Unbiased discovery of cancer pathways and therapeutics using Pathway Ensemble Tool and Benchmark.

Correctly identifying perturbed biological pathways is a critical step in uncovering basic disease mechanisms and developing much-needed therapeutic strategies. However, whether current tools are optimal for unbiased discovery of relevant pathways remains unclear. Here, we create "Benchmark" to critically evaluate existing tools and find that most function sub-optimally. We thus develop the "Pathway Ensemble Tool" (PET), which outperforms existing methods. Deploying PET, we identify prognostic pathways across 12 cancer types. PET-identified prognostic pathways offer additional insights, with genes within these pathways serving as reliable biomarkers for clinical outcomes. Additionally, normalizing these pathways using drug repurposing strategies represents therapeutic opportunities. For example, the top predicted repurposed drug for bladder cancer, a CDK2/9 inhibitor, represses cell growth in vitro and in vivo. We anticipate that using Benchmark and PET for unbiased pathway discovery will offer additional insights into disease mechanisms across a spectrum of diseases, enabling biomarker discovery and therapeutic strategies.

Genome-wide analyses reveals an association between invasive urothelial carcinoma in the Shetland sheepdog and NIPAL1.

Naturally occurring canine invasive urinary carcinoma (iUC) closely resembles human muscle invasive bladder cancer in terms of histopathology, metastases, response to therapy, and low survival rate. The heterogeneous nature of the disease has led to the association of large numbers of risk loci in humans, however most are of small effect. There exists a need for new and accurate animal models of invasive bladder cancer. In dogs, distinct breeds show markedly different rates of iUC, thus presenting an opportunity to identify additional risk factors and overcome the locus heterogeneity encountered in human mapping studies. In the association study presented here, inclusive of 100 Shetland sheepdogs and 58 dogs of other breeds, we identify a homozygous protein altering point mutation within the NIPAL1 gene which increases risk by eight-fold (OR = 8.42, CI = 3.12-22.71), accounting for nearly 30% of iUC risk in the Shetland sheepdog. Inclusion of six additional loci accounts for most of the disease risk in the breed and explains nearly 75% of the phenotypes in this study. When combined with sequence data from tumors, we show that variation in the MAPK signaling pathway is an overarching cause of iUC susceptibility in dogs.

Leading the pack: Best practices in comparative canine cancer genomics to inform human oncology.

Pet dogs develop spontaneous cancers at a rate estimated to be five times higher than that of humans, providing a unique opportunity to study disease biology and evaluate novel therapeutic strategies in a model system that possesses an intact immune system and mirrors key aspects of human cancer biology. Despite decades of interest, effective utilization of pet dog cancers has been hindered by a limited repertoire of necessary cellular and molecular reagents for both in vitro and in vivo studies, as well as a dearth of information regarding the genomic landscape of these cancers. Recently, many of these critical gaps have been addressed through the generation of a highly annotated canine reference genome, the creation of several tools necessary for multi-omic analysis of canine tumours, and the development of a centralized repository for key genomic and associated clinical information from canine cancer patients, the Integrated Canine Data Commons. Together, these advances have catalysed multidisciplinary efforts designed to integrate the study of pet dog cancers more effectively into the translational continuum, with the ultimate goal of improving human outcomes. The current review summarizes this recent progress and provides a guide to resources and tools available for comparative study of pet dog cancers.

cTULIP: application of a human-based RNA-seq primary tumor classification tool for cross-species primary tumor classification in canine.

