Objective: To investigate the clinicopathological and molecular characteristics of primary renal Ewing sarcoma. Methods: Seventeen cases of primary renal Ewing sarcoma were collected, diagnosed from January 2011 to May 2023 at Peking University Third Hospital (11 cases), Peking University Institute of Urology (4 cases), and Peking University Cancer Hospital (2 cases). The clinical, histopathological, and molecular features were studied using HE staining, immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and targeted next-generation sequencing (NGS). The prognostic correlations were assessed. Results: The cohort included 9 males and 8 females, aged 32 (24, 46) years. Presenting symptoms included hematuria and lumbar pain (13/17), right lower limb/buttock edema (1/17) and persistent low fever (1/17), and other two cases were incidentally detected by imaging in physical examination. All tumors showed renal venous/inferior vena cava tumor thrombosis and were treated with radical nephrectomy and thrombectomy. The tumor maximum diameters were 13.0 (9.0, 15.5) cm. Histologically, tumors showed morphology of diffuse sheets of small round cells with indistinct borders, round nuclei, fine chromatin, and frequent mitoses. Focal rosette-like structures were identified. The morphological heterogeneity was noted in 11 cases, including short spindle-shaped cells, gland-like structure formations, and hemangioma-like patterns. Renal pelvis invasion (5/17) and perirenal fat involvement (10/17) were identified. Immunohistochemistry showed diffuse positive for CD99 (17/17) and NKX2.2 (12/12), while FLI1 was positive in 6/12. Ki-67 indices ranged from 5% to 85%. FISH results confirmed EWSR1 rearrangement in all cases, and NGS (6/17) validated EWSR1-FLI1 fusions. Follow-up (3-84 months; 13/17) revealed metastases in 6 patients (adrenal gland, liver, bone, lung and peritoneum; 3-36 months post-surgery), of which 2 patients died, 1 patient with unknown time of death. The other 6 patients remained disease-free. All the metastatic cases showed elevated Ki-67 index (50%-70%) and necrosis. Conclusions: Primary renal Ewing sarcoma is an aggressive malignant tumor, often diagnosed at advanced stages with venous tumor thrombosis. The morphological heterogeneity and high rate of hemorrhage and necrosis are correlated with more invasive potential, suggesting prognostic significance.

Tropomyosin receptor kinase (TRK) fusions are actionable oncogenic drivers, and pan-TRK immunohistochemistry (IHC) serves as a reliable screening tool for NTRK gene aberrations. However, the expression profile and clinical significance of pan-TRK in meningiomas remain unclear. This study aimed to characterize pan-TRK expression, its correlation with clinicopathological features, and underlying NTRK rearrangement status in meningiomas. We retrospectively analyzed 70 primary intracranial tumor specimens, including 50 meningiomas (21 WHO grade 1, 26 grade 2, and 3 grade 3) and 20 non-meningioma CNS tumors (9 solitary fibrous tumors, 6 hemangioblastomas, and 5 schwannomas) from the First Affiliated Hospital of China Medical University (2020-2022). Pan-TRK IHC was performed using the Ventana EPR17341 antibody, with fluorescence in situ hybridization (FISH) validating NTRK gene aberrations in positive cases. Clinicopathological correlations were analyzed using chi-square/Fisher's exact tests and Spearman's rank correlation. Pan-TRK immunoreactivity was detected in 12/50 (24.0%) meningiomas, predominantly with cytoplasmic staining (75.0%). Positivity was significantly higher in high-grade (WHO 2/3) meningiomas (7/29, 46.7% versus 3/21, 14.3% in grade 1; p = 0.031) and tumors with Ki-67 index ≥5% (7/15, 46.7% versus 5/35, 14.3% in Ki-67 <5%; p = 0.027). No pan-TRK expression was observed in non-meningioma tumors (0/20). FISH confirmed NTRK gene aberrations in 2/12 (16.7%) pan-TRK-positive cases (both WHO 3 anaplastic meningiomas), with strong/moderate IHC staining correlating with aberrations. Pan-TRK is frequently expressed in meningiomas, particularly in high-grade and proliferative tumors, and may have preliminary utility in differentiating meningiomas from other CNS tumors. However, NTRK gene aberrations are rare, necessitating FISH or next-generation sequencing confirmation for pan-TRK-positive cases to identify candidates for TRK-targeted therapy. Further studies are needed to clarify the biological role of non-fusion-mediated pan-TRK overexpression in meningioma progression.

