Smooth muscle hamartoma of the lungs in a Wistar Hannover rat.

Hamartomas are tumor-like masses comprising disorganized normal tissue elements. To date, spontaneous hamartomas have been reported in several organs and tissues in rodents but not in the lungs. Here, we report the first case of a hamartoma in the lungs of a 108-week-old female Wistar Hannover rat. Grossly, a white spot, 7 mm in diameter, was observed on the costal surface of the left lung. Histopathologically, the nodular lesions adjacent to the bronchioles comprised mature smooth muscle cells. The lesion was not encapsulated and spread along the alveolar walls and ducts without compression of the surrounding tissue. In the nodules, elastic fibers enclosed small lumens lined with factor VIII-related antigen-positive endothelial cells. This structure suggested that the nodule mimicked an artery. Moreover, structural abnormalities were observed within the bronchioles and arterioles owing to the increased number of smooth muscle cells in the surrounding tissues. These features suggested that this was a case of tissue malformation rather than a neoplasm, leading to the diagnosis of a smooth muscle hamartoma of the lung.

Ectopic pancreatic acinar cell carcinoma in the thoracic cavity of F344 rat.

Ectopic pancreatic tissue can occasionally cause inflammation, hemorrhage, stenosis, and invagination, similar to normal pancreatic tissue; however, tumorigenesis is rare. This case report describes an ectopically observed pancreatic acinar cell carcinoma in the thoracic cavity of a female Fischer (F344/DuCrlCrlj) rat. Histopathologically, polygonal tumor cells with periodic acid-Schiff-positive cytoplasmic eosinophilic granules showed solid proliferation and infrequently formed acinus-like structures. Immunohistochemically, the tumor cells were positive for cytokeratin, trypsin, and human B-cell leukemia/lymphoma 10, which specifically reacted with pancreatic acinar cells, and negative for vimentin and human α-smooth muscle actin. Ectopic pancreas develops in the submucosa of the gastrointestinal tract; however, there are few reports of its development and neoplasia in the thoracic cavity. To the best of our knowledge, this is the first report of ectopic pancreatic acinar cell carcinoma in the thoracic cavity of a rat.

Gene expression analysis of antioxidant and DNA methylation on the rat liver after 4-week wood preservative chromated copper arsenate exposure.

Our previous 4-week repeated dose toxicity study showed that wood preservative chromated copper arsenate (CCA) induced hepatocellular hypertrophy accompanied by biochemical hepatic dysfunction and an increase in oxidative stress marker, 8-hydroxydeoxyguanosine, in female rats. To further explore the molecular mechanisms of CCA hepatotoxicity, we analyzed 10%-buffered formalin-fixed liver samples from female rats for cell proliferation, apoptosis, and protein glutathionylation and conducted microarray analysis on frozen liver samples from female rats treated with 0 or 80 mg/kg/day of CCA. Chemical analysis revealed that dimethylated arsenical was the major metabolite in liver tissues of male and female rats. CCA increase labeling indices of proliferating cell nuclear antigen and decrease terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling accompanied with increased expression of protein glutathionylation, indicating a decrease in glutathione (GSH) in hepatocytes of female rats. Microarray analysis revealed that CCA altered gene expression of antioxidants, glutathione-S-transferase (GST), heat shock proteins and ubiquitin-proteasome pathway, cell proliferation, apoptosis, DNA methylation, cytochrome P450, and glucose and lipid metabolism in female rats. Increased expression of GSTs, including Gsta2, Gsta3, Mgst1, and Cdkn1b (p27), and decreased expression of the antioxidant Mt1, and DNA methylation Dnmt1, Dnmt3a, and Ctcf were confirmed in the liver of female rats in a dose-dependent manner. Methylation status of the promoter region of the Mt1 was not evidently changed between control and treatment groups. The results suggested that CCA decreased GSH and altered the expression of several genes, including antioxidants, GST, and DNA methylation, followed by impaired cell proliferation in the liver of female rats.

Single-cell next-generation sequencing of circulating tumor cells in patients with neuroblastoma.