Introduction: The domestic dog, Canis familiaris, is quickly gaining traction as an advantageous model for use in the study of cancer, one of the leading causes of death worldwide. Naturally occurring canine cancers share clinical, histological, and molecular characteristics with the corresponding human diseases. Methods: In this study, we take a deep-learning approach to test how similar the gene expression profile of canine glioma and bladder cancer (BLCA) tumors are to the corresponding human tumors. We likewise develop a tool for identifying misclassified or outlier samples in large canine oncological datasets, analogous to that which was developed for human datasets. Results: We test a number of machine learning algorithms and found that a convolutional neural network outperformed logistic regression and random forest approaches. We use a recently developed RNA-seq-based convolutional neural network, TULIP, to test the robustness of a human-data-trained primary tumor classification tool on cross-species primary tumor prediction. Our study ultimately highlights the molecular similarities between canine and human BLCA and glioma tumors, showing that protein-coding one-to-one homologs shared between humans and canines, are sufficient to distinguish between BLCA and gliomas. Discussion: The results of this study indicate that using protein-coding one-to-one homologs as the features in the input layer of TULIP performs good primary tumor prediction in both humans and canines. Furthermore, our analysis shows that our selected features also contain the majority of features with known clinical relevance in BLCA and gliomas. Our success in using a human-data-trained model for cross-species primary tumor prediction also sheds light on the conservation of oncological pathways in humans and canines, further underscoring the importance of the canine model system in the study of human disease.

Development of an Anti-canine PD-L1 Antibody and Caninized PD-L1 Mouse Model as Translational Research Tools for the Study of Immunotherapy in Humans.

Immune checkpoint blockade therapy, one of the most promising cancer immunotherapies, has shown remarkable clinical impact in multiple cancer types. Despite the recent success of immune checkpoint blockade therapy, however, the response rates in patients with cancer are limited (∼20%-40%). To improve the success of immune checkpoint blockade therapy, relevant preclinical animal models are essential for the development and testing of multiple combination approaches and strategies. Companion dogs naturally develop several types of cancer that in many respects resemble clinical cancer in human patients. Therefore, the canine studies of immuno-oncology drugs can generate knowledge that informs and prioritizes new immuno-oncology therapy in humans. The challenge has been, however, that immunotherapeutic antibodies targeting canine immune checkpoint molecules such as canine PD-L1 (cPD-L1) have not been commercially available. Here, we developed a new cPD-L1 antibody as an immuno-oncology drug and characterized its functional and biological properties in multiple assays. We also evaluated the therapeutic efficacy of cPD-L1 antibodies in our unique caninized PD-L1 mice. Together, these in vitro and in vivo data, which include an initial safety profile in laboratory dogs, support development of this cPD-L1 antibody as an immune checkpoint inhibitor for studies in dogs with naturally occurring cancer for translational research. Our new therapeutic antibody and caninized PD-L1 mouse model will be essential translational research tools in raising the success rate of immunotherapy in both dogs and humans. Significance: Our cPD-L1 antibody and unique caninized mouse model will be critical research tools to improve the efficacy of immune checkpoint blockade therapy in both dogs and humans. Furthermore, these tools will open new perspectives for immunotherapy applications in cancer as well as other autoimmune diseases that could benefit a diverse and broader patient population.

Identification of a naturally-occurring canine model for early detection and intervention research in high grade urothelial carcinoma.

