Characterizing the molecular and immune landscape of canine bladder cancer.

Transitional cell carcinoma (TCC), also known as urothelial carcinoma, is the most common bladder cancer in humans and dogs. Approximately one-quarter of human TCCs are muscle-invasive and associated with a high risk of death from metastasis. Canine TCC (cTCC) tumours are typically high-grade and muscle-invasive. Shared similarities in risk factors, histopathology, and clinical presentation suggest that cTCC may serve as a model for the assessment of novel therapeutics that may inform therapies for human muscle-invasive TCC. The goal of this study was to characterize cTCC at the molecular level to identify drivers of oncogenesis and druggable targets. We performed whole exome sequencing (WES) of 11 cTCC tumours and three matched normal samples, identifying 583 variants in protein-coding genes. The most common variant was a V-to-E missense mutation in BRAF, identified in 4 out of 11 samples (36%) via WES. Sanger sequencing identified BRAF variants in 8 out of the same 11 cTCC samples, as well as in 22 out of 32 formalin-fixed paraffin embedded (FFPE) cTCC samples, suggesting an overall prevalence of 70%. RNA-Seq was performed to compare the gene expression profiles of cTCC tumours to normal bladder tissue. cTCC tumours exhibited up-regulation of genes involved in the cell cycle, DNA repair, and antiviral immunity. We also analysed the immune landscape of cTCC using immune gene signatures and immunohistochemical analysis. A subset of tumours had characteristics of a hot tumour microenvironment and exhibited high expression of signatures associated with complete response to PD-1/PD-L1 blockade in human bladder cancer.

MicroRNA Expression Changes and Integrated Pathways Associated With Poor Outcome in Canine Osteosarcoma.

MicroRNAs (miRNA) are small non-coding RNA molecules involved in post-transcriptional gene regulation. Deregulation of miRNA expression occurs in cancer, and miRNA expression profiles have been associated with diagnosis and prognosis in many cancers. Osteosarcoma (OS), an aggressive primary tumor of bone, affects ~10,000 dogs each year. Though survival has improved with the addition of chemotherapy, up to 80% of canine patients will succumb to metastatic disease. Reliable prognostic markers are lacking for this disease. miRNAs are attractive targets of biomarker discovery efforts due to their increased stability in easily obtained body fluids as well as within fixed tissue. Previous studies in our laboratory demonstrated that dysregulation of genes in aggressive canine OS tumors that participate in miRNA regulatory networks is reportedly disrupted in OS or other cancers. We utilized RT-qPCR in a 384-well-plate system to measure the relative expression of 190 miRNAs in 14 canine tumors from two cohorts of dogs with good or poor outcome (disease-free interval >300 or <100 days, respectively). Differential expression analysis in this subset guided the selection of candidate miRNAs in tumors and serum samples from larger groups of dogs. We ultimately identified a tumor-based three-miR Cox proportional hazards regression model and a serum-based two-miR model, each being able to distinguish patients with good and poor prognosis via Kaplan-Meier analysis with log rank test. Additionally, we integrated miRNA and gene expression data to identify potentially important miRNA-mRNA interactions that are disrupted in canine OS. Integrated analyses of miRNAs in the three-miR predictive model and disrupted genes from previous expression studies suggest the contribution of the primary tumor microenvironment to the metastatic phenotype of aggressive tumors.

RNase I Modulates Escherichia coli Motility, Metabolism, and Resistance.

Bacteria are constantly adapting to their environment by sensing extracellular factors that trigger production of intracellular signaling molecules, known as second messengers. Recently, 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) were identified in Escherichia coli and have emerged as possible novel signaling molecules. 2',3'-cNMPs are produced through endonucleolytic cleavage of short RNAs by the T2 endoribonuclease, RNase I; however, the physiological roles of RNase I remain unclear. Our transcriptomic analysis suggests that RNase I is involved in modulating numerous cellular processes, including nucleotide metabolism, motility, acid sensitivity, metal homeostasis, and outer membrane morphology. Through a combination of deletion strain and inhibitor studies, we demonstrate that RNase I plays a previously unknown role in E. coli stress resistance by affecting pathways that are part of the defense mechanisms employed by bacteria when introduced to external threats, including antibiotics. Thus, this work provides insight into the emerging roles of RNase I in bacterial signaling and physiology and highlights the potential of RNase I as a target for antibacterial adjuvants.

Chemical Profiling of A-to-I RNA Editing Using a Click-Compatible Phenylacrylamide.

Straightforward methods for detecting adenosine-to-inosine (A-to-I) RNA editing are key to a better understanding of its regulation, function, and connection with disease. We address this need by developing a novel reagent, N-(4-ethynylphenyl)acrylamide (EPhAA), and illustrating its ability to selectively label inosine in RNA. EPhAA is synthesized in a single step, reacts rapidly with inosine, and is "click"-compatible, enabling flexible attachment of fluorescent probes at editing sites. We first validate EPhAA reactivity and selectivity for inosine in both ribonucleosides and RNA substrates, and then apply our approach to directly monitor in vitro A-to-I RNA editing activity using recombinant ADAR enzymes. This method improves upon existing inosine chemical-labeling techniques and provides a cost-effective, rapid, and non-radioactive approach for detecting inosine formation in RNA. We envision this method will improve the study of A-to-I editing and enable better characterization of RNA modification patterns in different settings.

