Toxicologic Pathology最新文献

Microscopic observation data collected from approximately 1800 male and female Sprague Dawley (SD) control rats used on 104-week carcinogenicity studies performed at North American Labcorp Early Development, Inc, Madison, WI, were retrospectively evaluated for spontaneous nonneoplastic findings. This study provides incidence of the most common spontaneous nonneoplastic microscopic findings in each organ system of SD rats encountered during 104-week carcinogenicity studies. Some of the most common spontaneous background findings were cardiomyopathy; chronic progressive nephropathy; uterine cystic endometrial hyperplasia; prostate inflammation; pulmonary alveolar macrophage infiltrates; hepatocyte vacuolation, bile duct hyperplasia, and basophilic foci in the liver; pancreatic fibrosis; splenic extramedullary hematopoiesis and pigment; decreased lymphocytes and epithelial hyperplasia in the thymus; ventral brain compression; cystic degeneration and hyperplasia of the adrenal cortex; and mammary gland hyperplasia. The most common nonneoplastic findings in male SD rats were chronic progressive nephropathy (80.9%) and rodent progressive cardiomyopathy (73.2%). The most common nonnenoplastic findings in female SD rats were cystic degeneration of the adrenal cortex (64.7%) and ventral compression of the brain due to pituitary neoplasms (62.7%).

Enhanced histopathology of the immune system uses a precise, compartment-specific, and semi-quantitative evaluation of lymphoid organs in toxicology studies. The assessment of lymphocyte populations in tissues is subject to sampling variability and limited distinctive cytologic features of lymphocyte subpopulations as seen with hematoxylin and eosin (H&E) staining. Although immunohistochemistry is necessary for definitive characterization of T- and B-cell compartments, routine toxicologic assessments are based solely on H&E slides. Here, a deep learning (DL) model was developed using normal rats to quantify relevant compartments of the spleen, including periarteriolar lymphoid sheaths, follicles, germinal centers, and marginal zones from H&E slides. Slides were scanned, destained, dual labeled with CD3 and CD79a chromogenic immunohistochemistry, and rescanned to generate exact co-registered images that served as the ground truth for training and validation. The DL model identified individual splenic compartments with high accuracy (97.8% Dice similarity coefficient) directly from H&E-stained tissue. The DL model was utilized to study the normal range of lymphoid compartment area and cellularity. Future implementation of our DL model and expanding this approach to other lymphoid tissues have the potential to improve accuracy and precision in enhanced histopathology evaluation of the immune system with concurrent gains in time efficiency for the pathologist.

Replication-competent oncolytic virus (OV) therapies are a promising new modality for cancer treatment. However, they pose unique challenges for preclinical assessment, due in part to their tumor specificity and ability to self-replicate in vivo. Understanding biodistribution, immune cell responses, and potential effects of intratumoral replication on these outcomes are important aspects of the nonclinical profile for OVs. Herein, a single intravenous dose of vesicular stomatitis virus pseudotyped with the glycoprotein of lymphocytic choriomeningitis virus (VSV-GP), or a cargo-expressing variant (VSV-GP-[cargo]), was examined in both tumor-free and CT26.CL25.IFNAR^-/- syngeneic tumor-bearing mouse models. Biodistribution and immune cell responses were characterized using different molecular pathology methods, including a strand-specific in situ hybridization method to differentiate administered viral genomes from replicated or transcribed viral anti-genome RNA. We identified distinct patterns of viral biodistribution and replication across tumor and nontumor sites but no major differences in biodistribution, off-tumor cell tropism, or immune cell responses between tumor-free and tumor-bearing mouse models. Our findings characterize key cellular changes following systemic exposure to VSV-GP, provide a better understanding of a nonclinical permissive tumor model for OV assessment, and demonstrate how current molecular pathology methods can provide a bridge between traditional biodistribution and pathology readouts.

