remodeling, ultimately linking microbial dysbiosis to connective tissue destruction. Understanding these mechanisms may support the development of EMT-related biomarkers and targeted interventions aimed at preserving epithelial barrier stability in periodontitis. Full article

Background: Amyloidosis involving the heart is one of the types of the disease recognized in humans and has been previously described in dogs. To date, no data regarding the presence of amyloid in cardiac tissues of a large group of feline patients have been published. Our research aimed to analyze the presence and localization of amyloid in the atrial and ventricular cardiac tissue in retrospectively enrolled cats diagnosed with various types of primary cardiomyopathies, hyperthyroidism-induced cardiomyopathy, myocarditis, and generalized disorders. Methods: This study was conducted on atrial specimens obtained from 119 animals and on ventricular specimens obtained from 69 animals from that group. The atrial and ventricular specimens obtained from the enrolled animals were stained with Congo Red and evaluated in a light microscope and polarized light for the presence of amyloid deposits. Results: Five cases from the enrolled group turned out positive for amyloid deposits: three cats diagnosed with feline hyperthyroidism, one cat diagnosed with kidney glomerulonephritis, and one cat diagnosed with restrictive cardiomyopathy. In all positive cats, the amyloid deposits were present within the small intramural coronary arteries of the left ventricular free wall and interventricular septum and/or left and right atrium. No myocardial amyloid deposits were identified in the study group. Conclusions: In conclusion, cardiac coronary arterial amyloidosis, although infrequent, can be observed in cats. Full article

Background/Objectives: In metastatic colorectal cancer (mCRC) the evaluation of mismatch repair (MMR) and microsatellite instability (MSI) status is essential to identify patients eligible for treatment with immune-checkpoint inhibitors (ICI). This study aims to evaluate the potential utility of Comprehensive Genomic Profiling (CGP) in assessing MSI status, in addition to other immunotherapy-predictive biomarkers such as high tumor molecular burden (TMB) and the POLE and POLD1 mutations. Methods: A total of 138 mCRC tumor samples underwent a first-level molecular test (MMR status by immunohistochemistry, MSI by a melting-based PCR approach and RAS/BRAF mutational status by a small next-generation sequencing (NGS) panel) and second-level CGP analysis by the FoundationOne CDx assay. The prevalence of dMMR and MSI tumors was reported. Moreover, the concordance between the MMR and MSI status was determined, and discordant cases were discussed. Results: Twelve cases (8.7%) were MMR-deficient (dMMR); 10 showed high MSI and TMB (>10 mut/Mb). MSI status assessed by CGP and PCR was concordant in all cases except one MSH6-deficient tumor. Two dMMR cases were stable with low TMB. Moreover, in two MLH1/PMS2-deficient cases CGP revealed pathogenic alterations in the MSH2 and MSH6 genes; in both cases, the MLH1 promoter was hypermethylated. A high TMB was the only positive biomarker in 11 cases with a proficient MMR system and no MSI. Conclusions: MSI assessment by CGP analysis showed high concordance (98%) with MMR and was helpful in evaluating ICI eligibility in three out of twelve dMMR cases. Overall, compared to standard methods, analyzing a broader range of microsatellite loci and the simultaneous assessment of multiple predictive biomarkers by CGP may increase diagnostic accuracy and improve therapeutic assessment. Full article

Background/Objectives: Phospholipase C zeta (PLCZ1; PLCζ) is a sperm-specific enzyme responsible for the Ca²⁺ oscillations required for oocyte activation, and altered PLCζ expression has been associated with fertilization failure in assisted reproductive technologies, particularly intracytoplasmic sperm injection (ICSI). This study aimed to develop and analytically validate a flow cytometry–based protocol for PLCζ quantification in human spermatozoa. Methods: The assay was established using normozoospermic samples and included validated positive and negative technical controls. Antibody specificity was confirmed by Western blot analysis. A defined gating strategy was used to assess linearity between fluorescence intensity and PLCζ expression. Analytical performance was evaluated for precision, reproducibility, stability, and sensitivity, including applicability to low sperm concentrations. Results: A linear relationship between fluorescence intensity and PLCζ expression was demonstrated. The assay showed high precision, reproducibility, and stability, with consistent results in samples stored up to 24 h at room temperature or up to one week post-fixation at 4 °C. Sensitivity testing confirmed suitability for low sperm concentrations. Conclusions: This work provides a standardized and analytically validated framework for PLCζ quantification using flow cytometry. Although the assay measures protein expression rather than functional competence or subcellular localization, it establishes a solid analytical basis for future studies to define clinically relevant PLCζ thresholds and assess its value as a biomarker of fertilization capacity. Full article

