Deletion of Trps1 regulatory elements recapitulates postnatal hip joint abnormalities and growth retardation of Trichorhinophalangeal syndrome in mice.

Trichorhinophalangeal syndrome (TRPS) is a genetic disorder caused by point mutations or deletions in the gene-encoding transcription factor TRPS1. TRPS patients display a range of skeletal dysplasias, including reduced jaw size, short stature, and a cone-shaped digit epiphysis. Certain TRPS patients experience early onset coxarthrosis that leads to a devastating drop in their daily activities. The etiologies of congenital skeletal abnormalities of TRPS were revealed through the analysis of Trps1 mutant mouse strains. However, early postnatal lethality in Trps1 knockout mice has hampered the study of postnatal TRPS pathology. Here, through epigenomic analysis we identified two previously uncharacterized candidate gene regulatory regions in the first intron of Trps1. We deleted these regions, either individually or simultaneously, and examined their effects on skeletal morphogenesis. Animals that were deleted individually for either region displayed only modest phenotypes. In contrast, the Trps1Δint/Δint mouse strain with simultaneous deletion of both genomic regions exhibit postnatal growth retardation. This strain displayed delayed secondary ossification center formation in the long bones and misshaped hip joint development that resulted in acetabular dysplasia. Reducing one allele of the Trps1 gene in Trps1Δint mice resulted in medial patellar dislocation that has been observed in some patients with TRPS. Our novel Trps1 hypomorphic strain recapitulates many postnatal pathologies observed in human TRPS patients, thus positioning this strain as a useful animal model to study postnatal TRPS pathogenesis. Our observations also suggest that Trps1 gene expression is regulated through several regulatory elements, thus guaranteeing robust expression maintenance in skeletal cells.

Establishment and characterization of TK-ALCL1: a novel NPM-ALK-positive anaplastic large-cell lymphoma cell line.

TK-ALCL1, a novel anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALK+ ALCL) cell line, was established from the primary tumor site of a 59-year-old Japanese male patient. The immune profile of TK-ALCL1 corresponds to that seen typically in primary ALCL cells, i.e., positive for ALK, CD30, EMA, and CD4, but negative for CD2, CD3, CD5, CD8a, and EBV-related antigens. The rearrangement of the T cell receptor-gamma locus shows that TK-ALCL1 is clonally derived from T-lineage lymphoid cells. FISH and RT-PCR analysis revealed that TK-ALCL1 has the nucleophosmin (NPM)-ALK fusion transcript, which is typical for ALK+ ALCL cell lines. When TK-ALCL1 was subcutaneously inoculated into 6-week-old BALB/c Rag2-/-/Jak3-/- (BRJ) mice, it formed tumor masses within 4-6 weeks. Morphological, immunohistochemical, and molecular genetic investigations confirmed that the xenograft and the original ALCL tumor were identical. The ALK inhibitors Alectinib and Lorlatinib suppressed proliferation in a dose-dependent manner. Thus, TK-ALCL1 provides a useful in vitro and in vivo model for investigation of the biology of ALK+ ALCL and of novel therapeutic approaches targeting ALK.

Methylation analysis of DCC gene in saliva samples is an efficient method for non-invasive detection of superficial hypopharyngeal cancer.

BACKGROUND: Advances in upper gastrointestinal endoscopic technology have enabled early detection and treatment of hypopharyngeal cancer. However, in-depth pharyngeal observations require sedation and are invasive. It is important to establish a minimally invasive and simple evaluation method to identify high-risk patients. METHODS: Eighty-seven patients with superficial hypopharyngeal cancer and 51 healthy controls were recruited. We assessed the methylation status of DCC, PTGDR1, EDNRB, and ECAD, in tissue and saliva samples and verified the diagnostic accuracy by methylation analyses of their promoter regions using quantitative methylation-specific PCR. RESULTS: Significant differences between cancer and their surrounding non-cancerous tissues were observed in the methylation values of DCC (p = 0.003), EDNRB (p = 0.001), and ECAD (p = 0.043). Using receiver operating characteristic analyses of the methylation values in saliva samples, DCC showed the highest area under the curve values for the detection of superficial hypopharyngeal cancer (0.917, 95% confidence interval = 0.864-0.970), compared with those for EDNRB (0.680) and ECAD (0.639). When the cutoff for the methylation values of DCC was set at ≥0.163, the sensitivity to detect hypopharyngeal cancer was 82.8% and the specificity was 90.2%. CONCLUSIONS: DCC methylation in saliva samples could be a non-invasive and efficient tool for early detection of hypopharyngeal cancer in high-risk patients.