Detection of BCR::ABL1-like ALL using RT-PCR, a targeted RNA panel, and RNA sequencing.

Complex chromosomal rearrangements (CCRs) are rare chromosomal structural abnormalities that are often difficult to be detected. Chromosome conformation-based karyotyping (c-Moka) is an emerging technology designed to analyze the three-dimensional structure of chromosomes, thereby identifying various types of chromosomal structural variations. In our study, conventional karyotyping was performed for a gravida with a high risk of sex chromosome abnormality, as indicated by non-invasive prenatal testing. The analysis suggested a potential chromosome translocation at the terminals of chromosomes 18 and 19. Subsequently, fluorescence insitu hybridization (FISH) was performed at metaphase stage using a 19p13.3 probe. The signal was detected on other chromosome rather than chromosome 18, leading us to hypothesize that the subject might have CCRs that was not identifiable through conventional karyotyping and FISH. To further investigate, we employed c-Moka, which revealed CCRs involving chromosomes 10, 18 and 19. Ultimately, the karyotype of the subject was determined to be 46,XX,t(10;18;19)(q26.3;p11.21;p13.3). This study underscores that, compared to conventional karyotyping, c-Moka has significant advantages in detecting CCRs.

Identification of the inhibitors of apoptosis proteins (IAPs) gene family in Portunus trituberculatus and its immune defense under Vibrio parahaemolyticus stress.

Introduction Nuclear protein in testis (NUT) carcinoma (NC) is an extremely rare and highly aggressive tumor. Non-canonical NC patients frequently have unique clinicopathological characteristics in terms of location, molecular alteration, and outcome, with morphological overlap with other tumors. Here, we report and summarize the non-canonical clinicopathological features of three unusual NC patients harboring BRD3::NUTM1 or WWTR1::NUTM1 gene rearrangement. Methods The data of three patients with NC who were diagnosed between 2019 and 2022 at the Department of Pathology at West China Hospital or the Sichuan Kingmed Center for Clinical Laboratory were collected. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and RNA sequencing were performed. A literature review was conducted for NC patients with non-canonical features. Results All three NC patients were male and aged between 31 and 60 years. The primary sites were the gastric corpus, nasal cavity, and subcutaneous tissue of the calvaria vertex. Histologically, two patients harbored NC with glandular differentiation, and one harbored NC with small cell carcinoma-like morphology. IHC revealed positivity for Pan-CK (cytokeratin) (PCK) (3/3), Epithelial membrane antigen (EMA) (3/3), Cytokeratin 5/6 (CK5/6) (3/3), and P63 (2/3). The Ki-67 index ranged from 20% to 40%. The remaining IHC markers were negative. RNA sequencing revealed BRD3::NUTM1 fusion in two patients and WWTR1::NUTM1 fusion in one patient. Conclusion In our study, the clinicopathological features of NC in patients with rare molecular alterations are reported and summarized. As increasing numbers of NUTM1 -rearranged tumors with diverse histologic phenotypes have been reported, the traditional classification of NC as a purely epithelial malignancy may warrant reconsideration. Non-canonical NC needs to be further reported, integrated, and classified to precisely guide targeted therapy.

ALK protein expression and gene copy number alterations in triple-negative breast cancer: Clinical implications.

Detection and localization of a new Wolbachia from supergroup W in Oppiella nova (Acari: Oribatida: Oppiidae) with potential impacts on host life-history traits.

