Association of equine gammaherpesvirus-5 with facial lymphohistiocytic interface dermatitis in seven adult horses from the United States.

Equine herpesvirus-5 (EHV-5) is commonly found in healthy asymptomatic horses worldwide. Although a cause-and-effect relationship has not been thoroughly determined, this virus has been associated with several disease conditions including equine multinodular pulmonary fibrosis (EMPF) and 1 case of interface dermatitis. The authors searched the New York State Animal Health Diagnostic Center database for cases of equine interface dermatitis between 2007 and 2022. Ten cases were identified and scrutinized for viral inclusion bodies which were present in 5 of 10 cases. Two similar cases with interface dermatitis and viral inclusion bodies, which were not part of a retrospective search, were from the Oregon Veterinary Diagnostic Laboratory. The authors describe a total of 7 horses with dermatitis characterized by crusted, alopecic, non-pruritic, non-painful, irregular to annular areas over the face, most commonly the muzzle, for up to several years duration. Histologically, there was a CD3+ T lymphocyte-dominated lymphohistiocytic interface dermatitis with hydropic degeneration, apoptotic keratinocytes, and pigmentary incontinence. Keratinocytes within the upper stratum spinosum and stratum granulosum had glassy pale basophilic intranuclear inclusion bodies consistent with herpesvirus. The presence of EHV-5 was confirmed by quantitative polymerase chain reaction (qPCR) and in situ hybridization in 7 horses and by electron microscopy in 1 horse. One horse later developed EMPF and was euthanized. EHV-5 was not detected with qPCR from 5 control horses and 5 horses with interface dermatitis without histologic evidence of viral inclusion bodies. These are the first cases of facial interface dermatitis associated with EHV-5 reported in the United States.

CT features of malignant tubular genital tract tumors in seven goats.

Neoplasia of the tubular genital tract in goats, while rarely described, is most commonly reported as uterine adenocarcinoma, leiomyoma, or leiomyosarcoma. In this retrospective, single-center, case series, medical records were searched for goats with a computed tomography (CT) diagnosis of tubular genital mass and a definitive histologic (surgical biopsy or necropsy) diagnosis of malignant neoplasia. Data recorded from CT images were presence of peritoneal/retroperitoneal fluid, urinary tract obstruction, abdominal lymphadenomegaly, additional abdominal nodules/masses, and pulmonary nodules. For masses, maximum cross-sectional area, contrast enhancement, and uterine luminal fluid accumulation were also recorded. Seven goats met the inclusion criteria (leiomyosarcoma n = 5, adenocarcinoma n = 2). Both goats with adenocarcinoma had upper urinary tract obstruction, moderate to severe regional lymphadenopathy, peritoneal fluid, and peritoneal or hepatic nodules/masses; one goat with adenocarcinoma was discharged and subsequently euthanized, and the other had palliative mass debulking and was lost to follow up. Goats with leiomyosarcoma had infrequent, mild peritoneal fluid and mild sublumbar lymphadenopathy. Of the goats with leiomyosarcoma, two were euthanized at or near the time of CT imaging, two were euthanized at the time of surgery due to perceived mass non-resectability, and one had mass regression approximately four months post ovariohysterectomy but was subsequently lost to follow up. Five goats had pulmonary nodules, three of which had pathologic confirmation (pulmonary metastasis in a single patient with adenocarcinoma, and lungworm granulomas in two goats with leiomyosarcoma). Severe sublumbar lymphadenopathy and obstructive uropathy were sequelae in the two caprine patients with genital adenocarcinoma, and in none with leiomyosarcoma.

Loss of the aryl hydrocarbon receptor increases tumorigenesis in p53-deficient mice.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates cell fate via activation of a diverse set of genes. There are conflicting reports describing the role of AhR in cancer. AhR-knockout mice do not develop tumors spontaneously, yet the AhR can act as a tumor suppressor in certain contexts. Loss of tumor suppression by p53 is common in human cancer. To investigate AhR function in the absence of p53, we generated mice lacking both AhR and p53. Mice deficient for AhR and p53 had shortened lifespan, increased tumorigenesis, and an altered tumor spectrum relative to control mice lacking only p53. In addition, knockout of both AhR and p53 resulted in reduced embryonic survival and neonatal fitness. We also examined the consequences of loss of AhR in p53-heterozygous mice and observed a significantly reduced lifespan and enhanced tumor burden. These findings reveal an important role for the AhR as a tumor suppressor in the absence of p53 signaling and support the development of anti-cancer therapeutics that would promote the tumor suppressive actions of the AhR.