Circulating tumor cells (CTCs) derived from any tumor tissue could contribute to metastasis and resistance to cancer treatments. In this study, we performed single-cell next-generation sequencing of CTCs and evaluated their usefulness for characterizing tumor biology and the mechanisms of metastasis in neuroblastomas (NB). We aimed to isolate CTCs from 10 patients with NB at diagnosis before any treatments and four patients at relapse. GD2+ CD90+ CD45- CD235a- DAPI- cells were isolated as neuroblastoma CTCs using fluorescence-activated cell sorting. In five patients with advanced stages (M stage), DNA and RNA sequencing of CTCs at single-cell level were performed. NB CTCs were isolated from eight of the 10 patients at diagnosis and three of the four patients at relapse. More CTCs could be isolated from patients with advanced stages. In one patient, ALK mutation (p.F1174L), was identified in both tumor tissue and a CTC. In patients with MYCN amplification, this gene was amplified in 12 of 13 CTCs. Using single-cell RNA sequencing, angiogenesis-related and cell cycle-related genes together with CCND1 and TUBA1A genes were found to be upregulated in CTCs. In one patient, CTCs were divided into two subgroups showing different gene expression profiles. In one subgroup, cell cycle-related and proliferation-related genes were differentially upregulated compared with the other group. In conclusion, next-generation sequencing of CTCs at single-cell level might help to characterize the tumor biology and the mechanisms of metastasis in NB.

The potential of organoids in toxicologic pathology: role of toxicologic pathologists in in vitro chemical hepatotoxicity assessment.

The development of in vitro toxicity assessment methods using cultured cells has gained popularity for promoting animal welfare in animal experiments. Herein, we briefly discuss the current status of hepatoxicity assessment using human- and rat-derived hepatocytes; we focus on the liver organoid method, which has been extensively studied in recent years, and discuss how toxicologic pathologists can use their knowledge and experience to contribute to the development of in vitro chemical hepatotoxicity assessment methods for drugs, pesticides, and chemicals. We also propose how toxicological pathologists should assess toxicity regarding the putative distribution of undifferentiated and differentiated cells in the organoid when liver organoids are observed in hematoxylin and eosin-stained specimens. This was done while considering the usefulness and limitations of in vitro studies for toxicologic pathology assessment.

Single-cell DNA and RNA sequencing of circulating tumor cells.

Single-cell sequencing of circulating tumor cells can precisely represent tumor heterogeneity and provide useful information for cancer treatment and research. After spiking TGW neuroblastoma cells into blood derived from healthy volunteer, the cells were isolated by fluorescence-activated cell sorting. DNA and mRNA were amplified by four different whole-genome amplifications (WGA) and three whole-transcriptome amplifications (WTA) methods, followed by single-cell DNA and RNA sequencing. Multiple displacement amplification (MDA)-based WGA methods showed higher amplification efficiency than other methods with a comparable depth of coverage as the bulk sample. The uniformity of coverage greatly differed among samples (12.5-89.2%), with some samples evaluated by the MDA-based WGA method using phi29 DNA polymerase and random primers showing a high (> 80%) uniformity of coverage. The MDA-based WTA method less effectively amplified mRNA and showed non-specific gene expression patterns. The PCR-based WTA using template switching with locked nucleic acid technology accurately amplified mRNA from a single cell. Taken together, our results present a more reliable and adaptable approach for CTC profiling at the single-cell level. Such molecular information on CTCs derived from clinical patients will promote cancer treatment and research.

[A Slight Change of Cholangiography Revealed Papillary Carcinoma of the Duodenum after Endoscopic Sphincterotomy(EST)].

A 79-year-old woman with chillness and nausea was admitted to our hospital. CT findings displayed a common extended bile duct with stacked stones and duodenal diverticulosis. The diagnosis was cholangitis with choledocholithiasis. She underwent endoscopic retrograde cholangiopancreatography(ERCP)to remove the common bile duct stones. Thereafter, she developed cholangitis several times without any obvious cause of biliary obstruction. A careful follow-up was continued using ERCP, and finally, a slightly irregular edge of the distal common bile duct was observed. Subsequently, bile duct brush cytology revealed adenocarcinoma. The final diagnosis was distal cholangiocarcinoma. An operation was performed and the pathological diagnosis of papillary carcinoma of the duodenum invading the common bile duct was made. We reviewed the first ERCP image findings retrospectively and noticed an abnormal papillary of the duodenum. We could not evaluate the papilla after endoscopic sphincterotomy(EST). We learned 2 important things. The first is to carefully observe naïve papilla, and the second is to pay attention to a slight change of cholangiography.

Adversity Considerations for Thyroid Follicular Cell Hypertrophy and Hyperplasia in Nonclinical Toxicity Studies: Results From the 6th ESTP International Expert Workshop.