Integrating Contrast Colour Correction (CACC) and Convolutional Neural Networks (CNN) can help fish breeders in earlier classification and identification of Cryptocaryon fish disease (protozoan white spot disease). Disease identification accuracy is enhanced through such method by adaptive colour changes and CNN feature extracting ability, thereby boosting underwater image clarity. Unlike traditional rule-based systems that relies on expert knowledge despite being error-prone, existing methods focus on visual quality without classifying impact influence. Early disease identification is hampered in terms of efficiency due to machine learning methods reliant on abundant human expertise other than efficient feature extracting. An artificial intelligence (AI)-oriented computer model is introduced for existing research in overcoming limitations and eliminating subjectivity. The model employs a proprietary method of diagnosing fish disease through underwater images examination that yields objective outcome. Several CNN structures such as GoogleNet, ResNet-101, AlexNet, ResNet-50 as well as VGG-16 are tested on its performance. The current study shows integration of CACC with CNN through a set of 15000 images boosting up model performance in Cryptocaryon fish disease detection. The introduced novel method significantly enhances performance with 99.53% accuracy, 99.08% precision along with 100.00% recall. This efficient, accurate approach can significantly reduce the workload of experts and fish farmers while promoting sustainable aquaculture and healthier aquatic ecosystems. • Contrast-Adaptive Colour Correction (CACC) and Convolutional Neural Network (CNN) enhance fish images for accurate Cryptocaryon fish disease detection. • 6500 image datasets from National Fish Health Research aids AI training and testing. • One of the CNN architectures, ResNet50 outperforms others with 99.52 % accuracy. • 99 % accuracy across key metrics ensures early disease detection. • AI-driven approach supports sustainable aquaculture and reduces losses.

CircSSRP1 regulates SMAD3 to affect proliferation and apoptosis of human embryonic palatal mesenchymal cells in nonsyndromic cleft lip with or without cleft palate by sponging miR-708-5p.

To describe a rare case of a normal fertile male with the specific tissue distribution of 46,XX in the peripheral blood and gonadal chimerism confirmed by the novel next-generation sequencing (NGS) methodology. Cytogenetic analyses, low-pass copy number variation sequencing (CNV-seq) and NGS-based short tandem repeat (STR) tests. Routine peripheral blood karyotyping showed 46,XX. Fluorescence in situ hybridization (FISH) analysis of lymphocyte metaphase nuclei confirmed 46,XX and sex-determine region Y (SRY)-negative. Further CNV-seq revealed the presence of Y chromosome in the semen. NGS-based STR tests discovered low-level Y chromosome in the buccal mucosa and three alleles in the semen, suggesting that this chimerism is likely the outcome of a parthenogenetically activated oocyte fertilized by a Y and X sperm. The patient underwent intracytoplasmic sperm injection (ICSI) and resulted in a normal singleton pregnancy. Individuals with 46,XX/46,XY chimerism in the gonad can display normal phenotype and fertility. NGS has proved to be a promising alternative to conventional chimerism assays.

Traditional proximity biotinylation approaches require extensive genetic engineering or intricate purification steps. Here, we introduce FISH+, a ready-to-use RNA proximity labeling method that relies on the recruitment of peroxidase to RNA targets, and facilitates in situ proximity biotinylation in fixed cells. This method permits concurrent visualization of RNA molecules and identification of RNA-interacting proteins. Using this method, we visualized 45S and NEAT1 RNA, and captured their proximal proteins, demonstrating the capability to concurrently visualize RNA and identify proximal proteins. By targeting PNCTR (~36 copies per cell), we observed distinctly bright RNA dots, representing the combined biotinylation signals from both RNA and proximal proteins in situ. This indicates the potential of the FISH+ method for enhanced RNA visualization. We further generalized the FISH+ method to explore XIST-interacting proteins, a number of reported interactors were significantly enriched, such as SPEN, CIZ1 and RBM15. Using quantitative mass spectrometry, we show that FISH+ correctly identifies known RNA-protein interactions in the nucleus of human cells. Overall, we established a watch-and-catch punctate RNA method through the integration of RNA fluorescence in situ hybridization (FISH) with proximity biotinylation. This method provides additional spatial information for the characterization of RNA-centric interactions in fixed, genetically unperturbed samples.