DOG1 is a sensitive and specific immunohistochemical marker for diagnosis of canine gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) and leiomyosarcomas are histologically similar primary neoplasms commonly occurring in the gastrointestinal tract of dogs and humans. Immunohistochemical staining (IHC) is needed to differentiate between these 2 entities and positive reactivity for KIT (cluster of differentiation [CD]117) is regarded as the gold standard for diagnosis of canine GIST. Studies estimate 5-10% of human GISTs stain negative or only weakly positive for KIT and have identified DOG1 (discovered on gastrointestinal stromal tumors protein 1) as a highly sensitive and specific marker for human GISTs. The purpose of this study was to evaluate immunoreactivity of a commercially available DOG1 antibody for use in diagnosis of canine GISTs. Fifty-five primary mesenchymal gastrointestinal tumors with histologic features consistent with GIST or leiomyosarcoma were evaluated via IHC for KIT, DOG1, and desmin. A subset of tumors was additionally evaluated for reactivity for smooth muscle actin (SMA). Thirty-three tumors (60%) were diagnosed as GIST based on positive immunoreactivity for KIT or DOG1 regardless of reactivity for desmin or SMA. Most GISTs (32/33, 97.0%) had similar staining for both KIT and DOG1. DOG1 expression was identified in 2 tumors (1 study tumor and 1 additional tumor) negative for KIT and desmin that had histologic features consistent with KIT-negative, platelet-derived growth factor receptor-alpha (PDGFRA)-mutant human GISTs. Our results suggest that DOG1 has improved specificity and sensitivity to that of KIT for differentiating between canine GISTs and leiomyosarcomas. Inclusion of both DOG1 and KIT IHC in diagnostic panels will improve the accuracy of canine GIST diagnosis.

HES1, a target of Notch signaling, is elevated in canine osteosarcoma, but reduced in the most aggressive tumors.

BACKGROUND: Hairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling. Notch signaling and HES1 expression have been linked to growth and survival in a variety of human cancer types and have been associated with increased metastasis and invasiveness in human osteosarcoma cell lines. Osteosarcoma (OSA) is an aggressive cancer demonstrating both high metastatic rate and chemotherapeutic resistance. The current study examined expression of Notch signaling mediators in primary canine OSA tumors and canine and human osteosarcoma cell lines to assess their role in OSA development and progression. RESULTS: Reverse transcriptase - quantitative PCR (RT-qPCR) was utilized to quantify HES1, HEY1, NOTCH1 and NOTCH2 gene expression in matched tumor and normal metaphyseal bone samples taken from dogs treated for appendicular OSA at the Colorado State University Veterinary Teaching Hospital. Gene expression was also assessed in tumors from dogs with a disease free interval (DFI) of <100 days compared to those with a DFI > 300 days following treatment with surgical amputation followed by standard chemotherapy. Immunohistochemistry was performed to confirm expression of HES1. Data from RT-qPCR and immunohistochemical (IHC) experiments were analyzed using REST2009 software and survival analysis based on IHC expression employed the Kaplan-Meier method and log rank analysis. Unbiased clustered images were generated from gene array analysis data for Notch/HES1 associated genes. Gene array analysis of Notch/HES1 associated genes suggested alterations in the Notch signaling pathway may contribute to the development of canine OSA. HES1 mRNA expression was elevated in tumor samples relative to normal bone, but decreased in tumor samples from dogs with a DFI < 100 days relative to those with a DFI > 300 days. NOTCH2 and HEY1 mRNA expression was also elevated in tumors relative to normal bone, but was not differentially expressed between the DFI tumor groups. Survival analysis confirmed an association between decreased HES1 immunosignal and shorter DFI. CONCLUSIONS: Our findings suggest that activation of Notch signaling occurs and may contribute to the development of canine OSA. However, association of low HES1 expression and shorter DFI suggests that mechanisms that do not alter HES1 expression may drive the most aggressive tumors.

Lymphadenitis as a major element of disease in the guinea pig model of tuberculosis.

Guinea pigs infected by low dose aerosol with the H37Rv strain of Mycobacterium tuberculosis rapidly developed granulomatous lesions in the pulmonary parenchyma and within the intra-thoracic hilar lymph node cluster. Lung lesions showed no predilection for specific lobes and were perivascular, peribronchial and peribronchiolar throughout the subpleural, hilar and pulmonary parenchyma. Marked hilar lymph node enlargement was due to coalescing foci of subcapsular, paracortical and medullary granulomatous inflammation that progressed to necrosis that effaced normal lymph node architecture. Lymph node lesions became severe and progressed more rapidly than pulmonary lesions. Immunization with BCG 6 weeks prior to infection significantly reduced the lung and lymph node lesion burden as well as the progression to necrosis in both tissues. Lymph node inflammation in BCG immunized animals partially resolved and was replaced by fibroblasts and fibrous connective tissue while lesions from non-immunized animals continued to progress to necrosis. We discuss here the observation that the distribution and progression of lung and lymph node lesions in the guinea pig aerosol model of tuberculosis have considerable similarity to the naturally occurring disease in children.

Magnetic resonance imaging of pulmonary lesions in guinea pigs infected with Mycobacterium tuberculosis.

We utilized magnetic resonance imaging to visualize lesions in the lungs of guinea pigs infected by low-dose aerosol exposure to Mycobacterium tuberculosis. Lesions were prominent in such images, and colorized three-dimensional reconstructions of images revealed a very uniform distribution in the lungs. Lesion numbers after 1 month were approximately similar to the aerosol exposure algorithm, suggesting that each was established by a single bacterium. Numbers of lesions in unprotected and vaccinated animals were similar over the first month but increased thereafter in the control animals, indicating secondary lesion development. Whereas lesion sizes increased progressively in control guinea pigs, lesions remained small in BCG-vaccinated animals. A prominent feature of the disease pathology in unprotected animals was rapid and severe lymphadenopathy of the mediastinal lymph node cluster, which is paradoxical given the strong state of cellular immunity at this time. Further development of this technical approach could be very useful in tracking lesion size, number, and progression in the search for new tuberculosis vaccines.