Endoscopic and Histological Gastritis in University Students with Helicobacter pylori Infection.

Objective Although the characteristics of Helicobacter pylori infection have been extensively reported, there is a lack of consensus regarding its characteristics in young adults. The present study examined the endoscopic and histological characteristics of young adults who underwent eradication therapy for H. pylori infection. Methods We examined the H. pylori infection status of first-year students at Okayama University School of Medicine and Dentistry between 2014 and 2020. A total of 152 (6.8%) students who were positive for H. pylori antibody or pepsinogen tests were enrolled in the study. Among them, 107 students underwent endoscopy, and their biopsy samples were investigated. Seventy-five students were diagnosed with H. pylori infections. Results Of 75 H. pylori-positive patients, 57 (76.0%) had endoscopic atrophic gastritis, and 42 (56.0%) had histological atrophy. A few patients had severe atrophic gastritis. All 65 patients who underwent an eradication assessment were successfully treated. After successful eradication, 26 patients underwent endoscopic follow-up. The mean follow-up period was 32.9 months. A histological evaluation revealed that gastric antrum atrophy had subsided in 11 of 14 patients, and atrophy in the lesser curvature of the gastric body had subsided in 7 of 8 patients. Conclusion More than half of young adults with H. pylori infection had atrophic gastritis. We found mild atrophy in young adults, which subsided shortly after eradication treatment. This study provides a foundation for future studies to evaluate the validity of eradication therapy in preventing gastric cancer in patients.

Evaluation of a Deep Learning Model for Metastatic Squamous Cell Carcinoma Prediction From Whole Slide Images.

CONTEXT.­: Squamous cell carcinoma (SCC) is a histologic type of cancer that exhibits various degrees of keratinization. Identifying lymph node metastasis in SCC is crucial for prognosis and treatment strategies. Although artificial intelligence (AI) has shown promise in cancer prediction, applications specifically targeting SCC are limited. OBJECTIVE.­: To design and validate a deep learning model tailored to predict metastatic SCC in radical lymph node dissection specimens, using whole slide images (WSIs). DESIGN.­: Using the EfficientNetB1 architecture, a model was trained on 6587 WSIs (2413 SCC and 4174 nonneoplastic) from several hospitals, encompassing esophagus, head and neck, lung, and skin specimens. The training exclusively relied on WSI-level labels without annotations. We evaluated the model on a test set consisting of 541 WSIs (41 SCC and 500 nonneoplastic) of radical lymph node dissection specimens. RESULTS.­: The model exhibited high performance, with receiver operating characteristic curve areas under the curve between 0.880 and 0.987 in detecting SCC metastases in lymph nodes. Although true positives and negatives were accurately identified, certain limitations were observed. These included false positives due to germinal centers, dust cell aggregations, and specimen-handling artifacts, as well as false negatives due to poor differentiation. CONCLUSIONS.­: The developed artificial intelligence model presents significant potential in enhancing SCC lymph node detection, offering workload reduction for pathologists and increasing diagnostic efficiency. Continuous refinement is needed to overcome existing challenges, making the model more robust and clinically relevant.

A Pancreatic Collision Tumor Comprising Mantle Cell Lymphoma and Adenocarcinoma: A Case Report and Literature Review.

A collision tumor is a rare clinical condition where two different tumors occur synchronically within a lesion. Pancreatic collision tumors with mantle cell lymphoma (MCL) are extremely rare and have only been reported in one case to date. We herein report an elderly patient with MCL and adenocarcinoma of the pancreas with Ann Arbor stage IV and Union for International Cancer Control stage IIB, respectively. The patient received palliative therapy and died 23 months after the diagnosis. Further research and case studies are required to investigate whether or not MCL-derived cyclin D1 overexpression affects the occurrence/growth of adenocarcinomas.

