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The European Society of Toxicologic Pathology (ESTP) organized a panel of 24 international experts from many fields of toxicologic clinical pathology (e.g., industry, academia, and regulatory) that came together in 2021 to align the use of terminology to convey the importance of clinical pathology findings in preclinical toxicity studies. An additional goal consisted of how to identify important findings in standard and nonstandard clinical pathology associated endpoints. This manuscript summarizes the information and opinions discussed and shared at the ninth ESTP International Expert Workshop, April 5 to 6, 2022. In addition to terminology usage, the workshop considered topics related to the identification and conveyance of the importance of test item-related findings. These topics included sources of variability, comparators, statistics, reporting, correlations to other study data, nonstandard biomarkers, indirect/secondary findings, and an overall weight-of-evidence approach.
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Digitization of histologic slides brings with it the promise of enhanced toxicologic pathology practice through the increased application of computational methods. However, the development of these advanced methods requires access to substrate image data, that is, whole slide images (WSIs). Deep learning methods, in particular, rely on extensive training data to develop robust algorithms. As a result, pharmaceutical companies interested in leveraging computational methods in their digital pathology workflows must first invest in data infrastructure to enable data access for both data scientists and pathologists. The process of building robust image data resources is challenging and includes considerations of generation, curation, and storage of WSI files, and WSI access including via linked metadata. This opinion piece describes the collective experience of building resources for WSI data in the Roche group. We elaborate on the challenges encountered and solutions developed with the goal of providing examples of how to build a data resource for digital pathology analytics in the pharmaceutical industry.
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Algoritmos , Indústria FarmacêuticaRESUMO
Clofibrate is a known rodent hepatotoxicant classically associated with hepatocellular hypertrophy and increased serum activities of cellular alanine aminotransferase/aspartate aminotransferase (ALT/AST) in the absence of microscopic hepatocellular degeneration. At toxic dose, clofibrate induces liver and skeletal muscle injury. The objective of this study was to assess novel liver and skeletal muscle biomarkers following clofibrate administration in Wistar rats at different dose levels for 7 days. In addition to classical biomarkers, liver injury was assessed by cytokeratin 18 (CK18) cleaved form, high-mobility group box 1, arginase 1 (ARG1), microRNA 122 (miR-122), and glutamate dehydrogenase. Skeletal muscle injury was evaluated with fatty acid binding protein 3 (Fabp3) and myosin light chain 3 (Myl3). Clofibrate-induced hepatocellular hypertrophy and skeletal muscle degeneration (type I rich muscles) were noted microscopically. CK, Fabp3, and Myl3 elevations correlated to myofiber degeneration. Fabp3 and Myl3 outperformed CK for detection of myofiber degeneration of minimal severity. miR-122 and ARG1 results were significantly correlated and indicated the absence of liver toxicity at low doses of clofibrate, despite increased ALT/AST activities. Moreover, combining classical and novel biomarkers (Fabp3, Myl3, ARG1, and miR-122) can be considered a valuable strategy for differentiating increased transaminases due to liver toxicity from skeletal muscle toxicity.
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Anticolesterolemiantes/efeitos adversos , Biomarcadores/sangue , Doença Hepática Induzida por Substâncias e Drogas/patologia , Clofibrato/efeitos adversos , Fígado/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Animais , Anticolesterolemiantes/administração & dosagem , Arginase/sangue , Aspartato Aminotransferases/sangue , Bilirrubina/sangue , Colesterol/sangue , Colinesterases/sangue , Clofibrato/administração & dosagem , Creatinina/sangue , Relação Dose-Resposta a Droga , Proteína 3 Ligante de Ácido Graxo/sangue , Glutamato Desidrogenase/sangue , Queratina-18/sangue , Fígado/metabolismo , Masculino , MicroRNAs/sangue , Músculo Esquelético/metabolismo , Cadeias Leves de Miosina/sangue , Ratos , Ratos Wistar , Triglicerídeos/sangueRESUMO
The virtual control group (VCG) concept provides a potential opportunity to reduce animal use in drug development by replacing concurrent control groups (CCGs) in nonclinical toxicity studies. This work investigated the feasibility and reliability of using VCGs in place of CCGs. A historical control database (HCD), constructed from Genentech Inc. rat toxicity study data, was reviewed to understand trends and sources of variability in control animals over time, and to identify data curation requirements for assembling VCGs, e.g. alignment of units of measurement. Several endpoints were investigated and stratified against different study design parameters. Sex, route of administration, fasting status, and body weight at study initiation were among the parameters that were indicated as key matching criteria. With a high-level understanding of potential sources of variability, a retrospective proof-of-concept (POC) study was designed, evaluating a historical rat pilot toxicity study for test article-related changes. A masked interpretation of the study was conducted using its CCG, and two unique VCGs that were constructed from individual animal data pulled from our HCD. While the results of the microscopic pathology assessment and most endpoints were similar across the different control groups, the POC revealed the risk of using VCGs to interpret subtle test article-related changes in clinical pathology parameters. Within the context of our POC, it appears the use of a VCG is not completely equivalent to the CCG especially with clinical pathology parameters. Additional work is needed to understand the potential utility, and thus, viability of VCGs in other contexts.