Background: Early detection and intervention research is expected to improve the outcomes for patients with high grade muscle invasive urothelial carcinoma (InvUC). With limited patients in suitable high-risk study cohorts, relevant animal model research is critical. Experimental animal models often fail to adequately represent human cancer. The purpose of this study was to determine the suitability of dogs with high breed-associated risk for naturally-occurring InvUC to serve as relevant models for early detection and intervention research. The feasibility of screening and early intervention, and similarities and differences between canine and human tumors, and early and later canine tumors were determined. Methods: STs (n=120) ≥ 6 years old with no outward evidence of urinary disease were screened at 6-month intervals for 3 years with physical exam, ultrasonography, and urinalysis with sediment exam. Cystoscopic biopsy was performed in dogs with positive screening tests. The pathological, clinical, and molecular characteristics of the "early" cancer detected by screening were determined. Transcriptomic signatures were compared between the early tumors and published findings in human InvUC, and to more advanced "later" canine tumors from STs who had the typical presentation of hematuria and urinary dysfunction. An early intervention trial of an oral cyclooxygenase inhibitor, deracoxib, was conducted in dogs with cancer detected through screening. Results: Biopsy-confirmed bladder cancer was detected in 32 (27%) of 120 STs including InvUC (n=29, three starting as dysplasia), grade 1 noninvasive cancer (n=2), and carcinoma in situ (n=1). Transcriptomic signatures including druggable targets such as EGFR and the PI3K-AKT-mTOR pathway, were very similar between canine and human InvUC, especially within luminal and basal molecular subtypes. Marked transcriptomic differences were noted between early and later canine tumors, particularly within luminal subtype tumors. The deracoxib remission rate (42% CR+PR) compared very favorably to that with single-agent cyclooxygenase inhibitors in more advanced canine InvUC (17-25%), supporting the value of early intervention. Conclusions: The study defined a novel naturally-occurring animal model to complement experimental models for early detection and intervention research in InvUC. Research incorporating the canine model is expected to lead to improved outcomes for humans, as well as pet dogs, facing bladder cancer.

Targeted elastin-like polypeptide fusion protein for near-infrared imaging of human and canine urothelial carcinoma.

Cystoscopic visualization of bladder cancer is an essential method for initial bladder cancer detection and diagnosis, transurethral resection, and monitoring for recurrence. We sought to develop a new intravesical imaging agent that is more specific and sensitive using a polypeptide based NIR (near-infrared) probe designed to detect cells bearing epidermal growth factor receptors (EGFR) that are overexpressed in 80% of urothelial carcinoma (UC) cases. The NIR imaging agent consisted of an elastin like polypeptide (ELP) fused with epidermal growth factor (EGF) and conjugated to Cy5.5 to give Cy5.5-N24-EGF as a NIR contrast agent. In addition to evaluation in human cells and tissues, the agent was tested in canine cell lines and tissue samples with naturally occurring invasive UC. Flow cytometry and confocal microscopy were used to test cell-associated fluorescence of the probe in T24 human UC cells, and in K9TCC-SH (high EGFR expression) and K9TCC-Original (low EGF expression) canine cell lines. The probe specifically engages these cells through EGFR within 15 min of incubation and reached saturation within a clinically relevant 1 h timeframe. Furthermore, ex vivo studies with resected canine and human bladder tissues showed minimal signal from normal adjacent tissue and significant NIR fluorescence labeling of tumor tissue, in good agreement with our in vitro findings. Differential expression of EGFR ex vivo was revealed by our probe and confirmed by anti-EGFR immunohistochemical staining. Taken together, our data suggests Cy5.5-ELP-EGF is a NIR probe with improved sensitivity and selectivity towards BC that shows excellent potential for clinical translation.

The miRNome of canine invasive urothelial carcinoma.

Urothelial carcinoma (UC) comprises up to 2% of all naturally occurring neoplasia in dogs and can be challenging to diagnose. MicroRNAs (miRNAs) have been reported to be dysregulated in numerous diseases, including neoplasia. MiRNA expression has been evaluated in human UC, but there is limited information regarding the miRNA transcriptome of UC in dogs. Our study aimed to evaluate differential miRNA expression in bladder tissue collected from normal canine urothelium and canine invasive UC (iUC) to elucidate the dysregulated pathways in canine UC. Next-Generation RNA sequencing (RNA-Seq) was performed for dogs with UC (n = 29) and normal canine urothelium (n = 4). Raw RNA data were subjected to normalization, and pairwise comparison was performed using EdgeR with Benjamini-Hochberg FDR multiple testing correction (p < 0.05; >2-fold change) comparing tissue samples of normal urothelium to canine iUC samples. Principal component analysis and hierarchical cluster analysis were performed. MiRNA of FFPE tissue samples of separate iUC (n = 5) and normal urothelium (n = 5) were used to evaluate five miRNAs using RT-qPCR. Pathway analysis was performed utilizing miRWalk, STRING database, and Metascape utilizing KEGG pathways and GO terms databases. Twenty-eight miRNAs were differentially expressed (DE) by RNA-Seq. RT-qPCR confirmed that four miRNAs are significantly downregulated in UC compared to healthy urothelial samples (miR-105a, miR-143, miR-181a, and miR-214). Principal component analysis and hierarchical cluster analysis showed separation between miRNAs in iUC and the control group. The DE miRNAs are most often associated with gene silencing by miRNA, miRNAs in cancer, and miRNAs involved in DNA damage responses. Proteins involved include HRAS, KRAS, ARAF, RAF1, MAPK1, MAP2K1, MAPK3, FGFR3, EGFR, HBEGF, RASSF1, E2F2, E2F3, ERBB2, SRC, MMP1, and UP3KA. The differential expression of miRNAs in canine iUC compared to normal canine urothelial tissue indicates that these markers should be further evaluated for their potential role as diagnostic and therapeutic targets.

