Supplementary Methods. Supplementary Table 1 - Key Resources Including Sources of Biological Samples, Antibodies, and Key Reagents. Supplementary Table 2 -Correlation of FLT3 Expression Levels With Disease Characteristics at Initial Diagnosis. Supplementary Table 3 - Summary of Human Nonimmune Tissues Positive for FLT3 mRNA and/or Protein Expression in At Least One Platform. Supplementary Table 4. Cerebellum FLT3 RNA Analysis. Supplementary Table 5 - Binding Affinities of Experimental FLT3 BiTE® Molecule and AMG 427. Supplementary Table 6 - Cell Line Characterization, Cytotoxicity, and T-Cell Activation (Human). Supplementary Table 7 - Cynomolgus Monkey Pharmacokinetics for Experimental FLT3 BiTE® Molecule. Supplementary Table 8 - Cynomolgus Monkey Pharmacokinetics for AMG 427. Supplementary Table 9 - Cynomolgus Monkey FLT3 Primer and Probe Sequences. Supplementary Table 10 - Patient Characteristics. Supplementary Table 11 - Mass Spectrometry Results. Supplementary Figure 1 - Additional flow cytometry expression analysis for acute myeloid leukemia (AML) and healthy donor (HD) bone marrow (BM). Supplementary Figure 2 - Analysis of FLT3 expression on normal human bone marrow. Supplementary Figure 3 - Analysis of FLT3 transcript and protein in cerebellum revealed truncated transcripts. Supplementary Figure 4 - In vitro characterization of FLT3 BiTE® molecules. Supplementary Figure 5 - Experimental FLT3 BiTE® molecule activity in vivo. Supplementary Figure 6 - FLT3 BiTE® molecule in vitro activity with cynomolgus monkey effector cells. Supplementary Figure 7 - FLT3 expression in cynomolgus monkey blood and bone marrow. Supplementary Figure 8 - AMG 427 pharmacodynamics in cynomolgus monkey. Supplementary Figure 9 - (A) Representative populations of T cells (CD2+) and AML cells (CD33+) over time from a responder (Patient 4; Figure 6A). (B) PD-1 expression on CD3+ cells from a T-cell-dependent cellular cytotoxicity (TDCC) of human pan T cells co-cultured 5:1 with MV4-11 cells for 48 hours assessed by flow cytometry.

Bettina Brauchle 1,2,† , Rebecca L. Goldstein 3,† , Christine M. Karbowski 4 , Anja Henn 5 , Chi-Ming Li 3 , Veit L. Bücklein 1,2 , Christina Krupka 1,2 , Michael C. Boyle 4 , Priya Koppikar 4 , Sascha Haubner 1,2 , Joachim Wahl 5 , Christoph Dahlhoff 5 , Tobias Raum 5 , Matthew J. Rardin 3 , Christine Sastri 3 , Dan A. Rock 3 , Michael von Bergwelt-Baildon 1,2,6 , Brendon Frank 3 , Klaus H. Metzeler 2,6 , Ryan Case 3 , Matthias Friedrich 5 , Mercedesz Balazs 3 , Karsten Spiekermann 2,6,7 , Angela Coxon 5 , Marion Subklewe 1,2,6,* , Tara Arvedson 3,*

Abstract Despite advances in the treatment of acute myeloid leukemia (AML), novel therapies are needed to induce deeper and more durable clinical response. Bispecific T-cell Engager (BiTE) molecules, which redirect patient T cells to lyse tumor cells, are a clinically validated modality for hematologic malignancies. Due to broad AML expression and limited normal tissue expression, fms-related tyrosine kinase 3 (FLT3) is proposed to be an optimal BiTE molecule target. Expression profiling of FLT3 was performed in primary AML patient samples and normal hematopoietic cells and nonhematopoietic tissues. Two novel FLT3 BiTE molecules, one with a half-life extending (HLE) Fc moiety and one without, were assessed for T-cell–dependent cellular cytotoxicity (TDCC) of FLT3-positive cell lines in vitro, in vivo, and ex vivo. FLT3 protein was detected on the surface of most primary AML bulk and leukemic stem cells but only a fraction of normal hematopoietic stem and progenitor cells. FLT3 protein detected in nonhematopoietic cells was cytoplasmic. FLT3 BiTE molecules induced TDCC of FLT3-positive cells in vitro, reduced tumor growth and increased survival in AML mouse models in vivo. Both molecules exhibited reproducible pharmacokinetic and pharmacodynamic profiles in cynomolgus monkeys in vivo, including elimination of FLT3-positive cells in blood and bone marrow. In ex vivo cultures of primary AML samples, patient T cells induced TDCC of FLT3-positive target cells. Combination with PD-1 blockade increased BiTE activity. These data support the clinical development of an FLT3 targeting BiTE molecule for the treatment of AML.

