Genome-wide association study indicates novel associations of annexin A13 to secretory and GAS2L2 with mucous otitis media.

To evaluate the genetics of chronic nonsuppurative otitis media (OM). We performed a genome-wide association study of 429,599 individuals included in the FinnGen study using three different case definitions: combined chronic nonsuppurative OM (7034 cases) (included serous and mucous chronic OM), mucous chronic OM (5953 cases), and secretory chronic OM (1689 cases). Individuals without otitis media were used as controls (417,745 controls). We used immunohistochemistry (IHC) of the murine middle ear to evaluate the expression of annexin A13. Four loci were significantly associated (p < 1.7 × 10-8) with nonsuppurative OM. Three out of the four association signals included missense variants in genes that may play a role in otitis media pathobiology. According to our subtype-specific analyses, one novel locus, located near ANXA13, was associated with secretory OM. Three loci (near TNFRSF13B, GAS2L2, and TBX1) were associated with mucous OM. Immunohistochemistry of murine middle ear samples revealed annexin A13 expression at the apical pole of the Eustachian tube epithelium as well as variable intensity of the secretory cells of the glandular structure in proximity to the Eustachian tube. We demonstrated that secretory and mucous OM have distinct and shared genetic associations. The association of GAS2L2 with ciliary epithelium function and the pathogenesis of dysfunctional mucosa in mucous OM is suggested. The abundant expression of annexin A13 in the Eustachian tube epithelium, along with its role in apical transport for the binding and transfer of phospholipids, indicates the role of annexin A13 and phospholipids in Eustachian tube dysfunction.

Multi-Tissue Controls and Multiplex Immunocytochemistry in Pulmonary Cytology.

INTRODUCTION: The World Health Organization 2021 lung cancer classification highlights the central role of immunohistochemistry (IHC) in diagnostic pathology. Despite traditional IHC being essential, its limitation to one marker per tissue section brings challenges, particularly when facing cytological limitedly sized samples. To overcome these challenges, multiplex immunocytochemistry (mICC) techniques offer the simultaneous detection of multiple markers from a single section. These advances complement the highly complex imaging techniques that enable additional analyses of cellular interactions. METHODS: The present study outlines a comprehensive mICC methodology of an automated multiplex immunoperoxidase staining method and multiple tissue hybrid controls for ICC/mICC. Protocols are presented in detail and demonstrate a careful approach to optimizing various markers for diagnostic workup including immunotherapy. CONCLUSION: Multiplex IHC/ICC emerges as a transformative force in biomedical diagnostics and research. Beyond simultaneous marker detection, it unravels complexities within tissues - unveiling co-localization nuances, deciphering expression patterns, and enhancing understanding of cellular populations. As personalized treatments gain prominence, the study emphasizes the heightened importance of diagnostic tools and sample adequacy. The present methodological study, encapsulating an automated multiplex immunoperoxidase staining method, symbolizes a stride towards precision in pulmonary carcinoma diagnosis. Multi-tissue controls represent a key element in quality assurance in pathology laboratories.

IGF2BP3 Associates with Proliferative Phenotype and Prognostic Features in B-Cell Acute Lymphoblastic Leukemia.

The oncofetal protein insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) belongs to a family of RNA-binding proteins involved in localization, stability, and translational regulation of target RNAs. IGF2BP3 is used as a diagnostic and prognostic marker in several malignancies. Although the prognosis of pediatric B-cell acute lymphoblastic leukemia (B-ALL) has improved, a subgroup of patients exhibits high-risk features and suffer from disease recurrence. We sought to identify additional biomarkers to improve diagnostics, and we assessed expression of IGF2BP3 in a population-based pediatric cohort of B-ALL using a tissue microarray platform. The majority of pediatric B-ALL cases were positive for IGF2BP3 immunohistochemistry and were associated with an increased proliferative phenotype and activated STAT5 signaling pathway. Two large gene expression data sets were probed for the expression of IGF2BP3-the highest levels were seen among the B-cell lymphomas of a germinal center origin and well-established (KMT2A-rearranged and ETV6-RUNX1) and novel subtypes of B-ALL (e.g., NUTM1 and ETV6-RUNX1-like). A high mRNA for IGF2BP3 was associated with a proliferative "metagene" signature and a high expression of CDK6 in B-ALL. A low expression portended inferior survival in a high-risk cohort of pediatric B-ALL. Overall, our results show that IGF2BP3 shows subtype-specificity in expression and provides prognostic utility in high-risk B-ALL.