Drug-induced nephrotoxicity is a major challenge in drug discovery and development, accounting for nearly a quarter of severe adverse effects in current pharmacotherapy. Antimicrobial use may be associated with this problem, with one-third of nephrotoxicity related to these drugs. During the lead optimization stage of our antibacterial programs, nephrotoxicity was observed with renal tubule degeneration and tubular granular casts. To examine the nephrotoxicity mechanisms and triage compounds, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to investigate the compound distribution in rat kidney sections. MALDI-MSI has emerged as a powerful tool allowing for the spatial localization of drugs and metabolites directly from tissue surfaces without the need for labels. By comparing the renal distribution of toxic and non-toxic compounds, a correlation of preferential renal cortex and outer-medullar distribution with positive in vivo nephrotoxicity was discovered for most of the drug candidates being tested. This correlation facilitated the ranking of compounds to aid in the lead optimization process of antimicrobial drug discovery. We envision that MALDI-MSI can be used for drug-induced nephrotoxicity derisking during drug discovery and development when a correlation between tissue distribution and nephrotoxicity can be established.

Characterizing the expression of novel targets in normal and diseased tissues is a fundamental component of a target validation data package. Often these targets are presented to the pathology team for assessment with bulk or single-cell RNAseq data and limited to no spatial tissue expression data. In situ hybridization to detect mRNA (RNAscope) is a valuable tool to (1) identify cells that may express the target protein and to corroborate protein expression during immunohistochemical (IHC) assay development or (2) to use as surrogate for single-cell expression IHC when antibodies are not available. Chromogenic RNAscope in situ hybridization (CISH) can be performed on frozen or formalin-fixed, paraffin-embedded (FFPE) tissues. This CISH workflow starts with RNA qualification of the tissue (to assess RNA integrity) by measuring the expression of housekeeping genes. RNA-qualified tissues then undergo CISH for the target in question, and positive CISH signals are quantified in VisioPharm by a combination of color deconvolution, size gating, and dot density thresholding. This RNA workflow can complement IHC or standalone in target validation for spatial characterization of novel targets.

Mass spectrometry imaging (MSI) was used to investigate and provide insights into observed biliary pathology found in dogs and rats after administration of two different compounds. Both compounds were associated with peribiliary inflammatory infiltrates and proliferation of the bile duct epithelium. However, MSI revealed very different spatial distribution profiles for the two compounds: Compound A showed significant accumulation within the bile duct epithelium with a much higher concentration than in the parenchymal hepatocytes, while Compound T exhibited only a slight increase in the bile duct epithelium compared to parenchymal hepatocytes. These findings implicate cholangiocyte uptake and accumulation as a key step in the mechanism of biliary toxicity. In both cases, compounds are shown at the site of toxicity in support of a direct mechanism of toxicity on the biliary epithelium. MSI is a powerful tool for localizing small molecules within tissue sections and improvements in sensitivity have enabled localization down to the cellular level in some cases. MSI was also able to identify biomarker candidates of toxicity by differential analysis of ion profiles comparing treated and control cholangiocytes from tissue sections.

Off-target evaluation is essential in preclinical safety assessments of novel biotherapeutics, supporting lead molecule selection, endpoint selection in toxicology studies, and regulatory requirements for first-in-human trials. Off-target interaction of a therapeutic antibody and antibody derivatives has been historically assessed via the Tissue Cross-Reactivity (TCR) study, in which the candidate molecule is used as a reagent in immunohistochemistry (IHC) to assess binding of the candidate molecule to a panel of human tissue sections. The TCR approach is limited by the performance of the therapeutic as an IHC reagent, which is often suboptimal to outright infeasible. Furthermore, binding of the therapeutic in IHC conditions typically has poor in vitro to in vivo translation and lacks qualitative data of the identity of putative off-targets limiting the decisional value of the data. More recently, cell-based protein arrays (CBPA) that allow for screening against a large portion of the human membrane proteome and secretome have emerged as a complement, and likely a higher value alternative, to IHC-based off-target assessment. These arrays identify specific protein interactions and may be useful for testing nontraditional antibody-based therapeutic formats that are unsuitable for TCR studies. This article presents an overview of CBPA technologies in the context of TCR and off-target assessment studies. Selected case examples and strategic considerations covering a range of different modalities are presented.