Luteinizing Hormone Receptor Expression in Neoplastic Mast Cells Is Increased in Spayed and Neutered Dogs.

Luteinizing hormone receptors (LHRs) are expressed in canine lymphoma and hemangiosarcoma. We hypothesized that LHR would be expressed in canine mast cell tumors (MCTs) and that more neoplastic mast cells would express LHR in gonadectomized dogs compared with intact dogs. Eleven archived formalin-fixed paraffin-embedded cutaneous MCT tissue sections were processed using routine immunohistochemistry. For both the KIT protein and LHR, the percentage of positive cells for each staining pattern (I-III) was calculated. A Student's t test was used to compare the total percentage of positive cells expressing LHR and KIT in intact and gonadectomized dogs. A one-way analysis of variance was used to compare the percentage of cells within each staining pattern for LHR and KIT in intact and gonadectomized dogs. All MCT expressed LHR. MCT from gonadectomized dogs had a significantly higher percentage of LHR-positive mast cells (84.2 ± 8.7%) compared with MCTs from intact dogs (64.3 ± 4.2%). This is the first study to demonstrate the expression of LHR in canine MCTs and to report that LHR expression is increased in neoplastic mast cells from gonadectomized dogs compared with intact dogs. Future studies are planned to evaluate the functionality of the LHR in canine neoplastic mast cells.

Mast Cell Tumors and Histiocytomas in Domestic Goats and Diagnostic Utility of CD117/c-Kit and Iba1 Immunohistochemistry.

Cutaneous round cell tumors in goats present a diagnostic challenge. In this article, we provide a description of caprine cutaneous mast cell tumors (MCT) and histiocytomas, and report on the validation of anti-human antibodies to CD117/KIT and Iba1 by immunohistochemistry on a range of caprine tissues. Cells immunolabeled for CD117/KIT included resident mast cells in normal lung and skin, interstitial cells of Cajal (intestine), and neuronal cell bodies (brain). Cells immunolabeled for Iba1 included resident macrophages in many tissues including normal lung, dendritic cells (hemolymphatic tissues), Kupffer cells, and microglia. Of 5 cutaneous MCT, only one had metachromasia of cytoplasmic granules; however, neoplastic cells of all 5 MCT had positive immunolabeling for CD117/KIT. The CD117/KIT immunolabeling pattern was predominately focal paranuclear in 3 cases, and cytoplasmic or membranous in 1 case each. Two histiocytomas were identified and had strong positive immunolabeling for Iba1 but not CD117/KIT. All 7 cutaneous round cell tumors described herein occurred in goats less than 4 years of age; the 2 cutaneous histiocytomas were in goats less than 14 months of age. Neither of the cutaneous histiocytomas recurred within 24 months of surgical removal.

Targeting Peritumoral Lesions Identified by Computed Tomography and Magnetic Resonance Imaging in Feline Injection-Site Sarcomas for Microscopic Examination.

Peritumoral lesions identified during in vivo imaging of feline injection-site sarcoma (FISS) are frequently interpreted as neoplastic. We recently showed that most peritumoral imaging-identified lesions (PTIILs) in FISS are non-neoplastic. In this article, we describe a protocol to target PTIIL for microscopic examination and report on the protocol's performance. Ten client-owned cats with FISS were prospectively enrolled. A fiducial marker sutured onto the skin, centered on the palpable mass, served as reference point throughout the study. Each FISS and surrounding tissue was imaged in vivo by dual phase computed tomography angiography and multiple magnetic resonance imaging pulse sequences and each PTIIL documented. Subgross measurements obtained during trimming aided localization and identification of PTIIL during microscopy. Histologic findings were categorized by descending clinical relevance: neoplastic, equivocal, non-neoplastic, within normal limits (WNL). Based on in vivo imaging resolution limits, histologic findings were ≥3 mm in at least one dimension and ≥3 mm apart. Surgical margins served as control tissue for PTIILs. Eighty-one of 87 PTIIL were examined histologically; 13 were neoplastic, 16 equivocal, and 28 non-neoplastic; 24 had no identified histologic correlate. Two neoplastic and 10 equivocal findings were located outside of PTIILs but none of them were located in sections of surgical margins. Computation of a simple confusion matrix yielded fair sensitivity (70.4%) and low specificity (59.7%) for prediction of PTIIL by histologic findings. After combining instances of normal microanatomy with non-neoplastic histologic findings, specificity increased (85.1%) and sensitivity decreased (35.8%). The protocol is a blueprint for targeting PTIIL for microscopic examination but may benefit from further refinement.