This study explored the use of virtual control groups (VCGs) as a potential method to reduce the number of living control animals in drug development. The process involves replacing concurrent control groups with historical animal data in nonclinical toxicity studies. Several parameters were identified as crucial factors that must be aligned before the construction of VCGs. The VCG concept was tested using a historical rat toxicity study, comparing results against the conventional control group as well as two unique VCGs. Although results were similar in most cases, potential risks in interpreting subtle changes in clinical pathology parameters were identified. Further work is needed to fully elucidate VCGs' potential, and whether it is a viable alternative to current methods. The significance of this work lies in the possibility of reducing the number of animals used in testing, in support of the 3Rs (replace, reduce, and refine).
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The gastrointestinal tract may be a site of origin for α-synuclein pathology in idiopathic Parkinson's disease (PD). Disruption of the autophagy-lysosome pathway (ALP) may contribute to α-synuclein aggregation. Here we examined epigenetic alterations in the ALP in the appendix by deep sequencing DNA methylation at 521 ALP genes. We identified aberrant methylation at 928 cytosines affecting 326 ALP genes in the appendix of individuals with PD and widespread hypermethylation that is also seen in the brain of individuals with PD. In mice, we find that DNA methylation changes at ALP genes induced by chronic gut inflammation are greatly exacerbated by α-synuclein pathology. DNA methylation changes at ALP genes induced by synucleinopathy are associated with the ALP abnormalities observed in the appendix of individuals with PD specifically involving lysosomal genes. Our work identifies epigenetic dysregulation of the ALP which may suggest a potential mechanism for accumulation of α-synuclein pathology in idiopathic PD.
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Apêndice/metabolismo , Autofagia , Epigênese Genética , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Animais , Apêndice/química , Encéfalo/metabolismo , Encéfalo/patologia , Metilação de DNA , Feminino , Humanos , Lisossomos/química , Lisossomos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doença de Parkinson/genética , Doença de Parkinson/patologia , Agregados Proteicos , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Histiocytic sarcoma is the most frequent hematopoietic tumor in rats. We report here a histiocytic sarcoma infiltrating the liver, the spleen and the pancreas from a Wistar rat. In the liver, the tumor was associated with oval cell and bile duct hyperplasia. The cells looked like neoplastic histocytic cells described in this species but with some particularities (e.g. lack of multinucleated giant cells). At immunohistochemistry, neoplastic cells in the liver were vimentine positive but lysozyme and CD68 negative. In the kidney, lysozyme-positive cytoplasmic droplets were observed. We describe here an atypical case of histiocytic sarcoma in the rat and we compare the nature of these neoplastic cells to other species.