Prognostic effect of categorized tumor deposits in gastric cancer: A single-center retrospective study.

BACKGROUND: Tumor deposits are defined as all types of isolated cancer lesions without lymphocyte aggregates considered part of the lymph node. Tumor deposits have been reported as a negative prognostic factor. However, the survival significance of categorized tumor deposits is uncertain, particularly in gastric cancer. This study aimed to investigate the prognostic difference among categorized tumor deposits. METHODS: Patients who underwent gastrectomy for gastric cancer were enrolled. All tumor deposits were categorized into irregular nodule, irregular nodule star, smooth nodule, and vascular/neural invasion. There are some cases with more than 2 categorized tumor deposits. These cases were categorized as tumor deposit complex in the following analysis. We performed survival analysis between the patients with and without tumor deposits, and compared the survival among each categorized tumor deposit. RESULTS: Of 868 patients, there were 96 (11.1%) and 772 (88.9%) patients with and without tumor deposits. Vascular/neural invasion, smooth nodule, irregular nodule, irregular nodule star, and the tumor deposits complex was observed in 6 (6.3%), 15 (15.6%), 43 (44.8%), 1 (1.0%), and 31 (32.3%) patients. Patients with tumor deposits displayed poorer survival than those without; the 3-year overall survival: tumor deposits negative = 87.0%, tumor deposits positive = 53.2% (P < .001). Survival analysis revealed tumor deposits can be a prognostic risk factor (hazard ratio: 1.9854, 95% confidence interval: 1.393-2.830, P < .01). Irregular nodule and the tumor deposits complex demonstrated the worst prognosis (irregular nodule 3-year overall survival: 51.2%, tumor deposits complex 3-year overall survival: 41.9%, P = .001), whereas smooth nodule demonstrated better prognosis (smooth nodule 3-year overall survival: 80%). CONCLUSION: Tumor deposits exerted a negative survival effect in gastric cancer. Irregular nodule and the tumor deposits complex displayed a strong prognostic effect.

Histological and immunohistochemical studies of the fungiform and the circumvallate papillae through the life stages from 6- to 72-week-old Sprague-Dawley male rats.

Taste sensitivity decreases with age. Therefore, we investigated the histological and immunohistochemical changes in the receptive fields circumvallate papilla (CvP) and fungiform papilla (FfP) to explore the mechanism underlying age-related changes in taste sensitivity in 6- to 72-week-old rats. We analyzed papilla size, the thickness of the keratin layer of the papilla and stratified squamous epithelium, taste bud size, the keratin layer around the taste pores in the CvP and FfP, and the number and distribution of taste buds in the CvP coronal section. We further assessed the expression of marker proteins for Type II and III cells, phospholipase C subtype beta 2 (PLCß2), and synaptosomal-associated protein 25 (SNAP-25). The cellular activity of these taste cells was examined through co-localization with the senescence cell marker protein-30 (SMP30). There were no differences in the number of taste bud sections in the CvP among the age groups. However, the size of the CvP increased and the density of the taste bud area in the CvP area decreased with increasing age. In contrast, the number of cells with co-expression of SMP30, PLCß2, and SNAP-25 decreased with age. Furthermore, the morphological structures of the CvP, FfP, and taste buds in these regions changed with age, but not the overall taste bud number in the CvP coronal section. The decrease in cell count with co-expression of SMP30 and PLCß2, or SNAP-25 may indicate reduced cellular functions of taste cells with aging.

Modeling of intramembranous ossification using human pluripotent stem cell-derived paraxial mesoderm derivatives.