Diabetic retinopathy (DR) is a progressive retinal disorder and a leading cause of vision impairment worldwide affecting the livelihood of millions. Its pathogenesis is driven by chronic hyperglycemia-induced neuronal and microvascular injury, leading to capillary occlusion, increased vascular permeability, and the eventual formation of fragile neo vessels. These changes mark the progression from non-proliferative diabetic retinopathy (NPDR) to proliferative diabetic retinopathy (PDR). Diabetic macular edema (DME), characterized by blood–retinal barrier disruption and macular fluid accumulation, further contributes to vision loss. This review provides an integrative perspective on the cellular and molecular mechanisms of DR, highlighting both vascular and neuroglial contributions to retinal pathology. Current therapeutic approaches, including anti-VEGF agents and corticosteroids, offer symptomatic relief but are limited by the need for repeated administration and variability in patient response. Emerging evidence implicates the role of thioredoxin-interacting protein (TXNIP) as one of mediators of the disease progression. Strongly upregulated under hyperglycaemic stress, TXNIP induces oxidative damage, inflammation, and neuronal apoptosis, exacerbating neurovascular dysfunction. We explore potential therapeutic strategies such as gene therapy, TXNIP-targeted molecular interventions, and stem cell-based approaches aimed at achieving long-term modulation of disease mechanisms. This article thus attempts to address a comprehensive understanding of DR pathophysiology and innovative new strategies to improve long-term visual outcomes. Full article

Objectives: This study evaluated the impact of combining icariin with a bovine xenograft on the enhancement of early socket healing in Wistar rats. Methods: Male Wistar rats underwent incisive extraction and were randomized into three groups (n = 8/group): (1) control, (2) xenograft, and (3) icariin–xenograft. On days 7 and 14, the animals were decapitated and their mandibles were examined. Histological analysis was conducted to assess the collagen matrix and the expression of BMP-2 and HIF-1α. Results: The icariin–xenograft group exhibited superior outcomes compared to the xenograft-alone and control groups. Histological analysis showed an earlier arrangement of connective tissue and an improved collagen matrix outcome in the icariin-treated sockets. Immunohistochemistry revealed elevated BMP-2 and HIF-1α expression in the icariin–xenograft group, indicating enhanced osteogenic and angiogenic signaling. Conclusions: Icariin-enhanced xenografts speed up the repair of early extraction sockets by enhancing the development of the collagen matrix and increasing the activity of pathways that promote osteogenesis and angiogenesis in low-oxygen conditions. This bioactive grafting technology appears to be a cost-effective method for preserving sockets and performing regenerative therapy in dentistry. Full article

Background: Differentiating atypical endometrial hyperplasia, also known as endometrial intraepithelial neoplasia (EAH/EIN) from endometrial hyperplasia without atypia is crucial due to the higher risk of progression to endometrioid adenocarcinoma, associated with atypical lesions. Immunohistochemical markers such as PAX2 and PTEN have emerged as potential adjuncts to improve diagnostic accuracy in morphologically challenging cases. Objective: To evaluate the diagnostic value of PAX2 and PTEN expression in distinguishing atypical from endometrial hyperplasia without atypia in the Bulgarian population. Materials and Methods: A total of 96 endometrial hyperplasia cases (48 typical, 48 atypical) were included. Histopathological evaluation was performed on hematoxylin and eosin–stained sections, with two experienced pathologists confirming diagnoses according to the WHO criteria. Immunohistochemical analysis of PTEN and PAX2 was conducted on formalin-fixed, paraffin-embedded tissue sections. Results: PTEN expression loss was observed in 6% (3/48) of hyperplasia without atypia cases, compared with 81.3% (38/48) of EAH/EIN cases. For PAX2, strong nuclear staining was retained in 60% (29/48) of endometrial hyperplasia without atypia cases, with no complete loss of expression. In contrast, 64.6% (31/48) of EAH/EIN cases showed complete loss of PAX2 expression, while only 35.4% (17/48) preserved nuclear immunoreactivity. Together, these results highlight clear and consistent differences in PTEN and PAX2 expression between hyperplasia without atypia and EAH/EIN and may aid pathologists in distinguishing these two entities in routine diagnostic practice. Conclusions: Expression loss of PAX2 and PTEN is significantly associated with EAH/EIN. Immunohistochemical evaluation of these markers provides valuable adjunctive information for the diagnosis of morphologically ambiguous cases and may enhance diagnostic reproducibility and accuracy. Incorporating PAX2 and PTEN into routine assessment may guide appropriate clinical management and risk stratification of patients with endometrial hyperplasia. Full article

