Pathologist-trained machine learning classifiers developed to quantitate celiac disease features differentiate endoscopic biopsies according to modified marsh score and dietary intervention response.

BACKGROUND: Histologic evaluation of the mucosal changes associated with celiac disease is important for establishing an accurate diagnosis and monitoring the impact of investigational therapies. While the Marsh-Oberhuber classification has been used to categorize the histologic findings into discrete stages (i.e., Type 0-3c), significant variability has been documented between observers using this ordinal scoring system. Therefore, we evaluated whether pathologist-trained machine learning classifiers can be developed to objectively quantitate the pathological changes of villus blunting, intraepithelial lymphocytosis, and crypt hyperplasia in small intestine endoscopic biopsies. METHODS: A convolutional neural network (CNN) was trained and combined with a secondary algorithm to quantitate intraepithelial lymphocytes (IEL) with 5 classes on CD3 immunohistochemistry whole slide images (WSI) and used to correlate feature outputs with ground truth modified Marsh scores in a total of 116 small intestine biopsies. RESULTS: Across all samples, median %CD3 counts (positive cells/enterocytes) from villous epithelium (VE) increased with higher Marsh scores (Type 0%CD3 VE = 13.4; Type 1-3%CD3 VE = 41.9, p < 0.0001). Indicators of villus blunting and crypt hyperplasia were also observed (Type 0-2 villous epithelium/lamina propria area ratio = 0.81; Type 3a-3c villous epithelium/lamina propria area ratio = 0.29, p < 0.0001), and Type 0-1 crypt/villous epithelial area ratio = 0.59; Type 2-3 crypt/villous epithelial area ratio = 1.64, p < 0.0001). Using these individual features, a combined feature machine learning score (MLS) was created to evaluate a set of 28 matched pre- and post-intervention biopsies captured before and after dietary gluten restriction. The disposition of the continuous MLS paired biopsy result aligned with the Marsh score in 96.4% (27/28) of the cohort. CONCLUSIONS: Machine learning classifiers can be developed to objectively quantify histologic features and capture additional data not achievable with manual scoring. Such approaches should be further investigated to improve biopsy evaluation, especially for clinical trials.

A Phase 2 study of prexasertib (LY2606368) in platinum resistant or refractory recurrent ovarian cancer.

OBJECTIVE: High-grade serous ovarian cancer, the most frequent type of ovarian cancer, has a poor prognosis and novel treatments are needed for patients with platinum resistant/refractory disease. New therapeutic strategies targeting cell cycle checkpoints, including CHK1 inhibition with prexasertib, may help improve clinical response and overcome resistance. METHODS: Patients with ovarian cancer (N = 169) were assigned to 4 cohorts as part of the Phase 2 multicenter trial (NCT03414047): Cohort 1: platinum resistant, BRCA-wildtype with ≥3 lines prior therapy; Cohort 2: platinum resistant BRCA-wildtype with <3 lines prior therapy; Cohort 3: platinum resistant, BRCA-mutated with prior PARP inhibitor therapy; Cohort 4: platinum refractory, BRCA-mutated, or BRCA-wildtype with any number of prior therapy lines. The primary endpoint was objective response rate (ORR) and secondary endpoints included disease control rate (DCR), and safety. DNA from tumor biopsies was sequenced to identify biomarkers. RESULTS: The ORR in platinum resistant patients (Cohorts 1--3) was 12.1%, and 6.9% in platinum refractory patients. In platinum resistant patients, DCR was 37.1%, and consistent across cohorts. In platinum refractory patients, DCR was 31.0%. Consistent with the prexasertib mechanism of action, the most common treatment related adverse events of all grades included thrombocytopenia, neutropenia, fatigue, nausea, and anemia. CONCLUSIONS: Prexasertib demonstrated durable single agent activity in a subset of patients with recurrent ovarian cancer regardless of clinical characteristics, BRCA status, or prior therapies, including PARPi. There was no obvious correlation with genomic alterations in responders vs non-responders, emphasizing the need for alternative biomarker approaches for responder identification.