Pan-tissue transcriptome analysis of long noncoding RNAs in the American beaver Castor canadensis.

BACKGROUND: Long noncoding RNAs (lncRNAs) have roles in gene regulation, epigenetics, and molecular scaffolding and it is hypothesized that they underlie some mammalian evolutionary adaptations. However, for many mammalian species, the absence of a genome assembly precludes the comprehensive identification of lncRNAs. The genome of the American beaver (Castor canadensis) has recently been sequenced, setting the stage for the systematic identification of beaver lncRNAs and the characterization of their expression in various tissues. The objective of this study was to discover and profile polyadenylated lncRNAs in the beaver using high-throughput short-read sequencing of RNA from sixteen beaver tissues and to annotate the resulting lncRNAs based on their potential for orthology with known lncRNAs in other species. RESULTS: Using de novo transcriptome assembly, we found 9528 potential lncRNA contigs and 187 high-confidence lncRNA contigs. Of the high-confidence lncRNA contigs, 147 have no known orthologs (and thus are putative novel lncRNAs) and 40 have mammalian orthologs. The novel lncRNAs mapped to the Oregon State University (OSU) reference beaver genome with greater than 90% sequence identity. While the novel lncRNAs were on average shorter than their annotated counterparts, they were similar to the annotated lncRNAs in terms of the relationships between contig length and minimum free energy (MFE) and between coverage and contig length. We identified beaver orthologs of known lncRNAs such as XIST, MEG3, TINCR, and NIPBL-DT. We profiled the expression of the 187 high-confidence lncRNAs across 16 beaver tissues (whole blood, brain, lung, liver, heart, stomach, intestine, skeletal muscle, kidney, spleen, ovary, placenta, castor gland, tail, toe-webbing, and tongue) and identified both tissue-specific and ubiquitous lncRNAs. CONCLUSIONS: To our knowledge this is the first report of systematic identification of lncRNAs and their expression atlas in beaver. LncRNAs-both novel and those with known orthologs-are expressed in each of the beaver tissues that we analyzed. For some beaver lncRNAs with known orthologs, the tissue-specific expression patterns were phylogenetically conserved. The lncRNA sequence data files and raw sequence files are available via the web supplement and the NCBI Sequence Read Archive, respectively.

miRWoods: Enhanced precursor detection and stacked random forests for the sensitive detection of microRNAs.

MicroRNAs are conserved, endogenous small RNAs with critical post-transcriptional regulatory functions throughout eukaryota, including prominent roles in development and disease. Despite much effort, microRNA annotations still contain errors and are incomplete due especially to challenges related to identifying valid miRs that have small numbers of reads, to properly locating hairpin precursors and to balancing precision and recall. Here, we present miRWoods, which solves these challenges using a duplex-focused precursor detection method and stacked random forests with specialized layers to detect mature and precursor microRNAs, and has been tuned to optimize the harmonic mean of precision and recall. We trained and tuned our discovery pipeline on data sets from the well-annotated human genome, and evaluated its performance on data from mouse. Compared to existing approaches, miRWoods better identifies precursor spans, and can balance sensitivity and specificity for an overall greater prediction accuracy, recalling an average of 10% more annotated microRNAs, and correctly predicts substantially more microRNAs with only one read. We apply this method to the under-annotated genomes of Felis catus (domestic cat) and Bos taurus (cow). We identified hundreds of novel microRNAs in small RNA sequencing data sets from muscle and skin from cat, from 10 tissues from cow and also from human and mouse cells. Our novel predictions include a microRNA in an intron of tyrosine kinase 2 (TYK2) that is present in both cat and cow, as well as a family of mirtrons with two instances in the human genome. Our predictions support a more expanded miR-2284 family in the bovine genome, a larger mir-548 family in the human genome, and a larger let-7 family in the feline genome.

Inter-Institutional Collaboration for the Development of a Local Peer Observation Process to Enhance Teaching.