The European Society of Toxicologic Pathology organized an expert workshop in May 2018 to address adversity considerations related to thyroid follicular cell hypertrophy and/or hyperplasia (FCHH), which is a common finding in nonclinical toxicity studies that can have important implications for risk assessment of pharmaceuticals, food additives, and environmental chemicals. The broad goal of the workshop was to facilitate better alignment in toxicologic pathology and regulatory sciences on how to determine adversity of FCHH. Key objectives were to describe common mechanisms leading to thyroid FCHH and potential functional consequences; provide working criteria to assess adversity of FCHH in context of associated findings; and describe additional methods and experimental data that may influence adversity determinations. The workshop panel was comprised of representatives from the European Union, Japan, and the United States. Participants shared case examples illustrating issues related to adversity assessments of thyroid changes. Provided here are summary discussions, key case presentations, and panel recommendations. This information should increase consistency in the interpretation of adverse changes in the thyroid based on pathology findings in nonclinical toxicity studies, help integrate new types of biomarker data into the review process, and facilitate a more systematic approach to communicating adversity determinations in toxicology reports.

An extraskeletal osteosarcoma in the auricle of a Wistar Hannover rat.

An extraskeletal osteosarcoma was detected in the auricle of a 110-week-old female Wistar Hannover rat. Grossly, the tumor, measuring 15 mm in size, was observed in the subcutis as a solid and hard mass. Histologically, the majority of the mass comprised mature, compact bone. It was surrounded by neoplastic cells showing a variety of histologies, such as sarcoma, not otherwise specified, and myxosarcoma away from the bone-forming region. However, these different histological regions were considered to be components of a single bone tumor, based on the common expression of osterix and a similar mixture of constituent cells in each region. The tumor was diagnosed as an extraskeletal osteosarcoma because of the presence of infiltrative growth and abnormal mitosis and its development in the auricle without attachment to the skeleton. The present case is a rare histological type of an extraskeletal osteosarcoma with independent and different histological elements in rats.

Spontaneous malignant myoid thymoma in an aged female Fischer 344 rat.

A whitish mass approximately 30 mm in diameter was noted in the anterior mediastinum of a 67-week-old female Fischer 344 rat. Histopathologically, two types of tumor cells were identified on the basis of morphologic features: epithelial tumor cells with a tubular or cord-like growth pattern and rhabdomyosarcomatous tumor cells characterized by the presence of cross-striations. Immunohistochemically, the epithelial tumor cells reacted positively for cytokeratin AE1/AE3, and some reacted positively for p63, which is expressed in normal thymic epithelial cells. The rhabdomyosarcomatous tumor cells stained positively for desmin, sarcomeric actin, and S-100 protein, which coincides with the stainability of normal thymic myoid cells. Since the tumor was also found to have malignant features such as high proliferative activity, cytologic atypia, and necrotic behavior, it was diagnosed as a malignant myoid thymoma. We believe that this is the first case report of such a tumor in a rodent.

Pathological and Clinical Pathological Changes Induced by Four-week, Repeated-dose, Oral Administration of the Wood Preservative Chromated Copper Arsenate in Wistar Rats.

Chromated copper arsenate (CCA) is used as a wood preservative worldwide. Exposure to it may adversely affect human health. Some events have increased human exposure to CCA, including the Great East Japan Earthquake, which generated a large amount of lumber debris from CCA-treated woods. We elucidated the toxicity due to daily exposure to CCA over a 4-week period at doses of 0, 8, 40, and 80 mg/kg/day in Wistar Hannover rats. Chromium (Cr) and arsenic (As), but not copper, were detected in the plasma samples of rats treated with various doses of CCA. Males and females showed sedation, and males had poor body weight gain. The clinical pathologies observed in both sexes included hypochromic and microcytic anemia, hepatic and renal dysfunction, and changes in lipid and glucose levels. Histopathologically, males and females showed forestomach hyperkeratosis, mucosal epithelial hyperplasia in the small intestine, rectal goblet cell hypertrophy, and lipofuscin deposition in the proximal renal tubule. Females showed diffuse hepatocellular hypertrophy with increased 8-hydroxydeoxyguanosine levels. These results indicated that oral administration of CCA mainly affected hematopoietic, gastrointestinal, hepatic, and renal systems owing to the toxic effects of As and/or Cr. Major toxic effects were observed in both sexes receiving 40 and 80 mg/kg/day.

A spontaneous myoepithelial carcinoma in the mammary gland of an aged female ICR (CD-1) mouse.