Effects of 0.5% and 2.0% Sodium Lauryl Sulfate in Male CD-1 Mice From a 3-Month Oral Gavage Toxicity Study.

The tolerability of single daily gavage doses of 0.5% or 2.0% (wt/vol) sodium lauryl sulfate (SLS) in 11- to 12-week-old male CD-1 mice was evaluated in a study of 3 months in duration. Live-phase, gross necropsy, and histopathologic parameters were evaluated. Mortality of 14% occurred in mice administered formulations containing SLS. Clinical observations in mice administered SLS included abnormal respiration (audible, irregular, and/or labored), swollen abdomen, rough haircoat, hunched appearance, and hypoactivity. Necropsy findings in mice administered SLS consisted of enlarged intestines containing abnormal contents with gas. There were no instances of mechanical gavage-related injury. Histologic evaluation of the respiratory tract revealed injury to the nasal passages and nasopharynx, including, but not limited to, inflammation, exudate, apoptosis/necrosis of epithelium, and atrophy of epithelium or olfactory nerves. Collectively, the data indicated that under the experimental conditions of our 3-month study in male CD-1 mice, once-daily gavage administration of vehicle formulations containing SLS at 0.5% or 2.0% resulted in nasal injury and 14% mortality supportive of gastroesophageal reflux. Sponsors utilizing formulations containing SLS in toxicity studies in CD-1 mice should exclude gastroesophageal reflux as a confounding factor in studies with morbidity or mortality associated with respiratory distress or evidence of aerophagia.

Inhibition of myostatin prevents microgravity-induced loss of skeletal muscle mass and strength.

The microgravity conditions of prolonged spaceflight are known to result in skeletal muscle atrophy that leads to diminished functional performance. To assess if inhibition of the growth factor myostatin has potential to reverse these effects, mice were treated with a myostatin antibody while housed on the International Space Station. Grip strength of ground control mice increased 3.1% compared to baseline values over the 6 weeks of the study, whereas grip strength measured for the first time in space showed flight animals to be -7.8% decreased in strength compared to baseline values. Control mice in space exhibited, compared to ground-based controls, a smaller increase in DEXA-measured muscle mass (+3.9% vs +5.6% respectively) although the difference was not significant. All individual flight limb muscles analyzed (except for the EDL) weighed significantly less than their ground counterparts at the study end (range -4.4% to -28.4%). Treatment with myostatin antibody YN41 was able to prevent many of these space-induced muscle changes. YN41 was able to block the reduction in muscle grip strength caused by spaceflight and was able to significantly increase the weight of all muscles of flight mice (apart from the EDL). Muscles of YN41-treated flight mice weighed as much as muscles from Ground IgG mice, with the exception of the soleus, demonstrating the ability to prevent spaceflight-induced atrophy. Muscle gene expression analysis demonstrated significant effects of microgravity and myostatin inhibition on many genes. Gamt and Actc1 gene expression was modulated by microgravity and YN41 in opposing directions. Myostatin inhibition did not overcome the significant reduction of microgravity on femoral BMD nor did it increase femoral or vertebral BMD in ground control mice. In summary, myostatin inhibition may be an effective countermeasure to detrimental consequences of skeletal muscle under microgravity conditions.

Cross-Platform Comparison of Computer-assisted Image Analysis Quantification of In Situ mRNA Hybridization in Investigative Pathology.

