Scientific and Regulatory Policy Committee Points to Consider: Sampling, Processing, Evaluation, Interpretation, and Reporting of Test Article-Related Ganglion Pathology for Nonclinical Toxicity Studies.

Certain biopharmaceutical products consistently affect dorsal root ganglia, trigeminal ganglia, and/or autonomic ganglia. Product classes targeting ganglia include antineoplastic chemotherapeutics, adeno-associated virus-based gene therapies, antisense oligonucleotides, and anti-nerve growth factor agents. This article outlines "points to consider" for sample collection, processing, evaluation, interpretation, and reporting of ganglion findings; these points are consistent with published best practices for peripheral nervous system evaluation in nonclinical toxicity studies. Ganglion findings often occur as a combination of neuronal injury (e.g., degeneration, necrosis, and/or loss) and/or glial effects (e.g., increased satellite glial cell cellularity) with leukocyte accumulation (e.g., mononuclear cell infiltration or inflammation). Nerve fiber degeneration and/or glial reactions may be seen in nerves, dorsal spinal nerve roots, spinal cord, and occasionally brainstem. Interpretation of test article (TA)-associated effects may be confounded by incidental background changes or experimental procedure-related changes and limited historical control data. Reports should describe findings at these sites, any TA relationship, and the criteria used for assigning severity grades. Contextualizing adversity of ganglia findings can require a weight-of-evidence approach because morphologic changes of variable severity occur in ganglia but often are not accompanied by observable overt in-life functional alterations detectable by conventional behavioral and neurological testing techniques.

Scientific and Regulatory Policy Committee Technical Review: Biology and Pathology of Ganglia in Animal Species Used for Nonclinical Safety Testing.

Dorsal root ganglia (DRG), trigeminal ganglia (TG), other sensory ganglia, and autonomic ganglia may be injured by some test article classes, including anti-neoplastic chemotherapeutics, adeno-associated virus-based gene therapies, antisense oligonucleotides, nerve growth factor inhibitors, and aminoglycoside antibiotics. This article reviews ganglion anatomy, cytology, and pathology (emphasizing sensory ganglia) among common nonclinical species used in assessing product safety for such test articles (TAs). Principal histopathologic findings associated with sensory ganglion injury include neuron degeneration, necrosis, and/or loss; increased satellite glial cell and/or Schwann cell numbers; and leukocyte infiltration and/or inflammation. Secondary nerve fiber degeneration and/or glial reactions may occur in nerves, dorsal spinal nerve roots, spinal cord (dorsal and occasionally lateral funiculi), and sometimes the brainstem. Ganglion findings related to TA administration may result from TA exposure and/or trauma related to direct TA delivery into the central nervous system or ganglia. In some cases, TA-related effects may need to be differentiated from a spectrum of artifactual and/or spontaneous background changes.

Uterine Carcinomas in Tetrabromobisphenol A-exposed Wistar Han Rats Harbor Increased Tp53 Mutations and Mimic High-grade Type I Endometrial Carcinomas in Women.

Endometrial carcinoma is the most common gynecologic malignancy is the United States and accounts for 6% of all cancers in women. The disease is classified as type I or type II based on clinicopathologic and molecular features. It is a multifactorial disease with a number of risk factors, including environmental exposures. How environmental exposures, such as flame retardants, may affect the incidence of endometrial cancer is a topic of current and ongoing interest. Tetrabromobisphenol A (TBBPA) is a widely used brominated flame retardant found in a variety of household products. A recent 2-year National Toxicology Program carcinogenicity study found that exposure to TBBPA was associated with a marked increase in the development of uterine tumors, specifically uterine carcinomas, in Wistar Han rats. Molecularly, TBBPA-induced uterine carcinomas in Wistar Han rats were characterized by a marked increase in tumor protein 53 mutation compared to spontaneous uterine carcinomas, as well as overexpression of human epidermal growth factor receptor 2. Similar to spontaneous carcinomas, tumors in TBBPA-exposed rats were estrogen receptor-alpha positive and progesterone receptor negative by immunohistochemistry. The morphologic and molecular features of uterine carcinomas in TBBPA-exposed rats resemble those of high-grade type I tumors in women, and these data suggest that exposure to TBBPA may pose an increased cancer risk.