7032 Background: FMS-like tyrosine kinase 3 (FLT3) is a tyrosine-protein kinase involved in hematopoiesis. In acute myeloid leukemia (AML), higher FLT3 transcript levels, independent of the presence of FLT3 mutations, correlate with higher leukocyte counts and higher degrees of bone marrow infiltration by leukemic cells. FLT3 protein is detectable on the cell surface of more than 80% of leukemia isolates from adult AML patients whereas FLT3 mutations are present only in approximately one third of patients. AMG 553 is a novel, investigational, adoptive cellular immunotherapy for the treatment of relapsed refractory AML, consisting of autologous T cells genetically modified ex vivo to express a transmembrane chimeric antigen receptor (CAR) to target FLT3 protein on the surface of AML cells irrespective of FLT3 mutational status. Methods: The nonclinical safety evaluation of AMG 553 includes assessment of FLT3 expression in normal tissues; in vitro assessment of cytotoxicity against human cells with or without FLT3 expression; toxicology studies in cynomolgus monkeys administered autologous T-cells transduced with an anti-FLT3 CAR or administered anti-FLT3 bi-specific T-cell engager (BiTE) antibodies. Results: Assessment of FLT3 expression in normal tissues indicates that FLT3 protein is detected on the cell surface of a subpopulation of human bone marrow hematopoietic stem and progenitor cells; expression in other tissues is cytoplasmic and not anticipated to be accessible to AMG 553. AMG 553-induced cytotoxicity was only observed in a percentage of primary bone marrow CD34+ cells. There was no evidence of CAR-T cell-mediated toxicity, expansion, or persistence in cynomolgus monkeys likely due to restricted cell surface FLT3 protein expression in healthy animals which demonstrates the limited value of such studies for this target. All findings in toxicology studies with anti-FLT3 BiTEs were consistent with the targeted killing of cells expressing FLT3 on the plasma membrane. Conclusions: The nonclinical safety data support AMG 553 as a novel therapeutic to target FLT3 protein on AML cells irrespective of FLT3 mutational status, while only affecting a percentage of normal hematopoietic stem and progenitor cells.

Advancements in technology and digitization have ushered in novel ways of enhancing tissue-based research via digital microscopy and image analysis. Whole slide imaging scanners enable digitization of histology slides to be stored in virtual slide repositories and to be viewed via computers instead of microscopes. Easier and faster sharing of histologic images for teaching and consultation, improved storage and preservation of quality of stained slides, and annotation of features of interest in the digital slides are just a few of the advantages of this technology. Combined with the development of software for digital image analysis, digital slides further pave the way for the development of tools that extract quantitative data from tissue-based studies. This review introduces digital microscopy and pathology, and addresses technical and scientific considerations in slide scanning, quantitative image analysis, and slide repositories. It also highlights the current state of the technology and factors that need to be taken into account to insure optimal utility, including preanalytical considerations and the importance of involving a pathologist in all major steps along the digital microscopy and pathology workflow.

The title of the 2017 Society of Toxicologic Pathology symposium was Musculoskeletal System. A brief overview of the General Scientific Symposium is presented herein and describes the topics presented by each speaker. Symposium speakers addressed subjects pertinent to musculoskeletal system toxicologic pathology and drug development ranging from molecular biology of bone homeostasis to regulatory agency requirements and considerations for registration of bone therapeutics. This overview serves to summarize sessions and is intended as a guide to the individual submissions by speakers and symposium contributors.