The human microbiome is often presented as “the next genetics,” with the expectation that microbial profiles will explain complex diseases and yield new therapies. Yet for most conditions, it remains unclear whether microbiome changes act as causal drivers or primarily mirror underlying host biology and pathology. In this narrative review, we argue that microbiome causality is frequently overstated relative to the roles of host genetics and the environment, and we explore the implications for molecular pathology. We outline a simple framework in which the microbiome can act as (i) a primary driver, (ii) a conditional mediator or effect modifier or (iii) an association biomarker that mainly reflects upstream processes. We then use marital and household studies as natural experiments to test whether chronic diseases track more strongly with a shared microbiome or with a shared lifestyle and host susceptibility. Across metabolic, inflammatory, neurodegenerative and ageing-related outcomes, spouses show only low to modest disease concordance, which is difficult to reconcile with a universally strong, transmissible microbiome causality. Adult microbiomes instead appear mostly host-constrained and context-dependent, acting more as destabilisers of homeostasis and amplifiers of allostatic load than as independent disease-causing factors. For molecular pathology, this suggests that microbiome features are often most informative as biomarkers integrated alongside host genomics, immune context and histopathology, rather than as standalone targets. Study designs and diagnostic workflows should therefore jointly model the host genome, environment, behaviour and microbiome within broader systems medicine frameworks. Full article

Autoimmune diseases arise from the loss of antigen-specific tolerance, leading to chronic inflammation and tissue damage. Peptide-based therapeutics provide a precise strategy to restore immune balance by targeting autoreactive lymphocytes and antigen-presenting cells in tolerogenic contexts. These therapies induce regulatory T cells, modulate APC phenotypes, and can interfere with proinflammatory signaling. Advances in delivery technologies, including nanoparticles, lipid nanoparticles, hydrogels, and conjugates, improve peptide stability, co-deliver tolerogenic cues, and enable targeted antigen presentation. mRNA lipid nanoparticle platforms permit in situ expression of peptides or immunomodulatory molecules. Preclinical studies in models of type 1 diabetes, multiple sclerosis, and lupus demonstrate robust antigen-specific tolerance, while early-phase clinical trials show safety and mechanistic engagement. Insights from approved peptide therapies in allergy and other fields underscore the importance of epitope selection, delivery context, and biomarker-guided development. Collectively, these strategies suggest that rationally formulated, precisely targeted peptide therapeutics hold promise for achieving durable immune tolerance in autoimmune disease. Full article

Background/Objectives: Combining Whole Slide Imaging (WSI) and Artificial Intelligence (AI) in digital pathology (DP) is accelerating the field of diagnostic pathology by improving analysis metrics accuracy, reproducibility, and speed. AI applications in pathology include automated image capture, assessment and analysis, risk stratification, and prognostic prediction. This integration introduces significant challenges, including data quality, high computational demands, the ability to generalize across different settings, and a range of ethical considerations. This review provides an end-to-end roadmap covering WSI acquisition, preprocessing, and deep learning (DL) channels through tumor recognition, biomarker prediction, and evolving computational methods such as original models and combined learning, highlighting the specific challenges and opportunities of WSI-attached AI in pathology. Methods: This review provides a WSI-centric analysis that examines AI and DL applications specifically as they overlap with the acquisition, processing, and computational analysis of WSI. Therefore, this review aims to comprehensively examine the challenges and pitfalls associated with the use of WSI in AI-Based Digital Pathology. Results: Pre-analytical factors like how the tissue is prepared, staining, and scanning artifacts affect AI and contain possible post-analytical barriers such as the range of colors used, color standardization, and algorithm transparency. Furthermore, there may be bias found in the training datasets that can blur the ethical and legal boundaries alongside regulatory uncertainty. Conclusions: Even though there is an array of challenges, AI applied in DP can enhance the accuracy of medical diagnosis, encourage workflow efficiency, facilitate cross-collaboration for pediatric research, and enable research into rare diseases. Further development on the topic needs to focus on defining standard operating procedures and guidelines alongside dependable datasets through teamwork from various scientific fields. Full article

Background: Dengue is a growing mosquito-borne viral infection of global concern. It remains a major public health challenge in Nepal, where reliable biomarkers for disease staging and prognosis are lacking. In this study, we investigated circulating microRNA-1246 (miR-1246) as a potential diagnostic and prognostic marker in dengue infection. Methods: Serum samples from 21 dengue-positive patients and 20 healthy controls were analyzed by quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR), with RNU6 as an internal control. Results: Dengue patients showed markedly elevated miR-1246 levels, with a mean 47-fold increase compared to controls (p = 0.001). Expression varied by disease stage, peaking in IgM positive cases, declining in weakly positive IgM patients, and reaching the lowest levels in IgG positive convalescent cases, a pattern consistent with clinical parameters such as platelet recovery. Receiver Operating Characteristic (ROC) analysis further highlighted diagnostic potential, yielding an Area Under the Curve (AUC) of 0.79, sensitivity of 95.24%, and specificity of 60.00%. Conclusions: These findings imply that miR-1246 is drastically dysregulated during dengue infection and could be a useful biomarker for tracking the intensity and course of the illness. Full article