Ultrasonographic detection of apex nodules in the urinary bladder of Scottish Terriers.

An apex nodule was recently identified in the urinary bladder of Scottish Terriers being screened for bladder cancer at our institution. This prospective, single-center, case series study was performed to better characterize the apex nodule and assess the clinical importance of the nodule. Scottish Terriers ≥6 years of age with no evidence of urinary tract disease underwent urinary tract ultrasonography and urinalysis at 6-month intervals. In dogs with evidence of the apex nodule, ultrasound features such as location, margins, number, echogenicity, size, and shape of the lesion were recorded by a veterinary oncologist and veterinary radiologist. The apex nodule was identified in eight (6%) of 134 dogs in the absence of other detectable bladder disease. Features of the nodules included the following: one nodule per dog, triangular to an oval shape, smooth mucosal covering, well-defined margins, isoechoic to the bladder wall, 2-4 mm at the base, and 4-6 mm protruding into the bladder lumen. In five dogs undergoing multiple ultrasonographic examinations, the nodule did not appear to change over time (up to 3.5 years). Cystoscopy performed in three dogs revealed a column of tissue covered by normal mucosa protruding into the bladder lumen. Histological features consistent with a neoplastic growth were absent. Five dogs remained free of any bladder disease. Three dogs developed urothelial carcinoma at sites distant to the nodule at 8-53 months after the nodule was first observed. Findings indicated that incidental apex nodules could mimic neoplasia and other bladder diseases in Scottish Terriers.

Interleukin-6 and thrombopoietin concentrations in dogs with carcinoma with and without thrombocytosis.

BACKGROUND: Carcinoma-associated thrombocytosis involves tumor production of mediators such as interleukin-6 (IL-6) and thrombopoietin (TPO) that increase thrombopoiesis and may play a role in tumor evasion and metastasis. Carcinoma-associated thrombocytosis is described in people, but has not been described in dogs. HYPOTHESIS/OBJECTIVES: Evaluate the concentrations of IL-6 and TPO in dogs diagnosed with carcinoma with or without thrombocytosis. We hypothesized that IL-6 and TPO concentrations would be higher in dogs with carcinoma compared to healthy dogs, and that IL-6 and TPO concentrations would be higher in dogs with carcinoma and thrombocytosis when compared to dogs with carcinoma and normal platelet counts. ANIMALS: One-hundred sixteen dogs: 63 with carcinoma and 53 healthy control dogs. METHODS: Complete blood count was performed in all dogs, and they were stratified for sub-group analysis based on the presence or absence of thrombocytosis (platelet count > 500 103/µL). Serum TPO and IL-6 concentrations were measured by ELISA. Results of selected numeric variables were compared using Wilcoxon rank sum tests for pairwise comparisons. A value of P < .05 was considered significant. RESULTS: Twelve of the dogs with carcinoma (12/63, 19.0%) and none of the healthy control dogs (0%) had thrombocytosis. Thrombopoietin concentrations (median [range]) were significantly higher in dogs with carcinoma when compared to controls (87.42 pg/mL [0 to >600] vs 15.99 pg/mL [0 to >600], P < .001). Interleukin-6 concentrations (median [range]) were not different between dogs with carcinoma and healthy control dogs (9.70 pg/mL [0-181.53] vs 3.03 pg/mL [0-280.77], P = .15). In dogs with carcinoma, the TPO and IL-6 concentrations were not different between dogs with thrombocytosis and dogs with normal platelet count. CONCLUSIONS AND CLINICAL IMPORTANCE: Thrombopoietin concentrations were significantly higher in dogs with carcinoma, regardless of platelet count. Thrombopoietin is likely to be 1 of multiple factors that can impact platelet number, production, and consumption in dogs with carcinoma.