CD19 is an immunoglobulin family member expressed ubiquitously on B cell lineage cells, from the pre-B cell to the mature B cell stage, until downregulation during terminal differentiation into plasma cells. CD19 is important for B cell activation, maturation and proliferation, and is widely expressed on the majority of B cell neoplasms. The T cell engaging bispecific antibody construct (BiTE ® ) blinatumomab, which targets CD19, is approved for the treatment of relapsed or refractory B cell precursor acute lymphoblastic leukemia in adults and children in the US and has shown great promise for treating other CD19-positive hematological malignancies. The short serum half-life of blinatumomab necessitates administration by continuous IV infusion to maintain therapeutic serum concentrations. To extend the half-life and allow for a more convenient administration, a next generation BiTE ® antibody construct designated CD19 HLE BiTE ® has been generated. The CD19 HLE BiTE ® comprises a new CD19- and CD3-targeting tandem single-chain Fv antibody fused to an Fc domain. The biological properties of CD19 HLE BiTE ® were evaluated in vitro and in vivo . CD19 HLE BiTE ® binds with high affinity to both human and non-human primate (NHP) CD19 as well as CD3. In vitro experiments demonstrated that CD19 HLE BiTE ® can recruit and activate human and NHP T cells, mediating redirected lysis of CD19-expressing cells at half-maximal effective concentrations in the low picomolar range. The fusion to an Fc domain resulted in a serum half-life of 210 hours following a single intravenous administration of a 5 µg/kg dose in NHP. In a repeat-dose study CD19 HLE BiTE ® caused robust depletion of circulating B cells, B lineage bone marrow cells, and peripheral lymphoid organ B cells in NHPs, whilst no overt signs of toxicity were observed in clinical and laboratory examinations of the animals. These results demonstrate that CD19 HLE BiTE ® effectively eliminated CD19-positive cells in vitro and in vivo , and may be suitable for once weekly dosing in patients with CD19-positive malignancies. Disclosures Lorenczewski: Amgen: Employment, Equity Ownership. Friedrich: Amgen Research (Munich) GmbH: Employment, Equity Ownership. Kischel: AMGEN: Employment. Dahlhoff: Amgen Research (Munich): Employment, Equity Ownership. Anlahr: Amgen: Employment, Equity Ownership. Balazs: Amgen Inc.: Employment, Equity Ownership. Rock: Amgen, Inc.: Employment, Equity Ownership. Boyle: Amgen: Employment. Goldstein: Amgen: Employment. Coxon: Amgen, Inc.: Employment. Chapman-Arvedson: Amgen, Inc.: Employment, Equity Ownership.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Japan (JSTP), Europe (ESTP), Great Britain (BSTP) and North America (STP) to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The primary purpose of this publication is to provide a standardized nomenclature for characterizing lesions observed in the cardiovascular (CV) system of rats and mice commonly used in drug or chemical safety assessment. The standardized nomenclature presented in this document is also available electronically for society members on the internet (http://goreni.org). Accurate and precise morphologic descriptions of changes in the CV system are important for understanding the mechanisms and pathogenesis of those changes, differentiation of natural and induced injuries and their ultimate functional consequence. Challenges in nomenclature are associated with lesions or pathologic processes that may present as a temporal or pathogenic spectrum or when natural and induced injuries share indistinguishable features. Specific nomenclature recommendations are offered to provide a consistent approach.

SWI/SNF (switching/sucrose nonfermenting)-dependent chromatin remodeling establishes coordinated gene expression programs during development, yet important functional details remain to be elucidated. We show that the Brg1 (Brahma-related gene 1; Smarca4) ATPase is globally expressed at high levels during postimplantation development and its conditional ablation, beginning at gastrulation, results in increased apoptosis, growth retardation, and, ultimately, embryonic death. Global gene expression analysis revealed that genes upregulated in Rosa26CreERT2; Brg1(flox/flox) embryos (here referred to as Brg1(d/d) embryos to describe embryos with deletion of the Brg1(flox/flox) alleles) negatively regulate cell cycle progression and cell growth. In addition, the p53 (Trp53) protein, which is virtually undetectable in early wild-type embryos, accumulated in the Brg1(d/d) embryos and activated the p53-dependent pathways. Using P19 cells, we show that Brg1 and CHD4 (chromodomain helicase DNA binding protein 4) coordinate to control target gene expression. Both proteins physically interact and show a substantial overlap of binding sites at chromatin-accessible regions adjacent to genes differentially expressed in the Brg1(d/d) embryos. Specifically, Brg1 deficiency results in reduced levels of the repressive histone H3 lysine K27 trimethylation (H3K27me3) histone mark and an increase in the amount of open chromatin at the regulatory region of the p53 and p21 (Cdkn1a) genes. These results provide insights into the mechanisms by which Brg1 functions, which is in part via the p53 program, to constrain gene expression and facilitate rapid embryonic growth.