Although availability of automated platforms has proliferated, there is no standard practice for computer-assisted generation of scores for mRNA in situ hybridization (ISH) visualized by brightfield microscopic imaging on tissue sections. To address this systematically, an ISH for peptidylprolyl isomerase B (PPIB) (cyclophilin B) mRNA was optimized and applied to a tissue microarray of archival non-small cell lung carcinoma cases, and then automated image analysis for PPIB was refined across 4 commercially available software platforms. Operator experience and scoring results from ImageScope, HALO, CellMap, and Developer XD were systematically compared with each other and to manual pathologist scoring. Markup images were compared and contrasted for accuracy, the ability of the platform to identify cells, and the ease of visual assessment to determine appropriate interpretation. Comparing weighted scoring approaches using H-scores (Developer XD, ImageScope, and manual scoring) a correlation was observed (R value=0.7955), and association between the remaining 2 approaches (HALO and CellMap) was of similar value. ImageScope showed the highest R value in comparison with manual scoring (0.7377). Mean-difference plots showed that HALO produced the highest relative normalized values, suggesting higher relative sensitivity. ImageScope overestimated PPIB ISH signal at the high end of the range scores; however, this tendency was not observed in other platforms. HALO emerged with the highest number of favorable observations, no apparent systematic bias in score generation compared with the other methods, and potentially higher sensitivity to detect ISH. HALO may serve as a tool to empower teams of investigative pathology laboratory scientists to assist pathologists readily with quantitative scoring of ISH.

Merestinib (LY2801653) inhibits neurotrophic receptor kinase (NTRK) and suppresses growth of NTRK fusion bearing tumors.

Merestinib is an oral multi-kinase inhibitor targeting a limited number of oncokinases including MET, AXL, RON and MKNK1/2. Here, we report that merestinib inhibits neurotrophic receptor tyrosine kinases NTRK1/2/3 which are oncogenic drivers in tumors bearing NTRK fusion resulting from chromosomal rearrangements. Merestinib is shown to be a type II NTRK1 kinase inhibitor as determined by x-ray crystallography. In KM-12 cells harboring TPM3-NTRK1 fusion, merestinib exhibits potent p-NTRK1 inhibition in vitro by western blot and elicits an anti-proliferative response in two- and three-dimensional growth. Merestinib treatment demonstrated profound tumor growth inhibition in in vivo cancer models harboring either a TPM3-NTRK1 or an ETV6-NTRK3 gene fusion. To recapitulate resistance observed from type I NTRK kinase inhibitors entrectinib and larotrectinib, we generated NIH-3T3 cells exogenously expressing TPM3-NTRK1 wild-type, or acquired mutations G595R and G667C in vitro and in vivo. Merestinib blocks tumor growth of both wild-type and mutant G667C TPM3-NTRK1 expressing NIH-3T3 cell-derived tumors. These preclinical data support the clinical evaluation of merestinib, a type II NTRK kinase inhibitor (NCT02920996), both in treatment naïve patients and in patients progressed on type I NTRK kinase inhibitors with acquired secondary G667C mutation in NTRK fusion bearing tumors.

Variability in Platelet-Derived Growth Factor Receptor Alpha Antibody Specificity May Impact Clinical Utility of Immunohistochemistry Assays.

Aberrant regulation of the receptor tyrosine kinase platelet-derived growth factor alpha (PDGFRα) is implicated in several types of cancer. Inhibition of the PDGFRα pathway may be a beneficial therapy, and detection of PDGFRα in tumor biopsies may lead to insights about which patients respond to therapy. Exploratory or clinical biomarker use of PDGFRα IHC has been frequently reported, often with polyclonal antibody sc-338. An sc-338-based assay was systematically compared with anti-PDGFRα rabbit monoclonal antibody D13C6 using immunoblot profiling and IHC in formalin-fixed and paraffin-embedded human tumor cell lines. Application of sc-338 to blots of whole cell lysates showed multiple bands including some of unknown origin, whereas application of D13C6 resulted in a prominent band at the expected molecular mass of PDGFRα. The IHC assay using D13C6 showed appropriate staining in cell lines, whereas the assay using sc-338 suggested nonspecific detection of proteins. An optimized IHC assay using D13C6 showed a range of staining in the tumor stromal compartment in lung and ovarian carcinomas. These observations suggest that use of clone sc-338 produced unreliable results and should not be used for an IHC research grade assay. In addition, this precludes its use as a potential antibody for a clinical diagnostic tool.