Evaluation of Aroclor 1260 exposure in a mouse model of diet-induced obesity and non-alcoholic fatty liver disease.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants associated with non-alcoholic fatty liver disease (NAFLD) in epidemiologic studies. The purpose of this study was to evaluate the hepatic effects of a PCB mixture, Aroclor 1260, whose composition mimics human bioaccumulation patterns, in a mouse model of diet-induced obesity (DIO). Male C57Bl/6J mice were fed control diet or 42% high fat diet (HFD) and exposed to Aroclor 1260 (20mg/kg or 200mg/kg in corn oil) for 12weeks. A glucose tolerance test was performed; plasma/tissues were obtained at necropsy for measurements of adipocytokine levels, histology, and gene expression. Aroclor 1260 exposure was associated with decreased body fat in HFD-fed mice but had no effect on blood glucose/lipid levels. Paradoxically, Aroclor 1260+HFD co-exposed mice demonstrated increased hepatic inflammatory foci at both doses while the degree of steatosis did not change. Serum cytokines, ALT levels and hepatic expression of IL-6 and TNFα were increased only at 20mg/kg, suggesting an inhibition of pro-inflammatory cytokine production at the 200mg/kg exposure. Aroclor 1260 induced hepatic expression of cytochrome P450s including Cyp3a11 (Pregnane-Xenobiotic Receptor target) and Cyp2b10 (constitutive androstane receptor target) but Cyp2b10 inducibility was diminished with HFD-feeding. Cyp1a2 (aryl hydrocarbon Receptor target) was induced only at 200mg/kg. In summary, Aroclor 1260 worsened hepatic and systemic inflammation in DIO. The results indicated a bimodal response of PCB-diet interactions in the context of inflammation which could potentially be explained by xenobiotic receptor activation. Thus, PCB exposure may be a relevant "second hit" in the transformation of steatosis to steatohepatitis.

Evaluation of eIF4E expression in an osteosarcoma-specific tissue microarray.

The ability to define osteosarcoma (OS) patients at greatest risk for metastatic progression and nonresponsiveness to conventional therapy is currently not possible. Such biomarkers are needed to predict overall prognosis, probability of metastases at diagnosis, and response to chemotherapy. The tissue microarray (TMA) serves as a powerful tool for detecting and validating protein biomarkers across a variety of patients. We constructed a novel outcome-linked TMA to add to and address shortcomings of currently available OS tissue resources. To test the use of our TMA, we surveyed the expression of eukaryotic initiation factor 4E (eIF4E) in OS patients using immunohistochemistry. Aberrant regulation of translation initiation is a feature of many cancers. eIF4E is central to initiation of protein synthesis. Its expression and activity have been implicated in tumor formation and potentially malignant and/or metastatic progression in some carcinomas. We found that eIF4E was uniformly expressed in OS patient samples. No association was found between eIF4E and outcome in OS patients. This novel OS TMA provided a facile mechanism to assess the role of a relevant protein biomarker in OS.

Correlation between clinical outcome and growth factor pathway expression in osteogenic sarcoma.

BACKGROUND: Multiple cell-signaling ligands and receptors-including vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), endothelial growth factor (EGF), v-akt murine thymoma viral oncogene homolog (AKT), platelet-derived growth factor (PDGF), mitogen-activated protein kinase (MAPK), and 70-kilodalton (kD) protein S6 kinase (p70S6 kinase)-reportedly are variably expressed in osteogenic sarcoma. Expression of these proteins may have future implications for prognostication and targeted therapy. The objective of the current study was to determine the relation between clinical outcome and the expression of these proteins. METHODS: A paraffin-embedded microarray of 48 human osteogenic sarcoma tissue specimens was stained with the antibodies against VEGF, IGF, EGF, AKT, PDGF, MAPK, and p70S6 kinase. Staining for each protein included the total protein and, when applicable, the phosphorylated version of the protein. Immunohistochemical staining was then correlated with patient survival (overall survival [OS] and event-free survival [EFS]), histologic response to chemotherapy, and serum markers. RESULTS: There was a negative correlation between VEGF receptor 3 (VEGF-R3) and both OS and EFS. VEGF-B was correlated with a poor histologic response to chemotherapy. Serum markers were not correlated with any specific proteins. When using a P value of .05, multiple correlations were observed between proteins of various pathways. CONCLUSIONS: The current results suggested that the VEGF pathway is a critical signaling pathway in osteogenic sarcoma. These data have identified specific proteins within these pathways toward which future investigations should be directed to further clarify their prognostic potential.