Fluoro-Chromogenic Labelling for Detection of MCM2 to Assess Proliferation Activity in HER2-amplified Breast Carcinomas.

Minichromosome Maintenance Protein 2 (MCM2) is critical in initiating DNA replication during the cell division process. As expressed intensively in all phases of the active cell cycle, MCM2 has been proposed as a novel biomarker to determine cellular proliferation. We aimed at clarifying the prevalence and clinical significance of MCM2 in HER2-amplified breast cancer subtype. MCM2 expression was studied in 142 primary HER2-amplified breast carcinomas by applying a novel fluoro-chromogenic immunohistochemistry and tailored digital image analysis to determine labelling index (MCM2-LI). The presence of MCM2 was detected with HRP-conjugated polymer and visualized with 3, 3'-diaminobenzidine tetrahydrochloride, in cytokeratin (CK)-positive and Cy2-IgG-labelled breast cancer cells of epithelial origin. Stained slides were digitized by scanning sequentially under bright field (for MCM2) and fluorescence (for CK) illumination. Multilayer JPEG2000 images were analyzed with ImmunoRatio 2.5 (accessory in SlideVantage 1.2 software) utilizing its bright field and fluorescence image-blending mode to display MCM2-CK dual-positive cells. MCM2-LI was retrospectively compared with histopathologic characteristics and patients' clinical outcome. MCM2 protein-expressing cells (median MCM2-LI, 63.5%) were more frequent than those of Ki67 (median Ki67 labelling index, 33%). Significant correlations were found between high MCM2-LI, high Ki67 labelling index, negative hormone receptor (ER, PR) statuses, high grade of malignancy, and high cyclin E expression. MCM2-LI was not shown to be predictive of disease recurrence during the median follow-up of 5.3 years but was shown to be useful to distinguish aggressive-type HER2-amplified breast carcinomas with high malignancy grade and hormone receptor negativity. The fluoro-chromogenic double-labelling immunohistochemistry accompanied with digital image analysis provides an accurate carcinoma-specific determination of MCM2-LI on a single tumor section.

Bacterial signatures in thrombus aspirates of patients with myocardial infarction.

BACKGROUND: Infectious agents, especially bacteria and their components originating from the oral cavity or respiratory tract, have been suggested to contribute to inflammation in the coronary plaque, leading to rupture and the subsequent development of coronary thrombus. We aimed to measure bacterial DNA in thrombus aspirates of patients with ST-segment-elevation myocardial infarction and to check for a possible association between bacteria findings and oral pathology in the same cohort. METHODS AND RESULTS: Thrombus aspirates and arterial blood from patients with ST-segment-elevation myocardial infarction undergoing primary percutaneous coronary intervention (n=101; 76% male; mean age, 63.3 years) were analyzed with real-time quantitative polymerase chain reaction with specific primers and probes to detect bacterial DNA from several oral species and Chlamydia pneumoniae. The median value for the total amount of bacterial DNA in thrombi was 16 times higher than that found in their blood samples. Bacterial DNA typical for endodontic infection, mainly oral viridans streptococci, was measured in 78.2% of thrombi, and periodontal pathogens were measured in 34.7%. Bacteria-like structures were detected by transmission electron microscopy in all 9 thrombus samples analyzed; whole bacteria were detected in 3 of 9 cases. Monocyte/macrophage markers for bacteria recognition (CD14) and inflammation (CD68) were detected in thrombi (8 of 8) by immunohistochemistry. Among the subgroup of 30 patients with myocardial infarction examined by panoramic tomography, a significant association between the presence of periapical abscesses and oral viridans streptococci DNA-positive thrombi was found (odds ratio, 13.2; 95% confidence interval, 2.11-82.5; P=0.004). CONCLUSIONS: Dental infection and oral bacteria, especially viridans streptococci, may be associated with the development of acute coronary thrombosis.