A Novel Eg5 Inhibitor (LY2523355) Causes Mitotic Arrest and Apoptosis in Cancer Cells and Shows Potent Antitumor Activity in Xenograft Tumor Models.

Intervention of cancer cell mitosis by antitubulin drugs is among the most effective cancer chemotherapies. However, antitubulin drugs have dose-limiting side effects due to important functions of microtubules in resting normal cells and are often rendered ineffective by rapid emergence of resistance. Antimitotic agents with different mechanisms of action and improved safety profiles are needed as new treatment options. Mitosis-specific kinesin Eg5 represents an attractive anticancer target for discovering such new antimitotic agents, because Eg5 is essential only in mitotic progression and has no roles in resting, nondividing cells. Here, we show that a novel selective Eg5 inhibitor, LY2523355, has broad target-mediated anticancer activity in vitro and in vivo. LY2523355 arrests cancer cells at mitosis and causes rapid cell death that requires sustained spindle-assembly checkpoint (SAC) activation with a required threshold concentration. In vivo efficacy of LY2523355 is highly dose/schedule-dependent, achieving complete remission in a number of xenograft tumor models, including patient-derived xenograft (PDX) tumor models. We further establish that histone-H3 phosphorylation of tumor and proliferating skin cells is a promising pharmacodynamic biomarker for in vivo anticancer activity of LY2523355.

Myostatin Neutralization Results in Preservation of Muscle Mass and Strength in Preclinical Models of Tumor-Induced Muscle Wasting.

Skeletal muscle wasting occurs in a great majority of cancer patients with advanced disease and is associated with a poor prognosis and decreased survival. Myostatin functions as a negative regulator of skeletal muscle mass and has recently become a therapeutic target for reducing the loss of skeletal muscle and strength associated with clinical myopathies. We generated neutralizing antibodies to myostatin to test their potential use as therapeutic agents to attenuate the skeletal muscle wasting due to cancer. We show that our neutralizing antimyostatin antibodies significantly increase body weight, skeletal muscle mass, and strength in non-tumor-bearing mice with a concomitant increase in mean myofiber area. The administration of these neutralizing antibodies in two preclinical models of cancer-induced muscle wasting (C26 colon adenocarcinoma and PC3 prostate carcinoma) resulted in a significant attenuation of the loss of muscle mass and strength with no effect on tumor growth. We also show that the skeletal muscle mass- and strength-preserving effect of the antibodies is not affected by the coadministration of gemcitabine, a common chemotherapeutic agent, in both non-tumor-bearing mice and mice bearing C26 tumors. In addition, we show that myostatin neutralization with these antibodies results in the preservation of skeletal muscle mass following reduced caloric intake, a common comorbidity associated with advanced cancer. Our findings support the use of neutralizing antimyostatin antibodies as potential therapeutics for cancer-induced muscle wasting.

Identification of LY2510924, a novel cyclic peptide CXCR4 antagonist that exhibits antitumor activities in solid tumor and breast cancer metastatic models.

Emerging evidence demonstrates that stromal cell-derived factor 1 (SDF-1) and CXCR4, a chemokine and chemokine receptor pair, play important roles in tumorigenesis. In this report, we describe a small cyclic peptide, LY2510924, which is a potent and selective CXCR4 antagonist currently in phase II clinical studies for cancer. LY2510924 specifically blocked SDF-1 binding to CXCR4 with IC50 value of 0.079 nmol/L, and inhibited SDF-1-induced GTP binding with Kb value of 0.38 nmol/L. In human lymphoma U937 cells expressing endogenous CXCR4, LY2510924 inhibited SDF-1-induced cell migration with IC50 value of 0.26 nmol/L and inhibited SDF-1/CXCR4-mediated intracellular signaling. LY2510924 exhibited a concentration-dependent inhibition of SDF-1-stimulated phospho-ERK and phospho-Akt in tumor cells. Biochemical and cellular analyses revealed that LY2510924 had no apparent agonist activity. Pharmacokinetic analyses suggested that LY2510924 had acceptable in vivo stability and a pharmacokinetic profile similar to a typical small-molecular inhibitor in preclinical species. LY2510924 showed dose-dependent inhibition of tumor growth in human xenograft models developed with non-Hodgkin lymphoma, renal cell carcinoma, lung, and colon cancer cells that express functional CXCR4. In MDA-MB-231, a breast cancer metastatic model, LY2510924 inhibited tumor metastasis by blocking migration/homing process of tumor cells to the lung and by inhibiting cell proliferation after tumor cell homing. Collectively, the preclinical data support further investigation of LY2510924 in clinical studies for cancer.