The Red Harmful Plague in Times of Climate Change: Blooms of the Cyanobacterium Planktothrix rubescens Triggered by Stratification Dynamics and Irradiance.

Planktothrix rubescens is a harmful planktonic cyanobacterium, forming concentrated metalimnetic populations in deep oligo- and mesotrophic lakes, even after successful restoration. In Lake Zurich (Switzerland), P. rubescens emerged as a keystone species with annual mass developments since the 1970s. Its success was partly attributed to effects of lake warming, such as changes in thermal stratification and seasonal deep mixing. However, recent observations based on a biweekly monitoring campaign (2009-2020) revealed two massive breakdowns and striking seasonal oscillations of the population. Here, we disentangle positive from negative consequences of secular lake warming and annual variations in weather conditions on P. rubescens dynamics: (i) despite the high survival rates of overwintering populations (up to 25%) during three consecutive winters (2014-2016) of incomplete deep convective mixing, cyanobacterial regrowth during the following stratified season was moderate and not overshooting a distinct standing stock threshold. Moreover, we recorded a negative trend for annual population maxima and total population size, pointing to a potential nutrient limitation after a series of incomplete winter mixing. Thus, the predication of steadily increasing blooms of P. rubescens could not be confirmed for the last decade. (ii) The seasonal reestablishment of P. rubescens was strongly coupled with a timely formation of a stable metalimnion structure, where the first positive net growth in the following productive summer season was observed. The trigger for the vertical positioning of filaments within the metalimnion was irradiance and not maximal water column stability. Repetitive disruptions of the vernal metalimnion owing to unstable weather conditions, as in spring 2019, went in parallel with a massive breakdown of the standing stock and marginal regrowth during thermal stratification. (iii) Driven by light intensity, P. rubescens was entrained into the turbulent epilimnion in autumn, followed by a second peak in population growth. Thus, the typical bimodal growth pattern was still intact during the last decade. Our long-term study highlights the finely tuned interplay between climate-induced changes and variability of thermal stratification dynamics and physiological traits of P. rubescens, determining its survival in a mesotrophic temperate lake.

Phase I/II Trial of Vemurafenib in Dogs with Naturally Occurring, BRAF-mutated Urothelial Carcinoma.

BRAF-targeted therapies including vemurafenib (Zelboraf) induce dramatic cancer remission; however, drug resistance commonly emerges. The purpose was to characterize a naturally occurring canine cancer model harboring complex features of human cancer, to complement experimental models to improve BRAF-targeted therapy. A phase I/II clinical trial of vemurafenib was performed in pet dogs with naturally occurring invasive urothelial carcinoma (InvUC) harboring the canine homologue of human BRAF V600E The safety, MTD, pharmacokinetics, and antitumor activity were determined. Changes in signaling and immune gene expression were assessed by RNA sequencing and phosphoproteomic analyses of cystoscopic biopsies obtained before and during treatment, and at progression. The vemurafenib MTD was 37.5 mg/kg twice daily. Anorexia was the most common adverse event. At the MTD, partial remission occurred in 9 of 24 dogs (38%), with a median progression-free interval of 181 days (range, 53-608 days). In 18% of the dogs, new cutaneous squamous cell carcinoma and papillomas occurred, a known pharmacodynamic effect of vemurafenib in humans. Upregulation of genes in the classical and alternative MAPK-related pathways occurred in subsets of dogs at cancer progression. The most consistent transcriptomic changes were the increase in patterns of T lymphocyte infiltration during the first month of vemurafenib, and of immune failure accompanying cancer progression. In conclusion, the safety, antitumor activity, and cutaneous pharmacodynamic effects of vemurafenib, and the development of drug resistance in dogs closely mimic those reported in humans. This suggests BRAF-mutated canine InvUC offers an important complementary animal model to improve BRAF-targeted therapies in humans.