Vertebrates form their skeletal tissues from three distinct origins (the neural crest, paraxial mesoderm, and lateral plate mesoderm) through two distinct modes of ossification (intramembranous and endochondral ossification). Since the paraxial mesoderm generates both intramembranous and endochondral bones, it is thought to give rise to both osteoprogenitors and osteo-chondroprogenitors. However, it remains unclear what directs the paraxial mesoderm-derived cells toward these different fates in distinct skeletal elements during human skeletal development. To answer this question, we need experimental systems that recapitulate paraxial mesoderm-mediated intramembranous and endochondral ossification processes. In this study, we aimed to develop a human pluripotent stem cell (hPSC)-based system that models the human intramembranous ossification process. We found that spheroid culture of the hPSC-derived paraxial mesoderm derivatives generates osteoprogenitors or osteo-chondroprogenitors depending on stimuli. The former induced intramembranous ossification, and the latter endochondral ossification, in mouse renal capsules. Transcriptional profiling supported the notion that bone signatures were enriched in the intramembranous bone-like tissues. Thus, we developed a system that recapitulates intramembranous ossification, and that enables the induction of two distinct modes of ossification by controlling the cell fate of the hPSC-derived paraxial mesoderm derivatives.

Direct observation of epoxy resin blocks for renal biopsy by low-vacuum scanning electron microscopy.

To improve the resolution of low-vacuum scanning electron microscopy (LVSEM), the epoxy resin block for the transmission electron microscopy (TEM) was observed directly with LVSEM. After observing ultrathin sections from renal biopsies of IgA nephropathy, membranous nephropathy, lupus nephritis, diabetic nephropathy (DM), thin basement membrane disease (TBMD), Alport's syndrome, Fabry's disease, and renal amyloidosis, the epoxy resin blocks of the same sites were observed by LVSEM and compared. The LVSEM image of the epoxy resin block corresponds to the negative of the TEM image, and when the gradation is reversed, the LVSEM image was comparable to the TEM image. At a low magnification of 100 ×, the entire specimen, including the glomerulus, was obtained. LVSEM at 5000 × magnification was sufficient to identify paramesangial deposits in IgA nephropathy and subepithelial electron-dense deposits (EDD) and spikes in membranous nephropathy. Glomerular basement membrane thickening in DM and thinning in TBMD could be sufficiently diagnosed with LVSEM at 6000 ×. Accumulation of ceramide in Fabry's disease was easily identified, but amyloid fibril could not be identified by LVSEM. LVSEM of renal biopsy epoxy resin blocks can replace TEM up to moderate magnification.

Lithium carbonate accelerates the healing of apical periodontitis.

Apical periodontitis is a disease caused by bacterial invasions through the root canals. Our previous study reported that lithium chloride (LiCl) had a healing effect on apical periodontitis. The aim of this report is to investigate the healing properties and mechanism of lithium ion (Li+) for apical periodontitis using rat root canal treatment model. 10-week-old male Wistar rat's mandibular first molars with experimentally induced apical periodontitis underwent root canal treatment and were applied lithium carbonate (Li2CO3) containing intracanal medicament. Base material of the medicament was used as a control. Subject teeth were scanned by micro-CT every week and the periapical lesion volume was evaluated. The lesion volume of Li2CO3 group was significantly smaller than that of the control group. Histological analysis showed that in Li2CO3 group, M2 macrophages and regulatory T cells were induced in the periapical lesion. In situ hybridization experiments revealed a greater expression of Col1a1 in Li2CO3 group compared with the control group. At 24 h after application of intracanal medicament, Axin2-positive cells were distributed in Li2CO3 group. In conclusion, Li2CO3 stimulates Wnt/ß-catenin signaling pathway and accelerate the healing process of apical periodontitis, modulating the immune system and the bone metabolism.

Degree of pharyngeal deformation caused by pharyngeal endoscopic submucosal dissection is associated with the incidence of aspiration pneumonia.

