Inter-Rater and Intra-Rater Agreement in Scoring Severity of Rodent Cardiomyopathy and Relation to Artificial Intelligence-Based Scoring.

We previously developed a computer-assisted image analysis algorithm to detect and quantify the microscopic features of rodent progressive cardiomyopathy (PCM) in rat heart histologic sections and validated the results with a panel of five veterinary toxicologic pathologists using a multinomial logistic model. In this study, we assessed both the inter-rater and intra-rater agreement of the pathologists and compared pathologists' ratings to the artificial intelligence (AI)-predicted scores. Pathologists and the AI algorithm were presented with 500 slides of rodent heart. They quantified the amount of cardiomyopathy in each slide. A total of 200 of these slides were novel to this study, whereas 100 slides were intentionally selected for repetition from the previous study. After a washout period of more than six months, the repeated slides were examined to assess intra-rater agreement among pathologists. We found the intra-rater agreement to be substantial, with weighted Cohen's kappa values ranging from k = 0.64 to 0.80. Intra-rater variability is not a concern for the deterministic AI. The inter-rater agreement across pathologists was moderate (Cohen's kappa k = 0.56). These results demonstrate the utility of AI algorithms as a tool for pathologists to increase sensitivity and specificity for the histopathologic assessment of the heart in toxicology studies.

Case Report: Neuroblastoma-Like Schwannoma in a Domestic Short-Haired Cat.

An axillary mass was detected in a 6-year-old, neutered, male, domestic short-haired cat during a wellness exam. Gross examination following surgical removal revealed a discrete, deep subcutaneous, discoid mass that was between 0.5- and 0.7-cm-in-diameter and diffusely firm and white. Histologically, the mass was well-demarcated, partially encapsulated, and expanded the panniculus carnosus. It was composed of tightly packed, giant rosettes of radially arranged fusiform cells stacked in one to 10 layers with peripherally palisading nuclei and with centrally oriented, fibrillary, cytoplasmic processes, and collagenous fibers. Laminin immunoreactivity and ultrastructural examination highlighted a continuous basal lamina outside the plasma membrane of each neoplastic cell. Neoplastic cells were immunoreactive for GFAP, S100, periaxin, and Sox-10 and were immunonegative for synaptophysin, smooth muscle actin, and pancytokeratin. Collective findings were consistent with a diagnosis of neuroblastoma-like schwannoma. This is the first veterinary report of this rare variant of benign schwannoma.

Using Artificial Intelligence to Detect, Classify, and Objectively Score Severity of Rodent Cardiomyopathy.

Rodent progressive cardiomyopathy (PCM) encompasses a constellation of microscopic findings commonly seen as a spontaneous background change in rat and mouse hearts. Primary histologic features of PCM include varying degrees of cardiomyocyte degeneration/necrosis, mononuclear cell infiltration, and fibrosis. Mineralization can also occur. Cardiotoxicity may increase the incidence and severity of PCM, and toxicity-related morphologic changes can overlap with those of PCM. Consequently, sensitive and consistent detection and quantification of PCM features are needed to help differentiate spontaneous from test article-related findings. To address this, we developed a computer-assisted image analysis algorithm, facilitated by a fully convolutional network deep learning technique, to detect and quantify the microscopic features of PCM (degeneration/necrosis, fibrosis, mononuclear cell infiltration, mineralization) in rat heart histologic sections. The trained algorithm achieved high values for accuracy, intersection over union, and dice coefficient for each feature. Further, there was a strong positive correlation between the percentage area of the heart predicted to have PCM lesions by the algorithm and the median severity grade assigned by a panel of veterinary toxicologic pathologists following light microscopic evaluation. By providing objective and sensitive quantification of the microscopic features of PCM, deep learning algorithms could assist pathologists in discerning cardiotoxicity-associated changes.

Histology Atlas of the Developing Prenatal and Postnatal Mouse Central Nervous System, with Emphasis on Prenatal Days E7.5 to E18.5.