LY2875358, a neutralizing and internalizing anti-MET bivalent antibody, inhibits HGF-dependent and HGF-independent MET activation and tumor growth.

PURPOSE: MET, the receptor for hepatocyte growth factor (HGF), has been implicated in driving tumor proliferation and metastasis. High MET expression is correlated with poor prognosis in multiple cancers. Activation of MET can be induced either by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation or by HGF-dependent autocrine or paracrine mechanisms. EXPERIMENTAL DESIGN/RESULTS: Here, we report on LY2875358, a novel humanized bivalent anti-MET antibody that has high neutralization and internalization activities, resulting in inhibition of both HGF-dependent and HGF-independent MET pathway activation and tumor growth. In contrast to other bivalent MET antibodies, LY2875358 exhibits no functional agonist activity and does not stimulate biologic activities such as cell proliferation, scattering, invasion, tubulogenesis, or apoptosis protection in various HGF-responsive cells and no evidence of inducing proliferation in vivo in a monkey toxicity study. LY2875358 blocks HGF binding to MET and HGF-induced MET phosphorylation and cell proliferation. In contrast to the humanized one-armed 5D5 anti-MET antibody, LY2875358 induces internalization and degradation of MET that inhibits cell proliferation and tumor growth in models where MET is constitutively activated. Moreover, LY2875358 has potent antitumor activity in both HGF-dependent and HGF-independent (MET-amplified) xenograft tumor models. Together, these findings indicate that the mechanism of action of LY2875358 is different from that of the one-armed MET antibody. CONCLUSIONS: LY2875358 may provide a promising therapeutic strategy for patients whose tumors are driven by both HGF-dependent and HGF-independent MET activation. LY2875358 is currently being investigated in multiple clinical studies.

Immunohistochemical application of a highly sensitive and specific murine monoclonal antibody recognising the extracellular domain of the human hepatocyte growth factor receptor (MET).

AIMS: Development of novel targeted therapies directed against hepatocyte growth factor (HGF) or its receptor (MET) necessitates the availability of quality diagnostics to facilitate their safe and effective use. Limitations of some commercially available anti-MET antibodies have prompted development of the highly sensitive and specific clone A2H2-3. Here we report its analytical properties when applied by an automated immunohistochemistry method. METHODS AND RESULTS: Excellent antibody specificity was demonstrated by immunoblot, ELISA, and IHC evaluation of characterised cell lines including NIH3T3 overexpressing the related kinase MST1R (RON). Sensitivity was confirmed by measurements of MET in cell lines or characterised tissues. IHC correlated well with FISH and quantitative RT-PCR assessments of MET (P < 0.001). Good total agreement (89%) was observed with the anti-MET antibody clone SP44 using whole-tissue sections, but poor positive agreement (21-47%) was seen in tissue microarray cores. Multiple lots displayed appropriate reproducibility (R(2)  > 0.9). Prevalence of MET positivity by IHC was higher in non-squamous cell NSCLC, MET or EGFR amplified cases, and in tumours harbouring abnormalities in EGFR exon 19 or 21. CONCLUSIONS: The anti-MET antibody clone A2H2-3 displays excellent specificity and sensitivity. These properties make it suitable for clinical trial investigations and development as a potential companion diagnostic.