Background and study aims Endoscopic submucosal dissection (ESD) is one of the most minimally invasive treatments for superficial squamous cell cancer of the pharynx. However, aspiration pneumonia (AsP) associated with postoperative deformity of the pharynx may occur. The purpose of this study was to investigate the frequency of AsP and the degree of pharyngeal deformity after pharyngeal ESD. Patients and methods This was a retrospective observational study of patients who underwent pharyngeal ESD at Okayama University Hospital between 2006 and 2017. The degree of pharyngeal deformation was assessed using the pharyngeal deformation grade (PDG). The primary endpoint was the frequency of AsP as a long-term adverse event. Results Among the 52 patients enrolled, nine developed aspiration pneumonia, with a 3-year cumulative incidence of 9.0 % (95 % confidence interval [CI], 3.3 %-22.0 %). There were 16, 18, 16, and two patients that had PDG 0, 1, 2, and 3, respectively. Patients with a history of radiotherapy, as a treatment of head and neck cancer (44.4 % vs. 11.6 %; P  = 0.02) and the high PDG group (PDG 2 and 3) (77.8 % vs. 25.6 %; P  = 0.005) had a significantly higher incidence of AsP. The 3-year cumulative incidence rate of AsP after ESD in the high PDG group was significantly higher than that in the low PDG group (PDG 0 and 1) (23.9 % [95 %CI, 9.2.-49.5%] vs. 0 %; P  = 0.03). Conclusions The incidence of aspiration pneumonia in the long-term course after pharyngeal ESD was revealed. The incidence of aspiration pneumonia may be associated with pharyngeal deformity, but further studies are needed.

A Case of GATA3 Positive Pleomorphic Liposarcoma, Epithelioid Variant: A Diagnostic Pitfall.

Pleomorphic liposarcoma is a rare malignant adipocytic tumor showing undifferentiated pleomorphic sarcoma morphology with various degrees of epithelioid features. It is sometimes difficult to distinguish from carcinoma metastasis. Immunohistochemical panel is very important for differential diagnosis; however, there is a risk that unexpected staining could lead to misinterpretation. We report a pleomorphic liposarcoma, epithelioid variant, in an 88-year-old man, with tricky-positive staining for GATA3. Histological examination revealed a tumor with epithelioid morphology. The tumor consists of solid sheets of epithelioid tumor cells with focal aggregates of pleomorphic lipoblasts. Immunohistochemically, the adipocytic tumor cell areas were positive for S100 protein, and the epithelioid tumor cells showed CAM 5.2 positivity. GATA3 was diffusely positive. The combination of CAM 5.2 and GATA3 staining suggested the possibility of metastatic cancer, but systemic clinical examinations did not detect any presence of a primary tumor, including urinary bladder, breasts, and salivary glands. The pathological diagnosis of pleomorphic liposarcoma, epithelioid variant, was made because of the presence of malignant lipoblasts. Our report may contribute for differential diagnosis of pleomorphic liposarcoma, epithelioid variant, with unexpected positive immunoreaction for GATA3.

Evaluation of safety and efficacy of autologous oral mucosa-derived epithelial cell sheet transplantation for prevention of anastomotic restenosis in congenital esophageal atresia and congenital esophageal stenosis.

BACKGROUND: We performed the first autologous oral mucosa-derived epithelial cell sheet transplantation therapy in a patient with refractory postoperative anastomotic stricture in congenital esophageal atresia (CEA) and confirmed its safety. In this study, patients with CEA and congenital esophageal stenosis were newly added as subjects to further evaluate the safety and efficacy of cell sheet transplantation therapy. METHODS: Epithelial cell sheets were prepared from the oral mucosa of the subjects and transplanted into esophageal tears created by endoscopic balloon dilatation (EBD). The safety of the cell sheets was confirmed by quality control testing, and the safety of the transplantation treatment was confirmed by 48-week follow-up examinations. RESULTS: Subject 1 had a stenosis resected because the frequency of EBD did not decrease after the second transplantation. Histopathological examination of the resected stenosis revealed marked thickening of the submucosal layer. Subjects 2 and 3 did not require EBD for 48 weeks after transplantation, during which time they were able to maintain a normal diet by mouth. CONCLUSIONS: Subjects 2 and 3 were free of EBD for a long period of time after transplantation, confirming that cell sheet transplantation therapy is clearly effective in some cases. In the future, it is necessary to study more cases; develop new technologies such as an objective index to evaluate the efficacy of cell sheet transplantation therapy and a device to achieve more accurate transplantation; identify cases in which the current therapy is effective; and find the optimal timing of transplantation; and clarify the mechanism by which the current therapy improves stenosis. TRIAL REGISTRATION: UMIN, UMIN000034566, registered 19 October 2018, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000039393 .

Deep Learning Approach to Classify Cutaneous Melanoma in a Whole Slide Image.