Evaluation of the central nervous system (CNS) in the developing mouse presents unique challenges, given the complexity of ontogenesis, marked structural reorganization over very short distances in 3 dimensions each hour, and numerous developmental events susceptible to genetic and environmental influences. Developmental defects affecting the brain and spinal cord arise frequently both in utero and perinatally as spontaneous events, following teratogen exposure, and as sequelae to induced mutations and thus are a common factor in embryonic and perinatal lethality in many mouse models. Knowledge of normal organ and cellular architecture and differentiation throughout the mouse's life span is crucial to identify and characterize neurodevelopmental lesions. By providing a well-illustrated overview summarizing major events of normal in utero and perinatal mouse CNS development with examples of common developmental abnormalities, this annotated, color atlas can be used to identify normal structure and histology when phenotyping genetically engineered mice and will enhance efforts to describe and interpret brain and spinal cord malformations as causes of mouse embryonic and perinatal lethal phenotypes. The schematics and images in this atlas illustrate major developmental events during gestation from embryonic day (E)7.5 to E18.5 and after birth from postnatal day (P)1 to P21.

Combined Inhibition of Both p110α and p110ß Isoforms of Phosphatidylinositol 3-Kinase Is Required for Sustained Therapeutic Effect in PTEN-Deficient, ER+ Breast Cancer.

Purpose: Determine the roles of the PI3K isoforms p110α and p110ß in PTEN-deficient, estrogen receptor α (ER)-positive breast cancer, and the therapeutic potential of isoform-selective inhibitors.Experimental Design: Anti-estrogen-sensitive and -resistant PTEN-deficient, ER+ human breast cancer cell lines, and mice bearing anti-estrogen-resistant xenografts were treated with the anti-estrogen fulvestrant, the p110α inhibitor BYL719, the p110ß inhibitor GSK2636771, or combinations. Temporal response to growth factor receptor-initiated signaling, growth, apoptosis, predictive biomarkers, and tumor volumes were measured.Results: p110ß primed cells for response to growth factor stimulation. Although p110ß inhibition suppressed cell and tumor growth, dual targeting of p110α/ß enhanced apoptosis and provided sustained tumor response. The growth of anti-estrogen-sensitive cells was inhibited by fulvestrant, but fulvestrant inconsistently provided additional therapeutic effects beyond PI3K inhibition alone. Treatment-induced decreases in phosphorylation of AKT and Rb were predictive of therapeutic response. Short-term drug treatment induced tumor cell apoptosis and proliferative arrest to induce tumor regression, whereas long-term treatment only suppressed proliferation to provide durable regression.Conclusions: p110ß is the dominant PI3K isoform in PTEN-deficient, ER+ breast cancer cells. Upon p110ß inhibition, p110α did not induce significant reactivation of AKT, but combined targeting of p110α/ß most effectively induced apoptosis in vitro and in vivo and provided durable tumor regression. Because apoptosis and tumor regression occurred early but not late in the treatment course, and proliferative arrest was maintained throughout treatment, p110α/ß inhibitors may be considered short-term cytotoxic agents and long-term cytostatic agents. Clin Cancer Res; 23(11); 2795-805. ©2016 AACR.

Proceedings of the 2016 National Toxicology Program Satellite Symposium.

The 2016 annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri" was held in San Diego, CA, at the Society of Toxicologic Pathology's (STP) 35th annual meeting. The goal of this symposium was to present and discuss challenging diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers' talks, along with select images that were used by the audience for voting and discussion. Some lesions and topics covered during the symposium included malignant glioma and histiocytic sarcoma in the rodent brain; a new statistical method designed for histopathology data evaluation; uterine stromal/glandular polyp in a rat; malignant plasma cell tumor in a mouse brain; Schwann cell proliferative lesions in rat hearts; axillary schwannoma in a cat; necrosis and granulomatous inflammation in a rat brain; adenoma/carcinoma in a rat adrenal gland; hepatocyte maturation defect and liver/spleen hematopoietic defects in an embryonic mouse; distinguishing malignant glioma, malignant mixed glioma, and malignant oligodendroglioma in the rat; comparison of mammary gland whole mounts and histopathology from mice; and discussion of the International Harmonization of Nomenclature and Diagnostic Criteria collaborations.