Local peer observation of teaching is considered an important mechanism for instructors to improve the quality and effectiveness of their teaching, but there is an absence of uniformity to establish a best practice for this process in veterinary curricula. The Regional Teaching Academy (RTA) of the Consortium of Western Colleges of Veterinary Medicine is comprised of educational advocates from five western veterinary colleges with a common goal of enhancing the quality and effectiveness of education in veterinary medical curricula. Members of the RTA recognized this deficit in best practices for local peer observation (LPO) and formed a working group called "Local Peer Observation of Teaching." The goal was to meet a critical need for the enhancement of individual teaching skills by using a scholarly approach to develop robust methods for peer observation of teaching. Two rubric-based instruments were developed: one for large-group/didactic settings, and the second for small-group/clinical settings. Each is accompanied by pre- and post-observation worksheets which are considered instrumental to success. Results of a qualitative survey of instrument users' experiences are shared. Both observers and observees view the experiential learning from faculty peer colleagues very positively and the meaningful feedback is appreciated and incorporated by observees. Suggestions for implementation of the peer observation process are discussed, considering strengths and challenges. The purpose of this article is to describe in depth, the development process and output of the efforts of the Local Peer Observation of Teaching working group as a potential best practice guideline for peer observation.

Comparative mechanisms of PAH toxicity by benzo[a]pyrene and dibenzo[def,p]chrysene in primary human bronchial epithelial cells cultured at air-liquid interface.

Current assumption for assessing carcinogenic risk of polycyclic aromatic hydrocarbons (PAHs) is that they function through a common mechanism of action; however, recent studies demonstrate that PAHs can act through unique mechanisms potentially contributing to cancer outcomes in a non-additive manner. Using a primary human 3D bronchial epithelial culture (HBEC) model, we assessed potential differences in mechanism of toxicity for two PAHs, benzo[a]pyrene (BAP) and dibenzo[def,p]chrysene (DBC), compared to a complex PAH mixture based on short-term biosignatures identified from transcriptional profiling. Differentiated bronchial epithelial cells were treated with BAP (100-500 µg/ml), DBC (10 µg/ml), and coal tar extract (CTE 500-1500 µg/ml, SRM1597a) for 48 h and gene expression was measured by RNA sequencing or quantitative PCR. Comparison of BAP and DBC gene signatures showed that the majority of genes (~60%) were uniquely regulated by treatment, including signaling pathways for inflammation and DNA damage by DBC and processes for cell cycle, hypoxia and oxidative stress by BAP. Specifically, BAP upregulated targets of AhR, NRF2, and KLF4, while DBC downregulated these same targets, suggesting a chemical-specific pattern in transcriptional regulation involved in antioxidant response, potentially contributing to differences in PAH potency. Other processes were regulated in common by all PAH treatments, BAP, DBC and CTE, including downregulation of genes involved in cell adhesion and reduced functional measurements of barrier integrity. This work supports prior in vivo studies and demonstrates the utility of profiling short-term biosignatures in an organotypic 3D model to identify mechanisms linked to carcinogenic risk of PAHs in humans.

Elucidating the transcriptional program of feline injection-site sarcoma using a cross-species mRNA-sequencing approach.

BACKGROUND: Feline injection-site sarcoma (FISS), an aggressive iatrogenic subcutaneous malignancy, is challenging to manage clinically and little is known about the molecular basis of its pathogenesis. Tumor transcriptome profiling has proved valuable for gaining insights into the molecular basis of cancers and for identifying new therapeutic targets. Here, we report the first study of the FISS transcriptome and the first cross-species comparison of the FISS transcriptome with those of anatomically similar soft-tissue sarcomas in dogs and humans. METHODS: Using high-throughput short-read paired-end sequencing, we comparatively profiled FISS tumors vs. normal tissue samples as well as cultured FISS-derived cell lines vs. skin-derived fibroblasts. We analyzed the mRNA-seq data to compare cancer/normal gene expression level, identify biological processes and molecular pathways that are associated with the pathogenesis of FISS, and identify multimegabase genomic regions of potential somatic copy number alteration (SCNA) in FISS. We additionally conducted cross-species analyses to compare the transcriptome of FISS to those of soft-tissue sarcomas in dogs and humans, at the level of cancer/normal gene expression ratios. RESULTS: We found: (1) substantial differential expression biases in feline orthologs of human oncogenes and tumor suppressor genes suggesting conserved functions in FISS; (2) a genomic region with recurrent SCNA in human sarcomas that is syntenic to a feline genomic region of probable SCNA in FISS; and (3) significant overlap of the pattern of transcriptional alterations in FISS with the patterns of transcriptional alterations in soft-tissue sarcomas in humans and in dogs. We demonstrated that a protein, BarH-like homeobox 1 (BARX1), has increased expression in FISS cells at the protein level. We identified 11 drugs and four target proteins as potential new therapies for FISS, and validated that one of them (GSK-1059615) inhibits growth of FISS-derived cells in vitro. CONCLUSIONS: (1) Window-based analysis of mRNA-seq data can uncover SCNAs. (2) The transcriptome of FISS-derived cells is highly consistent with that of FISS tumors. (3) FISS is highly similar to soft-tissue sarcomas in dogs and humans, at the level of gene expression. This work underscores the potential utility of comparative oncology in improving understanding and treatment of FISS.