Basal and Luminal Molecular Subtypes in Naturally-Occurring Canine Urothelial Carcinoma are Associated with Tumor Immune Signatures and Dog Breed.

BACKGROUND: Improved therapies are needed for patients with invasive urothelial carcinoma (InvUC). Tailoring treatment to molecular subtypes holds promise, but requires further study, including studies in pre-clinical animal models. Naturally-occurring canine InvUC harbors luminal and basal subtypes, mimicking those observed in humans, and could offer a relevant model for the disease in people. OBJECTIVE: To further validate the canine InvUC model, clinical and tumor characteristics associated with luminal and basal subtypes in dogs were determined, with comparison to findings from humans. METHODS: RNA sequencing (RNA-seq) analyses were performed on 56 canine InvUC tissues and bladder mucosa from four normal dogs. Data were aligned to CanFam 3.1, and differentially expressed genes identified. Data were interrogated with panels of genes defining luminal and basal subtypes, immune signatures, and other tumor features. Subject and tumor characteristics, and outcome data were obtained from medical records. RESULTS: Twenty-nine tumors were classified as luminal and 27 tumors as basal subtype. Basal tumors were strongly associated with immune infiltration (OR 52.22, 95%CI 4.68-582.38, P = 0.001) and cancer progression signatures in RNA-seq analyses, more advanced clinical stage, and earlier onset of distant metastases in exploratory analyses (P = 0.0113). Luminal tumors were strongly associated with breeds at high risk for InvUC (OR 0.06, 95%CI 0.01 -0.37, P = 0.002), non-immune infiltrative signatures, and less advanced clinical stage. CONCLUSIONS: Dogs with InvUC could provide a valuable model for testing new treatment strategies in the context of molecular subtype and immune status, and the search for germline variants impacting InvUC onset and subtype.

Assessment of urinary 15-F2 -isoprostanes in dogs with urothelial carcinoma of the urinary bladder and other lower urinary tract diseases.

BACKGROUND: The 15-F2 -isoprostanes are by-products of oxidative stress and are increased in the urine of people with lower urinary tract diseases (LUTD), especially urinary neoplasia. Urothelial carcinoma (UC) is the most common urinary neoplasm in dogs. Earlier detection of UC by noninvasive means could lead to improved outcomes. Urinary 15-F2 -isoprostanes potentially could provide this means, but have not been evaluated in dogs with UC. OBJECTIVE: The objective of this study was to measure urinary 15-F2 -isoprostanes in dogs with UC and dogs with other LUTD. ANIMALS: One hundred seventeen dogs: 46 dogs with UC, 30 dogs with LUTD, and 25 control dogs. METHODS: Any dog that was presented with dysuria was eligible for inclusion. Diagnosis of UC was confirmed histologically. Urinalysis was performed in each case, and 15-F2 -isoprostanes quantified by gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) and normalized to urinary creatinine concentration. RESULTS: Dogs with urinary diseases (UC + LUTD) had higher median urinary 15-F2 -isoprostanes when compared to control dogs (5.92 ng/mg [range, 0.46-31.03] vs 3.73 [range, 1.8-7.98]; P = .02). Urinary 15-F2 -isoprostanes were similar in dogs with UC (5.33 ng/mg [range, 0.46-31.03]) compared to dogs with LUTD (6.29 ng/mg [range, 0.54-18.93]; P = .47) and control dogs (P = .06). Dogs with UC had higher qualitative measures of proteinuria (P = .004), hematuria (P = .01), and epithelial cells on urinalysis (P = .002) compared to the other groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Urinary F2 -isoprostanes are not useful for the detection of UC in dogs. Future research could evaluate urinary 15-F2 -isoprostanes as a marker of inflammation in disease progression and prognosis.