Hypereosinophilia of undetermined significance (HE-US) represents a rare subtype of hypereosinophilia (HE) defined by a persistent absolute eosinophil count (AEC) ≥ 1500/uL without an identifiable reactive or clonal cause, and importantly, without evidence of eosinophil-mediated end-organ damage. Long-term structured follow-up is essential due to its unpredictable outcome and the risk of transition into overt hypereosinophilic syndrome (HES) or occult malignancies. We report the case of a 47-year-old woman with severe, persistent blood hypereosinophilia (up to 13600/uL) accompanied by chronic inconstant fixed erythematous-violaceous skin lesions. Extensive diagnostic evaluation for secondary, autoimmune, and clonal etiologies was negative, including fluorescence in situ hybridization (FISH) for FIP1L1-PDGFRA mutation. Bone marrow biopsy showed granulocytic hyperplasia with eosinophils <10% and mild reticulin fibrosis (MF-1). Whole-body computed tomography (CT) revealed small generalized lymphadenopathy (≤ 11mm) without parenchymal infiltration. The patient declined lymph-node biopsy. Although skin biopsy revealed eosinophilic infiltration, it was characterized as a non-specific reactive pattern without objective evidence of eosinophil-mediated cytotoxicity. The case was provisionally diagnosed as HE-US, acknowledging that lymphoproliferative disease could not be definitively excluded in the absence of the lymph-node biopsy. Intermittent systemic corticosteroids provide rapid hematologic response, but prompted immediate recurrence when tapered. The reported case underscores the diagnostic complexity and difficulties in managing hypereosinophilia of undetermined significance in clinical practice. While current evidence suggests a generally benign course, rare progression to hypereosinophilic syndrome or other malignancies requires structured long-term monitoring and individualized therapeutic decision-making.

Fusobacterium nucleatum (F. nucleatum) is an oral commensal bacterium that acts as a pathobiont with pro-tumorigenic activity in various gastrointestinal cancers. However, its functional role, invasive capacity, and mechanistic contributions in cervical cancer remain largely unexplored. We identified F. nucleatum in cervical cancer tissues using bioinformatics and clinical 16S rRNA sequencing. Its spatial localization and intracellular presence were confirmed by fluorescence in situ hybridization (FISH) and transmission electron microscopy (TEM), respectively. Functional validation included in vitro assays for proliferation, migration, and apoptosis in cervical cancer cell lines, with bacterial invasion visualized by confocal microscopy, and in vivo tumor growth assessment in a xenograft model. The underlying mechanism involving high mobility group box 1 (HMGB1) and the NF-κB pathway was analyzed by western blot, qPCR, immunofluorescence, and ELISA. F. nucleatum was enriched in cervical cancer and correlated with poor patient survival. It invaded cervical cancer cells, promoted proliferation, migration, and invasion, suppressed apoptosis in vitro, and accelerated tumor growth in vivo. Mechanistically, infection triggered HMGB1 upregulation and specific activation of the canonical NF-κB pathway (via IκBα degradation, p65 phosphorylation/nuclear translocation), leading to selective secretion of IL-6/IL-8. Our study suggests that F. nucleatum is associated with cervical cancer malignancy, potentially acting through upregulation of HMGB1 and activation of the canonical NF-κB signaling pathway, thereby contributing to an altered tumor microenvironment. These findings reveal a previously unrecognized microbial-driven oncogenic mechanism in cervical cancer and highlight its potential as a prognostic marker and a therapeutic target. Not applicable in our manuscript.

Multiple myeloma (MM) is a heterogeneous plasma cell (PC) malignancy in both clinical presentation and genetic profile. Cytogenetic characterization, particularly by interphase fluorescence insitu hybridization (FISH), is critical for risk stratification and treatment decisions. FISH results depend on the PC infiltration rate in bone marrow (BM); therefore, CD138-positive immunoselection is required to overcome reduced FISH sensitivity due to low infiltration. We evaluated the performance of automated PC immunoselection compared to the manual method. We retrospectively analyzed data from 715 BM samples sent for routine FISH testing between 2019 and 2024. PCs were isolated manually (n = 351) or automatically (n = 364). BM infiltration was assessed by flow cytometry (FC) and/or cytomorphology. FISH analysis was performed using a primary panel with three DNA probes or an expanded panel with additional probes. Confirmed diagnoses were obtained from clinical records. FISH was successfully performed in 81% of samples, with automated processing achieving a higher success rate (86% vs. 75%, p < 0.001). Automated processing provided higher PC yields, enabling testing with expanded probe sets. Chromosomal abnormalities were detected in more than 90% of confirmed MM cases, regardless of isolation method. PC infiltration strongly predicted FISH success, with infiltration of ≥ 3% corresponding to an 80% probability of successful FISH. Automated immunoselection improves PC yield, enabling broader FISH testing compared to manual processing. Information on PC infiltration obtained by cytomorphology or FC, which indicates sample quality, can support quality assessment in the cytogenetics laboratory.