A human monoclonal antibody targeting the stem cell factor receptor (c-Kit) blocks tumor cell signaling and inhibits tumor growth.

Stem cell factor receptor (c-Kit) exerts multiple biological effects on target cells upon binding its ligand stem cell factor (SCF). Aberrant activation of c-Kit results in dysregulated signaling and is implicated in the pathogenesis of numerous cancers. The development of more specific and effective c-Kit therapies is warranted given its essential role in tumorigenesis. In this study, we describe the biological properties of CK6, a fully human IgG1 monoclonal antibody against the extracellular region of human c-Kit. CK6 specifically binds c-Kit receptor with high affinity (EC 50 = 0.06 nM) and strongly blocks its interaction with SCF (IC 50 = 0.41 nM) in solid phase assays. Flow cytometry shows CK6 binding to c-Kit on the cell surface of human small cell lung carcinoma (SCLC), melanoma, and leukemia tumor cell lines. Furthermore, exposure to CK6 inhibits SCF stimulation of c-Kit tyrosine kinase activity and downstream signaling pathways such as mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), in addition to reducing tumor cell line growth in vitro. CK6 treatment significantly decreases human xenograft tumor growth in NCI-H526 SCLC (T/C% = 57) and Malme-3M melanoma (T/C% = 58) models in vivo. The combination of CK6 with standard of care chemotherapy agents, cisplatin and etoposide for SCLC or dacarbazine for melanoma, more potently reduces tumor growth (SCLC T/C% = 24, melanoma T/C% = 38) compared with CK6 or chemotherapy alone. In summary, our results demonstrate that CK6 is a c-Kit antagonist antibody with tumor growth neutralizing properties and are highly suggestive of potential therapeutic application in treating human malignancies harboring c-Kit receptor.

Evaluation of hair loss in cats occurring after treatment with a topical flea control product.

BACKGROUND: A formulation containing 39.6% spinetoram resulted in a higher than anticipated number of reports of alopecia at the site of application in the first months following commercial product launch. HYPOTHESIS/OBJECTIVES: To determine the cause of the alopecia using histopathology, including assessment for inflammation, follicular findings of physical trauma (plucking/pulling behaviour) and changes in follicular cycling. ANIMALS: Twenty-four flea-free, male and female adult domestic short hair cats within a private research colony. METHODS: Cats were treated with a single application of 39.6% spinetoram on day 0; personnel were not blinded. Observations of the skin and hair coat began immediately and were repeated at 30 min and 1, 2, 3, 4, 6, 8 and 12 h post-application and then on subsequent days at the same time as initial dosing and at 2, 4, 6, 8 and 12 h after that time, until day 5. If hair thinning or loss was observed, a skin biopsy sample was collected. Two cats not exhibiting abnormalities were biopsied on day 6. RESULTS: Thirty-eight per cent of cats (nine of 24) developed hair thinning and alopecia of sufficient severity within 78 h post-application of the product to warrant skin biopsy. Abnormalities in the skin were limited to the application site and were consistent with physical trauma (pulling or plucking) to the hair. CONCLUSIONS AND CLINICAL IMPORTANCE: Microscopic changes in the hair follicles of affected cats were consistent with self-induced trauma or barbering behaviour. All changes were reversible and paralleled findings associated with well-established, topical flea control products.

Inhibition of tumor growth and metastasis in non-small cell lung cancer by LY2801653, an inhibitor of several oncokinases, including MET.