This article describes the results of comparisons of digitally scanned whole slide images (WSIs) and glass microscope slides for diagnosis of tissues under peer review by the National Toxicology Program. Findings in this article were developed as a result of the data collected from 6 pathology working groups (PWGs), 1 pathology peer review, and survey comments from over 25 participating pathologists. For each PWG, 6–14 pathologists examined 10–143 tissues per study from 6- and 9-month perinatal studies and 2-year carcinogenicity studies. Overall it was found that evaluation of WSIs is generally equivalent to using glass slides. Concordance of PWG consensus diagnoses based upon review of WSIs versus glass slides ranged from 74% to 100% (median 86%). The intra- and interobserver diagnostic variation did not appear to influence the conclusions of any study. Based upon user opinions collected from surveys, WSIs may be less optimal than glass slides for evaluation of subtle lesions, large complex lesions, small lesions in a large section of tissue, and foci of altered hepatocytes. These results indicate that, although there may be some limitations, the use of WSIs can effectively accomplish the objectives of a conventional glass slide review and definitely serves as a useful adjunct to the conduct of PWGs.

BRG1, a catalytic subunit of the SWI/SNF ATPase chromatin‐remodeling complex, plays a vital role in gene regulation and tumorigenesis. Germline SWI/SNF function loss results in early embryonic lethality at approximately E3.5 in the mouse. BRG1 necessity during development beyond the pre‐implantation stage, however, has not been defined. The myriad temporal genomic changes during development require chromatin remodeling to facilitate differential gene expression. We hypothesized that BRG1 deficiency during post‐implantation development will cause gastrulation failure. Using a tamoxifen inducible conditional mouse model, we show that BRG1 ablation beginning at gastrulation results in arrested growth and early embryonic death. Mutant embryos failed to grow; histologic analysis revealed several developmental defects such as lack of normal cardiac structure and disrupted neural fold architecture. Immunohistochemistry analysis revealed aberrant expression of cell cycle and apoptosis marker proteins in BRG1 mutants. Microarray analysis revealed BRG1 ablation increased expression of cell cycle regulators, particularly within the p53 pathway. Co‐IP analyses reveal BRG1 interacts with CHD4 to suppress p53 pathways in P19 cells. These results suggest BRG1 plays a critical role in genomic surveillance essential for cell survival after the pre‐implantation period for normal cellular survival, proliferation and differentiation.Grant Funding Source: Z01 ES071006‐13

Heart failure is a leading cause of death in humans, and stress is increasingly associated with adverse cardiac outcomes. Glucocorticoids are primary stress hormones, but their direct role in cardiovascular health and disease is poorly understood. To determine the in vivo function of glucocorticoid signaling in the heart, we generated mice with cardiomyocyte-specific deletion of the glucocorticoid receptor (GR). These mice are born at the expected Mendelian ratio, but die prematurely from spontaneous cardiovascular disease. By 3 mo of age, mice deficient in cardiomyocyte GR display a marked reduction in left ventricular systolic function, as evidenced by decreases in ejection fraction and fractional shortening. Heart weight and left ventricular mass are elevated, and histology revealed cardiac hypertrophy without fibrosis. Removal of endogenous glucocorticoids and mineralocorticoids neither augmented nor lessened the hypertrophic response. Global gene expression analysis of knockout hearts before pathology onset revealed aberrant regulation of a large cohort of genes associated with cardiovascular disease as well as unique disease genes associated with inflammatory processes. Genes important for maintaining cardiac contractility, repressing cardiac hypertrophy, promoting cardiomyocyte survival, and inhibiting inflammation had decreased expression in the GR-deficient hearts. These findings demonstrate that a deficiency in cardiomyocyte glucocorticoid signaling leads to spontaneous cardiac hypertrophy, heart failure, and death, revealing an obligate role for GR in maintaining normal cardiovascular function. Moreover, our findings suggest that selective activation of cardiomyocyte GR may represent an approach for the prevention of heart disease.