CT angiography and MRI imaging features do not predict the tumor type and grade of feline injection site sarcoma.

Computed tomographic angiography (CTA) and magnetic resonance imaging (MRI) have been described as methods for preoperative surgical planning in cats with feline injection site sarcomas (FISS), however, few published studies have compared these modalities. The objective of this retrospective, secondary analysis study was to determine if imaging features of FISS on CTA and MRI are predictive of neoplastic peritumoral projections. Archived data from a previous prospective study were retrieved for 10 cats with FISS. All cats had been evaluated in a single anesthetic episode with MRI and dual phase CT (CTA) imaging followed by surgical removal. Histopathological grading and targeted histopathology of imaging-identified peritumoral projections were performed. Two observers evaluated the CTA and MRI studies for FISS shape, margination, size, enhancement pattern, postcontrast uniformity, pre- and postcontrast margination, the number of muscles involved, mass mineralization, and bone lysis. Metal was present in the imaging field of three of 10 cats, resulting in one nondiagnostic MRI. Peritumoral projections were detected in all cats with both imaging modalities, and most were benign. At least one neoplastic peritumoral projection was detected in six cats using MRI, five cats using CTA, and three cats with both modalities. Higher grade FISS were larger than low grade using MRI, and FISS were larger using MRI. Other FISS imaging features using MRI and CTA were similar. Findings supported use of either MRI or CTA for detecting neoplastic peritumoral projections in cats with FISS. Authors recommend CTA for cats with known metallic objects in the scan field.

Activation of the Aryl Hydrocarbon Receptor by 10-Cl-BBQ Prevents Insulitis and Effector T Cell Development Independently of Foxp3+ Regulatory T Cells in Nonobese Diabetic Mice.

Aryl hydrocarbon receptor (AhR) activation by high-affinity ligands mediates immunosuppression in association with increased regulatory T cells (Tregs), making this transcription factor an attractive therapeutic target for autoimmune diseases. We recently discovered 10-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (10-Cl-BBQ), a nanomolar affinity AhR ligand with immunosuppressive activity and favorable pharmacologic properties. In this study, we tested the consequences of AhR activation in the NOD model. Oral 10-Cl-BBQ treatment prevented islet infiltration without clinical toxicity, whereas AhR-deficient NOD mice were not protected. Suppression of insulitis was associated with an increased frequency, but not total number, of Foxp3(+) Tregs in the pancreas and pancreatic lymph nodes. The requirement for Foxp3(+) cells in AhR-induced suppression of insulitis was tested using NOD.Foxp3(DTR) mice, which show extensive islet infiltration upon treatment with diphtheria toxin. AhR activation prevented the development of insulitis caused by the depletion of Foxp3(+) cells, demonstrating that Foxp3(+) cells are not required for AhR-mediated suppression and furthermore that the AhR pathway is able to compensate for the absence of Foxp3(+) Tregs, countering current dogma. Concurrently, the development of disease-associated CD4(+)Nrp1(+)Foxp3(-)RORγt(+) cells was inhibited by AhR activation. Taken together, 10-Cl-BBQ is an effective, nontoxic AhR ligand for the intervention of immune-mediated diseases that functions independently of Foxp3(+) Tregs to suppress pathogenic T cell development.

Divergent roles of p120-catenin isoforms linked to altered cell viability, proliferation, and invasiveness in carcinogen-induced rat skin tumors.

