Evaluation of the Use of Single- and Multi-Magnification Convolutional Neural Networks for the Determination and Quantitation of Lesions in Nonclinical Pathology Studies.

Digital pathology platforms with integrated artificial intelligence have the potential to increase the efficiency of the nonclinical pathologist's workflow through screening and prioritizing slides with lesions and highlighting areas with specific lesions for review. Herein, we describe the comparison of various single- and multi-magnification convolutional neural network (CNN) architectures to accelerate the detection of lesions in tissues. Different models were evaluated for defining performance characteristics and efficiency in accurately identifying lesions in 5 key rat organs (liver, kidney, heart, lung, and brain). Cohorts for liver and kidney were collected from TG-GATEs open-source repository, and heart, lung, and brain from internally selected R&D studies. Annotations were performed, and models were trained on each of the available lesion classes in the available organs. Various class-consolidation approaches were evaluated from generalized lesion detection to individual lesion detections. The relationship between the amount of annotated lesions and the precision/accuracy of model performance is elucidated. The utility of multi-magnification CNN implementations in specific tissue subtypes is also demonstrated. The use of these CNN-based models offers users the ability to apply generalized lesion detection to whole-slide images, with the potential to generate novel quantitative data that would not be possible with conventional image analysis techniques.

Proof of Concept: The Use of Whole-Slide Images (WSI) for Peer Review of Tissues on Routine Regulatory Toxicology Studies.

This Proof of Concept (POC) study was to assess whether assessment of whole slide images (WSI) of the 2 target tissues for a contemporaneous peer review can elicit concordant results to the findings generated by the Study Pathologist from the glass slides. Well-focused WSI of liver and spleen from 4 groups of mice, that had previously been diagnosed to be the target tissues by an experienced veterinary toxicologic pathologist examining glass slides, were independently reviewed by 3 veterinary pathologists with varying experience in assessment of WSIs. Diagnostic discrepancies were then reviewed by an experienced adjudicating pathologist. Assessment of microscopic findings using WSI showed concordance with the glass slides, with only slight discrepancy in severity grades noted. None of the lesions recorded by the Study pathologist were "missed" and no lesions were added by the pathologists evaluating WSIs, thus demonstrating equivalence of the WSI to glass slides for this study.

BSTP Review of 12 Case Studies Discussing the Challenges, Pathology, Immunogenicity, and Mechanisms of Inhaled Biologics.

The inhalation route is a relatively novel drug delivery route for biotherapeutics and, as a result, there is a paucity of published data and experience within the toxicology/pathology community. In recent years, findings arising in toxicology studies with inhaled biologics have provoked concern and regulatory challenges due, in part, to the lack of understanding of the expected pathology, mechanisms, and adversity induced by this mode of delivery. In this manuscript, the authors describe 12 case studies, comprising 18 toxicology studies, using a range of inhaled biotherapeutics (monoclonal antibodies, fragment antigen-binding antibodies, domain antibodies, therapeutic proteins/peptides, and an oligonucleotide) in rodents, nonhuman primates (NHPs), and the rabbit in subacute (1 week) to chronic (26 weeks) toxicology studies. Analysis of the data revealed that many of these molecules were associated with a characteristic pattern of toxicity with high levels of immunogenicity. Microscopic changes in the airways consisted of a predominantly lymphoid perivascular/peribronchiolar (PV/PB) mononuclear inflammatory cell (MIC) infiltrate, whereas changes in the terminal airways/alveoli were characterized by simple ("uncomplicated") increases in macrophages or inflammatory cell infiltrates ranging from mixed inflammatory cell infiltration to inflammation. The PV/PB MIC changes were considered most likely secondary to immunogenicity, whereas simple increases in alveolar macrophages were most likely secondary to clearance mechanisms. Alveolar inflammatory cell infiltrates and inflammation were likely induced by immune modulation or stimulation through pharmacologic effects on target biology or type III hypersensitivity (immune complex disease). Finally, a group of experts provide introductory thoughts regarding the adversity of inhaled biotherapeutics and the basis for reasonable differences of opinion that might arise between toxicologists, pathologists, and regulators.

Biologic Immunomodulatory Drugs and Infection in the Respiratory Tract of Nonhuman Primates.

Though rare due to measures and practices to control the risk, infections can occur in research and toxicology studies, especially in nonhuman primates (NHPs) exposed to xenobiotics, particularly immunomodulatory drugs. With such xenobiotics, immunocompromised or immunosuppressed animals will not be able to mount a protective response to infection by an opportunistic pathogen (bacteria, virus, parasite, or fungus) that might otherwise be nonpathogenic and remain clinically asymptomatic in immunocompetent animals. The respiratory tract is one of the most commonly affected systems in clinic, but also in toxicology studies. Pulmonary inflammation will be the main finding associated with opportunistic infections and may cause overt clinical disease with even early sacrifice or death, and may compromise or complicate the pathology evaluation. It is important to properly differentiate the various features of infection, to be aware of the range of possible opportunistic pathogens and how they may impact the interpretation of pathology findings. This review will present the most common bacterial, viral, parasitic, and fungal infections observed in the respiratory tract in NHPs during research and/or toxicology studies.

Implementing Reverse Phase Protein Array Profiling as a Sensitive Method for the Early Pre-Clinical Detection of Off-Target Toxicities Associated with Sunitinib Malate.