PURPOSE: Lung cancer is the leading cause of cancer-related death worldwide. Sustained activation, overexpression, or mutation of the MET pathway is associated with a poor prognosis in a variety of tumors, including non-small cell lung cancer (NSCLC), implicating the MET pathway as a potential therapeutic target for lung cancer. Previously, we reported on the development of LY2801653: a novel, orally bioavailable oncokinase inhibitor with MET as one of its targets. Here, we discuss the evaluation of LY2801653 in both preclinical in vitro and in vivo NSCLC models. Experimental Design/ RESULTS: Treatment with LY2801653 showed tumor growth inhibition in tumor cell lines and patient-derived tumor xenograft models as a single agent (37.4%-90.0% inhibition) or when used in combination with cisplatin, gemcitabine, or erlotinib (66.5%-86.3% inhibition). Mechanistic studies showed that treatment with LY2801653 inhibited the constitutive activation of MET pathway signaling and resulted in inhibition of NCI-H441 cell proliferation, anchorage-independent growth, migration, and invasion. These in vitro findings were confirmed in the H441 orthotopic model where LY2801653 treatment significantly inhibited both primary tumor growth (87.9% inhibition) and metastasis (64.5% inhibition of lymph node and 67.7% inhibition of chest wall). Tumor-bearing animals treated with LY2801653 had a significantly greater survival time (87% increase compared with the vehicle-treated mice). In the MET-independent NCI-H1299 orthotopic model, treatment with LY2801653 showed a significant inhibition of primary tumor growth but not metastasis. CONCLUSIONS: Collectively, these results support clinical evaluation of LY2801653 in NSCLCs and suggest that differences in the MET activation of tumors may be predictive of response.

Hematopoietic proliferative lesions in the spleen of rasH2 transgenic mice treated with MNU.

In this study, rasH2-Tg mice treated with N-methyl-N-nitrosurea (MNU) developed exuberant hematoproliferative changes in the spleen that included dysplasia and features of neoplasia. Hematoproliferative change was characterized as exuberant proliferation of hematopoietic cells within the spleen that distorted but did not displace normal splenic morphologic features. The hematopoietic cells were of mixed lineage, but one type, often erythroid, predominated. Cellular atypia was present in all mice with hematoproliferative change, and dysplasia was present in five of eight examined. Hematoproliferative neoplasia was characterized by similar cytologic features but also resulted in displacement/disruption of normal splenic architecture and increased numbers of unidentified blast cells. One case was differentiated toward myeloid proliferation, suggesting granulocytic leukemia. Affected mice had other neoplasms, such as lymphoma and anemia. These proliferative and dysplastic lesions of the spleen in rasH2-Tg mice treated with MNU require additional characterization to definitively differentiate them from the reactive hematopoiesis that can occur in response to inflammatory, neoplastic, or hematopoietic insults in mice.

Detection of left ventricular hypertrophy in rats administered a peroxisome proliferator-activated receptor alpha/gamma dual agonist using natriuretic peptides and imaging.

Chronic treatment with suprapharmacologic doses of peroxisome proliferator-activated receptor (PPAR) agonists has a known potential for causing left ventricular hypertrophy (LVH). The mechanism by which LVH develops is not well understood nor are biomarkers of it well characterized. Natriuretic peptides are important regulators of cardiac growth, blood volume, and arterial pressure and may be useful biomarkers of LVH and hemodynamic changes that precede it. We measured amino-terminal pro-atrial natriuretic peptide (NTproANP), amino-terminal pro-brain natriuretic peptide (NTproBNP), and cardiac troponin I (cTnI) concentrations in serum and plasma, as well as transcripts in left ventricular heart tissue for atrial natriuretic peptide precursor (Nppa), brain natriuretic peptide precursor (Nppb), and myosin heavy chain-beta (Myh7) as potential biomarkers of LVH induced by a PPARalpha/gamma dual agonist in Sprague-Dawley rats. We used magnetic resonance imaging, echocardiography, and hemodynamics to identify structural and functional cardiovascular changes related to the biomarkers. Heart-to-brain weight ratios (HW:BrW) were correlated with NTproANP, NTproBNP, and cTnI concentrations in serum as well as fold change in expression of Nppa and Nppb. LVH was characterized by increased left ventricular wall thickness and inner diameter, increased cardiac output, decreased arterial blood pressure, and increased heart rate. In these studies, each end point contributed to the early detection of LVH, the ability to monitor its progression, and demonstrated the ability of NTproANP concentration in serum to predict LVH and hemodynamic changes.