RNAseq expression patterns of canine invasive urothelial carcinoma reveal two distinct tumor clusters and shared regions of dysregulation with human bladder tumors.

BACKGROUND: Invasive urothelial carcinoma (iUC) is highly similar between dogs and humans in terms of pathologic presentation, molecular subtypes, response to treatment and age at onset. Thus, the dog is an established and relevant model for testing and development of targeted drugs benefiting both canine and human patients. We sought to identify gene expression patterns associated with two primary types of canine iUC tumors: those that express a common somatic mutation in the BRAF gene, and those that do not. METHODS: We performed RNAseq on tumor and normal tissues from pet dogs. Analysis of differential expression and clustering, and positional and individual expression was used to develop gene set enrichment profiles distinguishing iUC tumors with and without BRAFV595E mutations, as well as genomic regions harboring excessive numbers of dysregulated genes. RESULTS: We identified two expression clusters that are defined by the presence/absence of a BRAFV595E (BRAFV600E in humans) somatic mutation. BRAFV595E tumors shared significantly more dysregulated genes than BRAF wild-type tumors, and vice versa, with 398 genes differentiating the two clusters. Key genes fall into clades of limited function: tissue development, cell cycle regulation, immune response, and membrane transport. The genomic site with highest number of dysregulated genes overall lies in a locus corresponding to human chromosome 8q24, a region frequently amplified in human urothelial cancers. CONCLUSIONS: These data identify critical sets of genes that are differently regulated in association with an activating mutation in the MAPK/ERK pathway in canine iUC tumors. The experiments also highlight the value of the canine system in identifying expression patterns associated with a common, shared cancer.

A novel, safe, fast and efficient treatment for Her2-positive and negative bladder cancer utilizing an EGF-anthrax toxin chimera.

Bladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin-shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF-conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF-based technologies and other toxin-derivatives. In contrast to known agents, this EGF-toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC50 < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor-free mice and dogs with the agent resulted in no toxicity. In addition, the EGF-toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF-toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF-anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer.

Immune Checkpoint B7x (B7-H4/B7S1/VTCN1) is Over Expressed in Spontaneous Canine Bladder Cancer: The First Report and its Implications in a Preclinical Model.

BACKGROUND: B7x (B7-H4/B7S1/VTCN1), an inhibitory immune checkpoint molecule is a potential therapeutic target owing to its immunosuppressive effect and well-known expression in cancers. Immune checkpoints in canine bladder cancer are largely undefined. Here, we report the first evaluation on expression of B7x in spontaneous canine invasive bladder cancer, a novel model system for the study of invasive human urothelial carcinoma. OBJECTIVE: This work aims to study expression of immune checkpoint B7x in spontaneous canine invasive bladder cancer. METHODS: RNA-seq analysis was performed to determine B7x expression in tumor versus normal bladder. Gene ontology (GO) study was used to explore the biological role of B7x. B7x protein expression was evaluated by immunohistochemistry (IHC). TCGA and GTEx were used to examine B7x expression in 599 human bladder urothelial carcinoma (BLCA). RESULTS: RNA-seq analysis indicated 5.72 and 7.04 fold up regulation of B7x in tumors, using DESeq2 and edge R respectively (p < 0.00008). B7x was closely associated with immune processes in GO analysis. IHC results revealed 60% of cases as B7x positive. B7x intensity was scored as negative in 40% (n = 20/50), low in 24% (n = 12/50), medium in 14% (n = 7/50) and high in 22% (n = 11/50) of cases. In human BLCA, B7x expression was significantly associated with worse overall survival (p = 0.02). CONCLUSIONS: Our results suggest that B7x is over expressed in canine bladder cancer. Thus canine model can be vital in advancing the translational research on B7x, a new potential therapeutic target in human bladder cancer.