PURPOSE: The tyrosine kinase inhibitor (TKI) sunitinib is a multi-targeted agent approved across multiple cancer indications. Nevertheless, since approval, data has emerged to describe a worrisome side effect profile including hypertension, hand-foot syndrome, fatigue, diarrhea, mucositis, proteinuria, and (rarely) congestive heart failure. It has been hypothesized that the observed multi-parameter toxicity profile is related to "on-target" kinase inhibition in "off-target" tissues. EXPERIMENTAL DESIGN: To interrogate off-target effects in pre-clinical studies, a reverse phase protein array (RPPA) approach is employed. Mice are treated with sunitinib (40 mg kg-1 ) for 4 weeks, following which critical organs are removed. The Zeptosens RPPA platform is employed for protein expression analysis. RESULTS: Differentially expressed proteins associated with damage and/or stress are found in the majority of organs from treated animals. Proteins differentially expressed in the heart are associated with myocardial hypertrophy, ischaemia/reperfusion, and hypoxia. However, hypertrophy is not evidenced on histology. Mild proteinuria is observed; however, no changes in renal glomerular structure are visible via electron microscopy. In skin, proteins associated with cutaneous inflammation, keratinocyte hyper-proliferation, and increased inflammatory response are differentially expressed. CONCLUSIONS AND CLINICAL RELEVANCE: It is posited that pre-clinical implementation of a combined histopathological/RPPA approach provides a sensitive method to mechanistically elucidate the early manifestation of TKI on-target/organ off-target toxicities.

Histopathological Findings in Cynomolgus Macaques ( Macaca fascicularis) Consistent with Secondary Immunological Reaction to Biotherapeutics with an Emphasis on the CNS and Eye.

Biotherapeutics are pharmaceutical products derived from or synthesized by biological systems. Such molecules carry the potential for immunogenicity which may lead to adverse immune responses. The cynomolgus macaque ( Macaca fascicularis) is the species of choice in nonclinical safety assessment of biotherapeutics. The main aim of this study was to confirm whether mononuclear cell infiltrates at specific locations represent a generic effect of biotherapeutics, and therefore the result of their immunogenicity. Following a review of microscopic findings in studies conducted over a 10-year period at one test facility, 15% of biotherapeutics were reported to have such findings. The most commonly affected site was the choroid plexus and less frequently the meninges and ciliary body. The reporting of such findings as test article-related becomes more subjective as the severity and incidence decreases. To assess the accuracy of such associations, a mathematical approach was employed to determine the probability of obtaining the observed results by chance. There was good agreement between this approach and the original findings. In addition to an increased number and size of mononuclear cell infiltrates in the brain, biotherapeutic administration was strongly associated with the presence of plasma cells and eosinophils.

A Novel Positron Emission Tomography (PET) Approach to Monitor Cardiac Metabolic Pathway Remodeling in Response to Sunitinib Malate.

Sunitinib is a tyrosine kinase inhibitor approved for the treatment of multiple solid tumors. However, cardiotoxicity is of increasing concern, with a need to develop rational mechanism driven approaches for the early detection of cardiac dysfunction. We sought to interrogate changes in cardiac energy substrate usage during sunitinib treatment, hypothesising that these changes could represent a strategy for the early detection of cardiotoxicity. Balb/CJ mice or Sprague-Dawley rats were treated orally for 4 weeks with 40 or 20 mg/kg/day sunitinib. Cardiac positron emission tomography (PET) was implemented to investigate alterations in myocardial glucose and oxidative metabolism. Following treatment, blood pressure increased, and left ventricular ejection fraction decreased. Cardiac [18F]-fluorodeoxyglucose (FDG)-PET revealed increased glucose uptake after 48 hours. [11C]Acetate-PET showed decreased myocardial perfusion following treatment. Electron microscopy revealed significant lipid accumulation in the myocardium. Proteomic analyses indicated that oxidative metabolism, fatty acid ß-oxidation and mitochondrial dysfunction were among the top myocardial signalling pathways perturbed. Sunitinib treatment results in an increased reliance on glycolysis, increased myocardial lipid deposition and perturbed mitochondrial function, indicative of a fundamental energy crisis resulting in compromised myocardial energy metabolism and function. Our findings suggest that a cardiac PET strategy may represent a rational approach to non-invasively monitor metabolic pathway remodeling following sunitinib treatment.

Calcium lignosulphonate: re-evaluation of relevant endpoints to re-confirm validity and NOAEL of a 90-day feeding study in rats.

A 90-day feeding study in Han/Wistar rats with calcium lignosulphonate was evaluated by the EFSA. The study was considered to be inadequate due to potentially impaired health status of the animals based upon a high incidence of minimal lymphoid hyperplasia in mesenteric/mandibular lymph nodes and Peyer's patches, and minimal lymphoid cell infiltration in the liver in all animals. The EFSA Panel further disagreed with the conclusion that the treatment-related observation of foamy histiocytosis in mesenteric lymph nodes was non-adverse and asked whether this observation would progress to something more adverse over time. A PWG was convened to assess the sections of lymph nodes, Peyer's patches and liver. In addition, all lymphoid tissues were re-examined. The clinical pathology and animal colony health screening data were re-evaluated. The question whether the foamy histiocytosis could progress to an adverse finding with increasing exposure duration was addressed by read-across. In conclusion, the animals on the 90-day feeding study were in good health, the study was adequate for safety evaluation, and the foamy histiocytes in the mesenteric lymph nodes were not considered adverse, but rather an adaptive response that was considered unlikely to progress to an adverse condition with time. The NOAEL was re-affirmed to be 2000 mg/kgbw/d.