To investigate the toxicity and carcinogenic potential of indole-3-carbinol (I3C), the National Toxicology Program has conducted 13-week subchronic studies in Fisher 344 rats and B6C3F1 mice, and chronic 2-year bioassays in Sprague-Dawley rats and B6C3F1 mice. While the chronic study results are not yet available, subchronic study results and short-term special evaluations of interim sacrifices in the 2-year rat bioassay are presented. F344 rats were orally gavaged ≤300 mg I3C/kg body weight 5 days a week for 13 weeks. Rats treated with ≥150 mg/kg demonstrated a dose-related dilation of lymphatics (lymphangiectasis) of the duodenum, jejunum, and mesenteric lymph nodes. Material within dilated lacteals stained positively for Oil Red O and Sudan Black, consistent with lipid. Electron microscopic evaluation confirmed extracellular lipid accumulation within the villar lamina propria, lacteals, and within villar macrophages. Analyses of hepatic and pulmonary CYP1A enzymes demonstrated dose-dependent I3C induction of CYP1A1 and 1A2. B6C3F1 mice orally gavaged ≤250 mg I3C/kg body weight did not demonstrate histopathological changes; however, hepatic CYP induction was similar to that in rats. The histopathologic changes of intestinal lymphangiectasis and lipidosis in this study share similarities with intestinal lymphangiectasia as observed in humans and dogs. However, the resultant clinical spectrum of protein-losing enteropathy was not present.

The heart is increasingly recognized as a target for toxicity. As studies in laboratory rodents are commonly used to investigate the potential toxicity of various agents, the identification and characterization of lesions of cardiotoxicity is of utmost importance. Although morphologic criteria have been established for degenerative myocardial lesions in rats and mice, differentiation of spontaneously occurring lesions from toxin-induced or toxin-related lesions remains difficult. A retrospective light microscopic evaluation was performed on the hearts of F344 rats and B6C3F(1) mice from National Toxicology Program (NTP) studies of six chemicals identified in the NTP database in which treatment-induced myocardial toxicity was present. Two previously defined myocardial lesions were observed: "cardiomyopathy" that occurred spontaneously or as a treatment-related effect and "myocardial degeneration" that occurred as a treatment-related effect. Both lesions consisted of the same basic elements, beginning with myofiber degeneration and necrosis, with varying amounts of inflammation, interstitial cell proliferation, and eventual fibrosis. This observation is indicative of the heart's limited repertoire of responses to myocardial injury, regardless of the nature of the inciting agent. A prominent differentiating factor between spontaneous and treatment-induced lesions was distribution and lesion onset. Once the respective lesions had undergone fibrosis, however, they generally appeared morphologically indistinguishable.

Objective —To determine the effect of hyperimmunization with an Escherichia coli J5 bacterin on serum IgG2 concentration, incidence of clinical mastitis, and rate of survival to the end of the lactation period (ie, day 305) in adult lactating dairy cattle. Design —Randomized controlled trial. Animals —1,012 Holstein cows in their second lactation and greater. Procedures —All cows were given 3 doses of the J5 bacterin; cows in the hyperimmunization group were given an additional 3 doses during the first 3 months of lactation. Blood was collected from a small sample of cows to determine anti-J5 IgG2 concentrations. Results —Cows in the hyperimmunization group had higher mean serum anti-J5 IgG2 concentrations than did control cows 28 days after administration of the fourth, fifth, and sixth doses of the J5 bacterin. However, mean serum anti-J5 concentrations during the subsequent lactation were not significantly different between groups. The proportions of cows that developed clinical mastitis were not significantly different between groups. However, control cows were more likely to have severe clinical mastitis than were cows in the hyperimmunization group. The percentage of control cows that remained in the herd to day 305 was significantly lower than the percentage of cows in the hyperimmunization group that did. Conclusions and Clinical Relevance —Results suggested that hyperimmunization of mature lactating dairy cattle was associated with increased serum anti-J5 IgG2 concentrations and decreased incidence of severe clinical mastitis, but did not alter survival rate of cows that developed severe clinical mastitis.