Naturally-Occurring Invasive Urothelial Carcinoma in Dogs, a Unique Model to Drive Advances in Managing Muscle Invasive Bladder Cancer in Humans.

There is a great need to improve the outlook for people facing urinary bladder cancer, especially for patients with invasive urothelial carcinoma (InvUC) which is lethal in 50% of cases. Improved outcomes for patients with InvUC could come from advances on several fronts including emerging immunotherapies, targeted therapies, and new drug combinations; selection of patients most likely to respond to a given treatment based on molecular subtypes, immune signatures, and other characteristics; and prevention, early detection, and early intervention. Progress on all of these fronts will require clinically relevant animal models for translational research. The animal model(s) should possess key features that drive success or failure of cancer drugs in humans including tumor heterogeneity, genetic-epigenetic crosstalk, immune cell responsiveness, invasive and metastatic behavior, and molecular subtypes (e.g., luminal, basal). Experimental animal models, while essential in bladder cancer research, do not possess these collective features to accurately predict outcomes in humans. These key features, however, are present in naturally-occurring InvUC in pet dogs. Canine InvUC closely mimics muscle-invasive bladder cancer in humans in cellular and molecular features, molecular subtypes, immune response patterns, biological behavior (sites and frequency of metastasis), and response to therapy. Thus, dogs can offer a highly relevant animal model to complement other models in research for new therapies for bladder cancer. Clinical treatment trials in pet dogs with InvUC are considered a win-win-win scenario; the individual dog benefits from effective treatment, the results are expected to help other dogs, and the findings are expected to translate to better treatment outcomes in humans. In addition, the high breed-associated risk for InvUC in dogs (e.g., 20-fold increased risk in Scottish Terriers) offers an unparalleled opportunity to test new strategies in primary prevention, early detection, and early intervention. This review will provide an overview of canine InvUC, summarize the similarities (and differences) between canine and human InvUC, and provide evidence for the expanding value of this canine model in bladder cancer research.

An ultrasound based platform for image-guided radiotherapy in canine bladder cancer patients.

BACKGROUND AND PURPOSE: Ultrasound (US) is a non-invasive, non-radiographic imaging technique with high spatial and temporal resolution that can be used for localizing soft-tissue structures and tumors in real-time during radiotherapy (RT) (inter- and intra-fraction). A comprehensive approach incorporating an in-house 3D-US system within RT is presented. This system is easier to adopt into existing treatment protocols than current US based systems, with the aim of providing millimeter intra-fraction alignment errors and sensitivity to track intra-fraction bladder movement. MATERIALS AND METHODS: An in-house integrated US manipulator and platform was designed to relate the computed tomographic (CT) scanner, 3D-US and linear accelerator coordinate systems. An agar-based phantom with measured speed of sound and densities consistent with tissues surrounding the bladder was rotated (0-45°) and translated (up to 55 mm) relative to the US and CT coordinate systems to validate this device. After acquiring and integrating CT and US images into the treatment planning system, US-to-US and US-to-CT images were co-registered to re-align the phantom relative to the linear accelerator. RESULTS: Statistical errors from US-to-US registrations for various patient orientations ranged from 0.1 to 1.7 mm for x, y, and z translation components, and 0.0-1.1° for rotational components. Statistical errors from US-to-CT registrations were 0.3-1.2 mm for the x, y and z translational components and 0.1-2.5° for the rotational components. CONCLUSIONS: An ultrasound-based platform was designed, constructed and tested on a CT/US tissue-equivalent phantom to track bladder displacement with a statistical uncertainty to correct and track inter- and intra-fractional displacements of the bladder during radiation treatments.