Although the histopathological diagnosis of cutaneous melanocytic lesions is fairly accurate and reliable among experienced surgical pathologists, it is not perfect in every case (especially melanoma). Microscopic examination-clinicopathological correlation is the gold standard for the definitive diagnosis of melanoma. Pathologists may encounter diagnostic controversies when melanoma closely mimics Spitz's nevus or blue nevus, exhibits amelanotic histopathology, or is in situ. It would be beneficial if diagnosing cutaneous melanocytic lesions can be automated by using deep learning, particularly when assisting surgical pathologists with their workloads. In this preliminary study, we investigated the application of deep learning for classifying cutaneous melanoma in whole-slide images (WSIs). We trained models via weakly supervised learning using a dataset of 66 WSIs (33 melanomas and 33 non-melanomas). We evaluated the models on a test set of 90 WSIs (40 melanomas and 50 non-melanomas), achieving ROC-AUC at 0.821 for the WSI level and 0.936 for the tile level by the best model.

Inference of core needle biopsy whole slide images requiring definitive therapy for prostate cancer.

BACKGROUND: Prostate cancer is often a slowly progressive indolent disease. Unnecessary treatments from overdiagnosis are a significant concern, particularly low-grade disease. Active surveillance has being considered as a risk management strategy to avoid potential side effects by unnecessary radical treatment. In 2016, American Society of Clinical Oncology (ASCO) endorsed the Cancer Care Ontario (CCO) Clinical Practice Guideline on active surveillance for the management of localized prostate cancer. METHODS: Based on this guideline, we developed a deep learning model to classify prostate adenocarcinoma into indolent (applicable for active surveillance) and aggressive (necessary for definitive therapy) on core needle biopsy whole slide images (WSIs). In this study, we trained deep learning models using a combination of transfer, weakly supervised, and fully supervised learning approaches using a dataset of core needle biopsy WSIs (n=1300). In addition, we performed an inter-rater reliability evaluation on the WSI classification. RESULTS: We evaluated the models on a test set (n=645), achieving ROC-AUCs of 0.846 for indolent and 0.980 for aggressive. The inter-rater reliability evaluation showed s-scores in the range of 0.10 to 0.95, with the lowest being on the WSIs with both indolent and aggressive classification by the model, and the highest on benign WSIs. CONCLUSION: The results demonstrate the promising potential of deployment in a practical prostate adenocarcinoma histopathological diagnostic workflow system.

Kruppel-like Factors in Skeletal Physiology and Pathologies.

Kruppel-like factors (KLFs) belong to a large group of zinc finger-containing transcription factors with amino acid sequences resembling the Drosophila gap gene Krüppel. Since the first report of molecular cloning of the KLF family gene, the number of KLFs has increased rapidly. Currently, 17 murine and human KLFs are known to play crucial roles in the regulation of transcription, cell proliferation, cellular differentiation, stem cell maintenance, and tissue and organ pathogenesis. Recent evidence has shown that many KLF family molecules affect skeletal cells and regulate their differentiation and function. This review summarizes the current understanding of the unique roles of each KLF in skeletal cells during normal development and skeletal pathologies.

Transfer Learning for Adenocarcinoma Classifications in the Transurethral Resection of Prostate Whole-Slide Images.

The transurethral resection of the prostate (TUR-P) is an option for benign prostatic diseases, especially nodular hyperplasia patients who have moderate to severe urinary problems that have not responded to medication. Importantly, incidental prostate cancer is diagnosed at the time of TUR-P for benign prostatic disease. TUR-P specimens contain a large number of fragmented prostate tissues; this makes them time consuming to examine for pathologists as they have to check each fragment one by one. In this study, we trained deep learning models to classify TUR-P WSIs into prostate adenocarcinoma and benign (non-neoplastic) lesions using transfer and weakly supervised learning. We evaluated the models on TUR-P, needle biopsy, and The Cancer Genome Atlas (TCGA) public dataset test sets, achieving an ROC-AUC up to 0.984 in TUR-P test sets for adenocarcinoma. The results demonstrate the promising potential of deployment in a practical TUR-P histopathological diagnostic workflow system to improve the efficiency of pathologists.