We report a female Crlj:CD1(ICR) mouse with a spontaneous mammary gland tumor composed of biphasic tumor cells, i.e., epithelioid and spindle-shaped myoepithelial cells. Macroscopically, a subcutaneous mass, approximately 3 cm in diameter was found in the lumbodorsal region. Histopathologically, the epithelioid cells proliferated in an alveolar or nest-like growth pattern, occasionally forming glandular-like structures. On the other hand, the spindle-shaped cells proliferated in a sarcomatous pattern. Normal mammary gland was observed in the vicinity of the tumor. Both types of tumor cells showed immunoreactivity for cytokeratin (wide spectrum screening), vimentin, S100, and p63. In addition, the epithelioid cells and spindle-shaped cells were immunopositive for glial fibrillary acidic protein and smooth muscle actin, respectively. Moderate atypia, high proliferative activity, massive necrosis, and partial infiltration to the surrounding tissues were also observed. We made a diagnosis of myoepithelial carcinoma, which is extremely rare in ICR mice.

Detection of respiratory allergies caused by environmental chemical allergen via measures of hyper-activation and degranulation of mast cells in lungs of NC/Nga mice.

Respiratory allergy triggered by exposure to environmental chemical allergen is a serious problem in many Asian countries and has the potential to cause severe health problems. Here, we aimed to elucidate the pathogenic mechanisms of this disease and develop an in vivo detection method for respiratory allergy induced by environmental chemical allergen. Both BALB/c and NC/Nga mice were sensitized topically for 3 weeks and were then subjected to inhalation challenge with pulverized trimellitic anhydride into particles measuring 2-µm in diameter. On the day after the final challenge, all mice were sacrificed, and IgE levels, immunocyte counts, and cytokine levels in the serum, hilar lymph nodes, and bronchoalveolar lavage fluid were measured. We also monitored the expression of genes encoding pro-inflammatory cytokines in the lung. We found that all endpoints were significantly increased in mice of both strains subjected to trimellitic anhydride inhalation as compared with the respective control groups. However, worsening of respiratory status was noted only in NC/Nga mice. Interestingly, type 2 helper T-cell reactions were significantly increased in BALB/c mice compared with that in NC/Nga mice. In contrast, the number of mast cells, levels of mast cell-related cytokine/chemokines, and production of histamine in NC/Nga mice were significantly higher than those in BALB/c mice. Thus, environmental chemical allergen induced respiratory allergy in NC/Nga mice in terms of functional and inflammatory symptoms. Furthermore, mast cells may be involved in the aggravation of airway allergic symptoms induced by environmental chemical allergens.

Significant upregulation of cytokine secretion from T helper type 9 and 17 cells in a NC/Nga mouse model of ambient chemical exposure-induced respiratory allergy.

It has been reported that ambient chemical exposure is closely associated with respiratory allergies. We attempted to develop an original protocol for detecting ambient chemical exposure-induced respiratory allergy in different strains of mice. In the process of comparing allergic potency of these mice, we observed that NC/Nga mice showed significant upregulation of respiratory allergic symptoms as well as specific type of cytokine secretions. The main purpose of this study was to investigate the mechanism underlying these phenomena in NC/Nga mice in comparison with BALB/c mice. For the model of respiratory allergy, female BALB/c and NC/Nga mice were sensitized and challenged with trimellitic anhydride. Clinical observation, IgE and immunocyte counts, and cytokine profile in the serum, lymph nodes, and bronchoalveolar lavage fluid were recorded. We also monitored the expression of genes encoding pro-inflammatory cytokines in the lung. We found that worsening of respiratory status was noted only in NC/Nga mice, whereas Th2 reactions were significantly increased in BALB/c mice compared with NC/Nga mice. In contrast, the levels of Th9 and Th17-derived cytokines in NC/Nga mice were significantly higher than those in BALB/c mice. Thus, Th9 and Th17 may be involved in the aggravation of respiratory allergic symptoms induced by ambient chemicals.

Characterizing "Adversity" of Pathology Findings in Nonclinical Toxicity Studies: Results from the 4th ESTP International Expert Workshop.

The identification of adverse health effects has a central role in the development and risk/safety assessment of chemical entities and pharmaceuticals. There is currently a need for better alignment regarding how nonclinical adversity is determined and characterized. The European Society of Toxicologic Pathology (ESTP) therefore coordinated a workshop to review available definitions of adversity, weigh determining and qualifying factors of adversity based on case examples, and recommend a practical approach to define and characterize adversity in toxicology reports, to serve as a valuable prerequisite for future organ- or lesion-specific workshops planned by the ESTP.

Toxicity and Carcinogenicity of Dichlorodiphenyltrichloroethane (DDT).