Quantitative profiling of RNA modifications enriched in non-membrane-bound cellular structures using APEX-RNA-MS.

Artificial intelligence-assisted screening for NTRK fusion-positive salivary gland tumors: A novel digital pathology workflow.

Our objectives were to describe molecular profiling in a real-life cohort of patients with radioiodine-resistant (RAI-R) differentiated or poorly differentiated thyroid cancer (DTC or PDTC) treated with lenvatinib and to focus on factors potentially influencing the quality of tissue samples for molecular analysis, including the impact of storage time, defined as the interval between tissue collection and molecular testing. We retrospectively included all lenvatinib-treated RAI-R DTC or PDTC patients tested with DNA- and/or RNA-based next-generation sequencing (NGS) in our center, also analyzing the results of fluorescence in situ hybridization (FISH) for RET fusions if the sample did not satisfy quality criteria for RNA-based NGS analysis. We investigated differences in terms of histotype, biopsy site, or storage time between adequate and inadequate samples for RNA-based NGS. At least one gene alteration was detected in 50% of the cohort (18 out of 36 patients); RAS and BRAF were the most frequent mutations, while gene fusions accounted for 5.6% of cases. Tissue samples were more frequently adequate for DNA-based NGS compared to RNA-NGS analysis (93.9% vs. 58.3%, p < 0.001). The median storage time was significantly longer in the case of inadequate samples for RNA-based NGS compared with adequate specimens (41.5 vs. 9.5 months, p = 0.016); samples archived for ≥3 years led more frequently to an inadequate result. Advanced RAI-R TC candidates for systemic therapy often harbor gene alterations. An adequate result was less frequently achieved in cases of RNA-based NGS than in DNA-based NGS, especially if the interval between tissue collection and molecular analysis was longer; nevertheless, the limited cohort size precludes definitive conclusions.

Understanding gene expression within defined neuronal populations is essential for dissecting the cellular and molecular diversity of the brain. mRNA assays provide a direct readout of gene expression, capturing transcriptional changes that may precede or occur independently of protein abundance, whereas protein assays reflect the cumulative effects of translation, modification, and degradation. Moreover, in histological analysis, immunohistochemical protein detection results in visually diffuse labeling, which makes it difficult to quantitatively assess levels and locations of expression at high resolution. Here, we present a protocol that allows for mRNA detection in single neuronal cell types with a high degree of sensitivity and anatomical resolution. This protocol combines fluorescent in situ hybridization (FISH) with immunohistochemistry (IHC) on the same tissue section. Briefly, FISH is carried out by ACDBio RNAscope® fluorescent in situ hybridization technology, which involves processing the tissue sections, followed by signal amplification. This involves target retrieval, probe hybridization, and signal enhancement. Then, the tissue section is processed for IHC, which involves blocking nonspecific sites and incubation with primary antibodies, followed by development of a fluorescent signal with secondary antibodies. Typically, visual mRNA detection with FISH can be seen as individual puncta, whereas targeting the protein with an antibody results in filled cells or processes. The variation in staining pattern allows for the quantification of distinct mRNA transcripts within different neuronal populations, which renders co-localization analyses easy and efficient. Key features • This protocol combines ACDBio RNAscopeTM technology with standard immunohistochemistry techniques. • Visual simultaneous detection of mRNA and protein expression within murine brain tissue to identify mRNA transcripts within specific neuronal subpopulations. • Using a custom macro code for FIJI/ImageJ for an efficient analysis of total mRNA expression or mRNA co-localization with neuronal subtypes. • This protocol can be used to detect any mRNA and protein combination given appropriate antigen retrieval and tissue preparation within any tissue type of interest.

A single-nucleus transcriptomic atlas of early marine shrimp embryogenesis reveals cell-type specification and eye pigment transport mechanisms.