Ultrasensitive cross-species measurement of cardiac troponin-I using the Erenna immunoassay system.

Serum cardiac troponin-I (cTnI) has been validated as a biomarker for cardiotoxicity in numerous animal models; however, owing to sensitivity issues cTnI concentrations in healthy, resting animals used in toxicology studies have not been established. Serum from healthy and isoproterenol hydrochloride (iso)-treated rats, dogs, and monkeys were assayed using the Erenna system. The Erenna cTnI assay provided sensitivity < 1 ng/L across human, rat, dog, and monkey cTnI. Linear responses (R(2)= 0.99) were observed for all species. Precision studies yielded interassay CVs of curve fit quantification from 2% to 4% between 1.6 and 5000 ng/L, and 23% at 0.78 ng/L. Strong correlation (R(2)= 0.99) was obtained between Erenna and Beckman Access cTnI. Concentrations of cTnI in healthy animals ranged from 1 to 9 ng/L. In longitudinal studies of iso-treated animals, the concentrations of cTnI in the control vehicle-treated groups were 10-20 ng/L for rats (N = 10) and predose values of 2-3 ng/L for dogs (N = 3). Measured with the Erenna assay system, cTnI was quantifiable at all time intervals tested in all animals treated with iso. The Erenna system provides sensitive measurement of cTnI in rats, dogs, and monkeys, makes it possible to determine small changes from normal concentrations, and provides cTnI values from small volumes of serum.

Fabp3 as a biomarker of skeletal muscle toxicity in the rat: comparison with conventional biomarkers.

Fatty acid binding protein 3 (Fabp3) has been used as a serological biomarker of cardiac injury, but its utility as a preclinical biomarker of injury to skeletal muscle is not well described. Fabp3 concentrations were determined for tissues from Sprague-Dawley rats and found to occur at highest concentrations in cardiac muscle and in skeletal muscles containing an abundance of type I fibers, such as the soleus muscle. Soleus is also a primary site of skeletal muscle (SKM) injury caused by lipid-lowering peroxisome proliferator-activated receptor alpha (PPAR-alpha) agonists. In rats administered repeat doses of a PPAR-alpha agonist, the kinetics and amplitude of plasma concentrations of Fabp3 were consistent with plasma compound concentrations and histopathology findings of swollen, hyalinized, and fragmented muscle fibers with macrophage infiltration. Immunohistochemical detection of Fabp3 revealed focal depletion of Fabp3 protein from injured SKM fibers which is consistent with increased serum Fabp3 concentrations in treated rats. We then assessed the predictivity of serological Fabp3 for SKM necrosis in short duration toxicology studies. Rats were treated with various doses of 27 different compounds, and the predictivity of serological biomarkers was assessed relative to histology in individual rats and in treatment groups. Under these study conditions, Fabp3 was the most useful individual biomarker based on concordance, sensitivity, positive and negative predictive values, and false negative rate. In addition, the combination of Fabp3 and aspartate aminotransferase (AST) had greater diagnostic value than the conventional combination of creatine kinase-MM isoenzyme (CK) and AST.

The rat mammary gland: morphologic changes as an indicator of systemic hormonal perturbations induced by xenobiotics.

The development and morphology of the rat mammary gland are dependent upon several hormones including estrogens, androgens, progesterone, growth hormone and prolactin. In toxicology studies, treatment with xenobiotics may alter these hormones resulting in changes in the morphology of reproductive tissues such as the mammary gland. In the rat, male and female mammary glands exhibit striking morphologic differences that can be altered secondary to hormonal perturbations. Recognizing these morphologic changes can help the pathologist predict potential xenobiotic-induced perturbations in the systemic hormonal milieu. This review examines the development of the rat mammary gland and the influence of sex hormones on the morphology of the adult male and female rat mammary gland. Specific case examples from the literature and data from our laboratory highlight the dynamic nature of the rat mammary gland in response to hormonal changes.