Dichlorodiphenyltrichloroethane (DDT) is still used in certain areas of tropics and subtropics to control malaria and other insect-transmitted diseases. DDT and its metabolites have been extensively studied for their toxicity and carcinogenicity in animals and humans and shown to have an endocrine disrupting potential affecting reproductive system although the effects may vary among animal species in correlation with exposure levels. Epidemiologic studies revealed either positive or negative associations between exposure to DDT and tumor development, but there has been no clear evidence that DDT causes cancer in humans. In experimental animals, tumor induction by DDT has been shown in the liver, lung, and adrenals. The mechanisms of hepatic tumor development by DDT have been studied in rats and mice. DDT is known as a non-genotoxic hepatocarcinogen and has been shown to induce microsomal enzymes through activation of constitutive androstane receptor (CAR) and to inhibit gap junctional intercellular communication (GJIC) in the rodent liver. The results from our previously conducted 4-week and 2-year feeding studies of p,p'-DDT in F344 rats indicate that DDT may induce hepatocellular eosinophilic foci as a result of oxidative DNA damage and leads them to hepatic neoplasia in combination with its mitogenic activity and inhibitory effect on GJIC. Oxidative stress could be a key factor in hepatocarcinogenesis by DDT.

Spontaneous extraskeletal osteosarcoma with various histological growth patterns in the abdominal wall of an ICR mouse.

Extraskeletal osteosarcoma is extremely rare in mice. This case report demonstrates a spontaneous murine extraskeletal osteosarcoma that exhibited various histological growth patterns in an ICR mouse. At necropsy, the tumor mass was located in the abdominal wall and was 45 × 30 × 25 mm in size. Histopathologically, the tumor showed the following four growth patterns: a solid pattern of polygonal cells embedded in an osteoid eosinophilic matrix with calcification, an irregular sheet pattern of short spindle cells accompanying some eosinophilic multinucleated cells, a fascicular pattern of spindle cells and a cystic pattern lined by short spindle cells. Immunohistochemically, most of the tumor cells were positive for vimentin, proliferating cell nuclear antigen and osterix. The multinucleated cells mentioned above were desmin positive and were regarded as regenerative striated muscles but not tumor cells. Since no clear continuity with normal bone tissues was observed, the tumor was diagnosed as an "extraskeletal osteosarcoma."

Recommendations from the INHAND Apoptosis/Necrosis Working Group.

Historically, there has been confusion relating to the diagnostic nomenclature for individual cell death. Toxicologic pathologists have generally used the terms "single cell necrosis" and "apoptosis" interchangeably. Increased research on the mechanisms of cell death in recent years has led to the understanding that apoptosis and necrosis involve different cellular pathways and that these differences can have important implications when considering overall mechanisms of toxicity, and, for these reasons, the separate terms of apoptosis and necrosis should be used whenever differentiation is possible. However, it is also recognized that differentiation of the precise pathway of cell death may not be important, necessary, or possible in routine toxicity studies and so a more general term to indicate cell death is warranted in these situations. Morphological distinction between these two forms of cell death can sometimes be straightforward but can also be challenging. This article provides a brief discussion of the cellular mechanisms and morphological features of apoptosis and necrosis as well as guidance on when the pathologist should use these terms. It provides recommended nomenclature along with diagnostic criteria (in hematoxylin and eosin [H&E]-stained sections) for the most common forms of cell death (apoptosis and necrosis). This document is intended to serve as current guidance for the nomenclature of cell death for the International Harmonization of Nomenclature and Diagnostic Criteria Organ Working Groups and the toxicologic pathology community at large. The specific recommendations are:Use necrosis and apoptosis as separate diagnostic terms.Use modifiers to denote the distribution of necrosis (e.g., necrosis, single cell; necrosis, focal; necrosis, diffuse; etc.).Use the combined term apoptosis/single cell necrosis whenThere is no requirement or need to split the processes, orWhen the nature of cell death cannot be determined with certainty, orWhen both processes are present together. The diagnosis should be based primarily on the morphological features in H&E-stained sections. When needed, additional, special techniques to identify and characterize apoptosis can also be used.

Poorly differentiated salivary gland carcinoma with prominent squamous metaplasia in a pregnant Wistar Hannover rat.

A subcutaneous pale brown-colored mass was observed macroscopically in the ventral neck of a 16-week-old Wistar rat on day 18 of gestation. The mass was well demarcated from the adjacent tissues with partial invasion into connective tissues. Necrosis and hemorrhage were evident throughout the mass. The mass comprised a diffuse sheet and a nest-like structure of epithelial cells with prominent squamous metaplasia. The neoplastic cells tested immunopositive for keratin, vimentin, glial fibrillary acidic protein and p63. A portion of the neoplastic cells exhibited a similar immunoreaction of prominin-1 to the ductal and acinar cells in normal submandibular and parotid glands. Collectively, the tumor was diagnosed as a poorly differentiated carcinoma derived from epithelial/myoepithelial lineages in the submandibular and/or parotid glands.