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) targets multiple organs for tumorigenesis in the rat, including the colon and the skin. PhIP-induced skin tumors were subjected to mutation screening, which identified genetic changes in Hras (7/40, 17.5%) and Tp53 (2/40, 5%), but not in Ctnnb1, a commonly mutated gene in PhIP-induced colon tumors. Despite the absence of Ctnnb1 mutations, ß-catenin was overexpressed in nuclear and plasma membrane fractions from PhIP-induced skin tumors, coinciding with loss of p120-catenin from the plasma membrane, and the appearance of multiple p120-catenin-associated bands in the nuclear extracts. Real-time RT-PCR revealed that p120-catenin isoforms 1 and 4 were upregulated in PhIP-induced skin tumors, whereas p120-catenin isoform 3 was expressed uniformly, compared with adjacent normal-looking tissue. In human epidermoid carcinoma and colon cancer cells, transient transfection of p120-catenin isoform 1A enhanced the viability and cell invasion index, whereas transient transfection of p120-catenin isoform 4A increased cell viability and cell proliferation. Knockdown of p120-catenin revealed a corresponding reduction in the expression of ß-catenin and a transcriptionally regulated target, Ccnd1/Cyclin D1. Co-immunoprecipitation experiments identified associations of ß-catenin with p120-catenin isoforms in PhIP-induced skin tumors and human cancer cell lines. The results are discussed in the context of therapeutic strategies that might target different p120-catenin isoforms, providing an avenue to circumvent constitutively active ß-catenin arising via distinct mechanisms in skin and colon cancer.

Dibenzo[def,p]chrysene transplacental carcinogenesis in wild-type, Cyp1b1 knockout, and CYP1B1 humanized mice.

The cytochrome P450 (CYP) 1 family is active toward numerous environmental pollutants, including polycyclic aromatic hydrocarbons (PAHs). Utilizing a mouse model, null for Cyp1b1 and expressing human CYP1B1, we tested the hypothesis that hCYP1B1 is important for dibenzo[def,p]chrysene (DBC) transplacental carcinogenesis. Wild-type mCyp1b1, transgenic hCYP1B1 (mCyp1b1 null background), and mCyp1b1 null mice were assessed. Each litter had an equal number of siblings with Ahrb-1/d and Ahrd/d alleles. Pregnant mice were dosed (gavage) on gestation day 17 with 6.5 or 12 mg/kg of DBC or corn oil. At 10 months of age, mortality, general health, lymphoid disease and lung tumor incidence, and multiplicity were assessed. hCYP1B1 genotype did not impact lung tumor multiplicity, but tended to enhance incidence compared to Cyp1b1 wild-type mice (P = 0.07). As with Cyp1b1 in wild-type mice, constitutive hCYP1B1 protein is non-detectable in liver but was induced with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Wild-type mice were 59% more likely to succumb to T-cell Acute Lymphoblastic Leukemia (T-ALL). Unlike an earlier examination of the Ahr genotype in this model (Yu et al., Cancer Res, 2006;66:755-762), but in agreement with a more recent study (Shorey et al., Toxicol Appl Pharmacol, 2013;270:60-69), this genotype was not associated with lung tumor incidence, multiplicity, or mortality. Sex was not significant with respect to lung tumor incidence or mortality but males exhibited significantly greater multiplicity. Lung tumor incidence was greater in mCyp1b1 nulls compared to wild-type mice. To our knowledge, this is the first application of a humanized mouse model in transplacental carcinogenesis. © 2016 Wiley Periodicals, Inc.

Ablation of epidermal RXRα in cooperation with activated CDK4 and oncogenic NRAS generates spontaneous and acute neonatal UVB induced malignant metastatic melanomas.

