J. Mol. Pathol., Volume 7, Issue 1 (March 2026) – 9 articles

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