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Sarcoma Histiocítico/patologia , Neoplasias Hepáticas/patologia , Neoplasias Pancreáticas/patologia , Neoplasias Esplênicas/patologia , Animais , Antígenos CD/biossíntese , Antígenos de Diferenciação Mielomonocítica/biossíntese , Sarcoma Histiocítico/metabolismo , Imuno-Histoquímica , Rim/metabolismo , Rim/patologia , Neoplasias Hepáticas/metabolismo , Masculino , Muramidase/biossíntese , Neoplasias Pancreáticas/metabolismo , Ratos , Ratos Wistar , Neoplasias Esplênicas/metabolismo , Vimentina/biossínteseRESUMO
Mammary gland adenomyoepitheliomas are benign complex mammary gland tumors composed of neoplastic cells of epithelial and myoepithelial origins, described in many species (humans, dogs, cats, rats) and rarely in mice. We report here an adenomyoepithelioma in a C57BL/6 female mouse. Histologically, tubes and cords formed by neoplastic epithelial cells were separated by bundles of neoplastic myoepithelial cells in a clear and partially mucinous matrix. The tumor displayed characteristics of a benign neoplastic proliferation with a compressive growth pattern, and moderate cellular pleomorphism and mitotic index. At immunohistochemistry, the epithelial cells were strongly cytokeratin positive; the myoepithelial cells were weakly cytokeratin positive and strongly smooth muscle actin positive. This is to our knowledge, the first report of a mammary gland adenomyoepithelioma in a C57BL/6 mouse.
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Neoplasias Mamárias Animais/patologia , Mioepitelioma/patologia , Mioepitelioma/veterinária , Animais , Feminino , Imuno-Histoquímica , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mioepitelioma/metabolismoRESUMO
A number of drugs can cause precipitates within renal tubules leading to crystal nephropathy. Crystal nephropathy is usually an exposure-related finding and is not uncommon in preclinical studies, where high doses are tested. An understanding of the nature of precipitates is important for human risk assessment and further development. Our aim was to investigate the ability of various imaging techniques to detect the presence of drugs or metabolites in renal crystals. We applied matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) imaging, Raman and infrared microspectroscopy, scanning electron microscopy coupled with energy dispersive X-ray (SEM/EDX) spectroscopy and standard histopathology to cases of drug-induced crystal nephropathy, induced in rodents and primates by 4 compounds. MALDI-FTICR MS imaging enabled the identification of the drug-related crystal content in all 4 cases of nephropathy, without reference material and with high accuracy. Crystals were composed of unchanged parent drug and/or metabolites. Similar results were obtained using Raman and infrared microspectroscopy for 2 compounds. In the absence of reference standards of metabolites, Raman and infrared microspectroscopy showed that the crystals consisted of components similar, but not identical, to the administered drug for the other compounds, a limitation for these techniques. SEM/EDX showed which counter ions were colocalized with the identified drug-related material, complementing the MALDI-FTICR MS findings. Therefore, we recommend MALDI-FTICR MS as a first-line methodology to characterize crystal nephropathies. Raman and infrared microspectroscopy may be useful when MALDI-FTICR MS imaging cannot be applied. SEM/EDX could be considered as a complementary technology.
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Injúria Renal Aguda/diagnóstico por imagem , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico por imagem , Rim/efeitos dos fármacos , Preparações Farmacêuticas/química , Animais , Cristalização , Avaliação Pré-Clínica de Medicamentos , Rim/diagnóstico por imagem , Macaca fascicularis , Camundongos , Estrutura Molecular , Preparações Farmacêuticas/análise , Ratos , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Infravermelho , Análise Espectral RamanRESUMO
Incidences of neoplastic lesions were evaluated in untreated Hannover Wistar Rats RjHan: WI (470 males and 470 females) used as control animals in eight carcinogenicity studies. All these studies were performed in a similar environment either for the in vivo and the postmortem evaluation. The major neoplastic lesions were found in the endocrine, integumentary and reproductive systems. Pituitary adenoma was the most frequent neoplasm and occurred in 33.9% of the males and 54.6% of the female rats. The other most frequent tumors in males were thyroid C-cell adenoma (8.6%), pancreatic islet cell adenoma (8.1%), subcutaneous fibrosarcoma (6.6%), subcutaneous fibroma (4.7%), benign pheochromocytoma (3.4%), and cutaneous keratoacanthoma (3.4%). In females, the other highest incidences were mammary fibroadenoma (29%), uterine endometrial stromal polyp (18.1%), mammary adenocarcinoma (14.2%), mammary fibroadenoma with atypia (13.7%), thyroid C-cell adenoma (7.5%), benign thymoma (3.7%), and subcutaneous fibrosarcoma (3.6%). All these data were compared to previously published historical control data. This retrospective analysis was undergone in order to illustrate the result of a stable organization which guarantees a robust historical data base for neoplastic and non neoplastic findings.