BACKGROUND: Understanding the underlying molecular mechanisms involved in the formation of cutaneous malignant melanoma is critical for improved diagnosis and treatment. Keratinocytic nuclear receptor Retinoid X Receptor α (RXRα) has a protective role against melanomagenesis and is involved in the regulation of keratinocyte and melanocyte homeostasis subsequent acute ultraviolet (UV) irradiation. METHODS: We generated a trigenic mouse model system (RXRα ep-/- | Tyr-NRAS Q61K | CDK4 R24C/R24C ) harboring an epidermal knockout of Retinoid X Receptor α (RXRα ep-/- ), combined with oncogenic NRAS Q61K (constitutively active RAS) and activated CDK4 R24C/R24C (constitutively active CDK4). Those mice were subjected to a single neonatal dose of UVB treatment and the role of RXR α was evaluated by characterizing the molecular and cellular changes that took place in the untreated and UVB treated trigenic RXRα ep-/- mice compared to the control mice with functional RXRα. RESULTS: Here we report that the trigenic mice develops spontaneous melanoma and exposure to a single neonatal UVB treatment reduces the tumor latency in those mice compared to control mice with functional RXRα. Melanomas from the trigenic RXRα ep-/- mice are substantial in size, show increased proliferation, exhibit increased expression of malignant melanoma markers and exhibit enhanced vascularization. Altered expression of several biomarkers including increased expression of activated AKT, p21 and cyclin D1 and reduced expression of pro-apoptotic marker BAX was observed in the tumor adjacent normal (TAN) skin of acute ultraviolet B treated trigenic RXRα ep-/- mice. Interestingly, we observed a significant increase in p21 and Cyclin D1 in the TAN skin of un-irradiated trigenic RXRα ep-/- mice, suggesting that those changes might be consequences of loss of functional RXRα in the melanoma microenvironment. Loss of RXRα in the epidermal keratinocytes in combination with oncogenic NRAS Q61K and CDK4 R24C/R24C mutations in trigenic mice led to significant melanoma invasion into the draining lymph nodes as compared to controls with functional RXRα. CONCLUSIONS: Our study demonstrates the protective role of keratinocytic RxRα in (1) suppressing spontaneous and acute UVB-induced melanoma, and (2) preventing progression of the melanoma to malignancy in the presence of driver mutations like activated CDK4 R24C/R24C and oncogenic NRAS Q61K .

A miRNA signature for an environmental heterocyclic amine defined by a multi-organ carcinogenicity bioassay in the rat.

Heterocyclic amines (HCAs) produced during high-temperature cooking have been studied extensively in terms of their genotoxic/genetic effects, but recent work has implicated epigenetic mechanisms involving non-coding RNAs. Colon tumors induced in the rat by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) have altered microRNA (miRNA) signatures linked to dysregulated pluripotency factors, such as c-Myc and Krüppel-like factor 4 (KLF4). We tested the hypothesis that dysregulated miRNAs from PhIP-induced colon tumors would provide a "PhIP signature" for use in other target organs obtained from a 1-year carcinogenicity bioassay in the rat. Downstream targets that were corroborated in the rat were then investigated in human cancer datasets. The results confirmed that multiple let-7 family members were downregulated in PhIP-induced skin, colon, lung, small intestine, and Zymbal's gland tumors, and were associated with c-myc and Hmga2 upregulation. PhIP signature miRNAs with the profile mir-21high/mir-126low/mir-29clow/mir-215low/mir-145low were linked to reduced Klf4 levels in rat tumors, and in human pan-cancer and colorectal cancer. It remains to be determined whether this PhIP signature has predictive value, given that more than 20 different genotoxic HCAs are present in the human diet, plus other agents that likely induce or repress many of the same miRNAs. Future studies should define more precisely the miRNA signatures of other HCAs, and their possible value for human risk assessment.

Lymphoma Classification in Goats.

Lymphoma has been described in individual cases for goats but not systematically characterized in a larger cohort. This study aimed to subtype caprine lymphoma based on topographic and subgross distribution, immunophenotype, and cellular morphology following the World Health Organization classification system for hematopoietic tumors in domestic animals. Fifteen caprine lymphoma cases were assessed with 6 submitted as biopsy and 9 for postmortem examination. Goats were predominantly young adult (median 3 years) and dwarf breeds (Pygmy and Pygora). The sexes were similarly represented. Nuclear size was measured relative to red blood cells (RBCs) and then adjusted for species-specific differences and designated small (<3× RBCs), intermediate (3-4× RBCs), or large (>4× RBCs). Using immunohistochemistry, 11 of 15 (73%) goats had T-cell lymphoma (TCL; CD3 positive, CD79α negative) and 4 of 15 (27%) had B-cell lymphoma (BCL; CD79α positive, CD3 negative). A multicentric distribution was most common. TCL generally involved the thoracic cavity and/or neck, suggestive of thymic origin or homing. TCLs were further classified as lymphoblastic lymphomas (3/11; 27%), large granular lymphocyte lymphoma (1/11; 9%), diffuse small lymphocytic lymphomas (3/11; 27%), or peripheral/mature T-cell lymphoma (PTCL) not otherwise specified (4/11 [36%], of which 3 were high grade and 1 intermediate grade). In 1 goat with PTCL, lymph nodes had either paracortical expansion or diffuse infiltrates suggesting transition from nodular to diffuse PTLC. BCLs were classified as diffuse large B-cell lymphoma (2/4; 50%) or B-cell lymphocytic lymphoma intermediate type (2/4; 50%). In contrast to dogs and horses, lymphomas in goats are predominantly TCL and frequently involve the mediastinum.