The PI3K/mTOR dual inhibitor P7170 demonstrates potent activity against endocrine-sensitive and endocrine-resistant ER+ breast cancer.

Activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway has been implicated in anti-estrogen resistance in breast cancer. We tested the therapeutic potential of the novel PI3K/mTOR dual inhibitor P7170 in a panel of anti-estrogen-sensitive and anti-estrogen-resistant models of ER+ breast cancer. Estrogen receptor-positive (ER+) breast cancer cells were treated ±P7170. Fresh cores from primary ER+/HER2- tumors from two patients were treated ±P7170 ex vivo. Mice bearing breast cancer xenografts were randomized to treatment with vehicle, fulvestrant, P7170, or combinations, and tumor volumes were measured. Tissues and cells were analyzed for markers of pathway activity, cell viability, and apoptosis. In cell lines, P7170 exhibited IC50 values in the range of 0.9-7 nM and induced apoptosis. P7170 potently inhibited mTOR activity (≤ 25 nM) and inhibited PI3K at higher concentrations (≥ 200 nM). P7170 completely inhibited MCF-7 tumor growth, significantly inhibited growth of fulvestrant-resistant T47D tumors, and suppressed tumor cell proliferation but did not induce apoptosis. While P7170 inhibits PI3K and mTOR in ER+/HER2- human breast cancer cells and tumors ex vivo, in vivo data indicate that the primary mechanism of P7170 anti-tumor action is inhibition of mTOR and cell proliferation. P7170 is a novel agent worthy of further investigation for the treatment of ER+ breast cancer.

The inflammasome sensor, NLRP3, regulates CNS inflammation and demyelination via caspase-1 and interleukin-18.

Inflammation is increasingly recognized as an important contributor to a host of CNS disorders; however, its regulation in the brain is not well delineated. Nucleotide-binding domain, leucine-rich repeat, pyrin domain containing 3 (NLRP3) is a key component of the inflammasome complex, which also includes ASC (apoptotic speck-containing protein with a card) and procaspase-1. Inflammasome formation can be triggered by membrane P2X(7)R engagement leading to cleavage-induced maturation of caspase-1 and interleukin-1ß (IL-1ß)/IL-18. This work shows that expression of the Nlrp3 gene was increased >100-fold in a cuprizone-induced demyelination and neuroinflammation model. Mice lacking the Nlrp3 gene (Nlrp3(-/-)) exhibited delayed neuroinflammation, demyelination, and oligodendrocyte loss in this model. These mice also showed reduced demyelination in the experimental autoimmune encephalomyelitis model of neuroinflammation. This outcome is also observed for casp1(-/-) and IL-18(-/-) mice, whereas IL-1ß(-/-) mice were indistinguishable from wild-type controls, indicating that Nlrp3-mediated function is through caspase-1 and IL-18. Additional analyses revealed that, unlike the IL-1ß(-/-) mice, which have been previously shown to show delayed remyelination, Nlrp3(-/-) mice did not exhibit delayed remyelination. Interestingly, IL-18(-/-) mice showed enhanced remyelination, thus providing a possible compensatory mechanism for the lack of a remyelination defect in Nlrp3(-/-) mice. These results suggest that NLRP3 plays an important role in a model of multiple sclerosis by exacerbating CNS inflammation, and this is partly mediated by caspase-1 and IL-18. Additionally, the therapeutic inhibition of IL-18 might decrease demyelination but enhance remyelination, which has broad implications for demyelinating diseases.

MHC class II exacerbates demyelination in vivo independently of T cells.

We have shown previously the importance of MHC class II for central nervous system remyelination; however, the function of MHC class II during cuprizone-induced demyelination has not been examined. Here, we show that I-A(beta)-/- mice exhibit significantly reduced inflammation and demyelination. RAG-1(1/1) mice are indistinguishable from controls, indicating T cells may not play a role. The role of MHC class II depends on an intact cytoplasmic tail that leads to the production of IL-1beta, TNF-alpha, and nitric oxide, and oligodendrocyte apoptosis. Thus, the function of MHC class II cytoplasmic tail appears to increase microglial proliferation and activation that exacerbates demyelination.