Quantification of surgical margin length changes after excision of feline injection site sarcomas-A pilot study.

OBJECTIVE: To evaluate degree length change of lateral surgical margins at various stages of processing for histological examination and quantify the length change between grossly normal surgical margins (GNSM) and pathologist-reported histologic tumor-free margin (HTFM) in widely resected feline injection site sarcoma (FISS) specimens. STUDY DESIGN: Prospective clinical study. ANIMALS: Five client-owned cats with injection site sarcomas. METHODS: All cats underwent wide surgical excision (35-55 mm gross lateral margins, 2 fascial planes deep). Gross normal lateral margin measurements from tumor edge were recorded in 4 directions (cranial, caudal, dorsal, ventral) at 5 time points: intra-op (in vivo GNSM); immediately following excision (ex vivo GNSM); following formalin fixation (post-fixation GNSM); after trimming and mounting on glass slides (on-slide GNSM); and HTFM at the narrowest point from the HTFM from the same slides used for on-slide GNSM. Percent change in lateral margin length from in vivo measurements was quantified at each time point and compared using 1-way repeated measures ANOVA. RESULTS: The largest mean decrease in percent GNSM length occurred immediately after excision (in vivo to ex vivo GNSM = 29%; P = .016). Formalin fixation, trimming, and mounting on slides did not result in additional significant changes in length. Mean HTFM length was significantly decreased compared to both in vivo GNSM (33%; P = .014) and on-slide GNSM (7%; P = .024). CONCLUSION: Significant decreases in surgical margin length in FISS specimens occur immediately following excision (prior to formalin fixation). Subgross evaluation of tumor-free margins from on-slide GNSM to HTFM overestimates the actual (histologic) tumor-free margins.

Antiproliferative effects of masitinib and imatinib against canine oral fibrosarcoma in vitro.

BACKGROUND: Canine oral fibrosarcoma (COF) is one of the most common oral tumors in dogs and carries a guarded prognosis due to a lack of effective systemic therapeutic options. Mastinib and imatinib are two commonly used tyrosine kinase inhibitors (TKIs) in veterinary oncology but their potential efficacy against COF is uncharacterized. To begin investigating the rationale for use of these TKIs against COF, the present study tested for the presence TKI targets PDGFR-α, PDGFR-ß, Kit, and VEGFR-2 and examined the in vitro effects on cell viability after TKI treatment alone or with doxorubicin. Immunohistochemistry for PDGFR-α, PDGFR-ß, Kit, and VEGFR-2 was performed in 6 COF tumor biopsies. Presence of these same receptors within 2 COF cell lines was probed by reverse transcription-polymerase chain reaction and, for those with mRNA detected, confirmed via western blot. Effects on cell viability were assessed using an MTS assay after masitinib or imatinib treatment alone (0-100 µM), or in combination with doxorubicin (0-3000 nM doxorubicin). Anti-PDGFRB siRNA knockdown was performed and the effect on cell viability quantified. RESULTS: Expression of the TKI targets evaluated was similar between the 2 COF cell lines and the 6 COF tumor biopsies: PDGFR-α and PDGFR-ß were detected in neoplastic cells from most COF tumor biopsies (5/6 and 6/6, respectively) and were present in both COF cell lines; KIT and KDR were not detected in any sample. Masitinib and imatinib IC50 values ranged from 7.9-33.4 µM, depending on the specific TKI and cell line tested. The addition of doxorubicin resulted in synergistic cytotoxicity with both TKIs. Anti-PDGFRB siRNA transfection reduced PDGFR-ß protein expression by 77% and 67% and reduced cell viability by 24% (p < 0.0001) and 28% (0 = 0.0003) in the two cell lines, respectively. CONCLUSIONS: These results provide rationale for further investigation into the use of TKIs, possibly in combination with doxorubicin, as treatment options for COF.