Canine urothelial carcinoma: a pilot study of microRNA detection in formalin-fixed, paraffin-embedded tissue samples and in normal urine.

We assessed the effects of fixation time in formalin and inclusion of surrounding tissue on microRNA (miRNA) cycle quantification (Cq) values in formalin-fixed, paraffin-embedded (FFPE) urothelial carcinoma (UC) tissue (n = 3), and the effect of conditions on miRNAs in urine from 1 healthy dog. MiRNAs were extracted using commercial kits and quantified using miRNA-specific fluorometry in normal bladder tissue scrolls, UC tissue cores, and bladder muscularis tissue cores from 4 FFPE bladder sections (3 UCs, 1 normal), plus 1 UC stored in formalin for 1, 8, 15, and 22 d before paraffin-embedding. Urine was collected from a healthy dog on 4 occasions; 1-mL aliquots were stored at 20, 4, -20, and -80°C for 4, 8, 24, and 48 h, and 1 and 2 wk. For both FFPE tissue and urine, we used reverse-transcription quantitative real-time PCR (RT-qPCR) to quantify miR-143, miR-152, miR-181a, miR-214, miR-1842, and RNU6B in each tissue or sample, using miR-39 as an exogenous control gene. The Cq values were compared with ANOVA and t-tests. The time of tissue-fixation in formalin did not alter miRNA Cq values; inclusion of the muscularis layer resulted in a statistically different miRNA Cq profile for miR-152, miR-181a, and RNU6B in bladder tissue. MiRNAs in acellular urine were stable for up to 2 wk regardless of the storage temperature. Our findings support using stored FFPE and urine samples for miRNA detection; we recommend measuring miRNA only in the tissue of interest in FFPE sections.

Serum thymidine kinase 1 activity as a prognostic biomarker in dogs with chemotherapy-treated diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is frequently treated with chemotherapy incorporating cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP), which induces remission in 80% to 95% of cases. However, not all dogs derive meaningful benefit from CHOP, and prognostic factors for dogs with DLBCL are poorly defined. Serum thymidine kinase 1 (TK1) activity, a marker of tumour cell proliferation, has shown promising initial results as a prognostic biomarker in dogs with multicentric lymphomas. The purpose of this study was to determine if baseline serum TK1 activity is associated with clinical outcome in dogs with CHOP-treated DLBCL. Baseline serum TK1 activity was measured in banked sera from 98 dogs with CHOP-treated DLBCL using a commercially available ELISA kit. Data on other potential prognostic factors were abstracted retrospectively from electronic medical records. Multivariable statistical methods were used to identify associations between TK1 and other potential prognostic factors with progression-free survival (PFS) and attainment of complete remission. TK1 activity at baseline was not associated with PFS (p = .299) or attainment of complete remission (p = .910) following CHOP chemotherapy. Of the other prognostic factors analysed, only purebred (vs. mixed breed) status (HR 8.81, 95% CI 1.68-46.30, p = .010), attainment of complete (vs. partial) remission (HR 0.09, 95% CI 0.02-0.49, p = .006), and baseline serum C-reactive protein concentration (HR 1.19, 95% CI 1.07-1.32, p = .001) were independently associated with PFS. Based on these findings, baseline serum TK1 activity does not appear to be a useful prognostic biomarker in dogs with CHOP-treated DLBCL.

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.

Greater baseline serum C-reactive protein concentrations are associated with reduced survival in dogs receiving cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy for primary nodal diffuse large B-cell lymphoma.

Prognostic factors for dogs with diffuse large B-cell lymphoma (DLBCL) are poorly characterized. Prior reports suggest that dogs with a systemic inflammatory response at the time of lymphoma diagnosis experience inferior survival times. However, no specific biomarkers of inflammation have been identified as prognostic indicators in dogs with DLBCL. Baseline C-reactive protein (CRP) concentrations were measured in banked sera from 91 dogs with chemotherapy-treated DLBCL using a commercially available laboratory assay. Associations between baseline serum CRP concentrations and other variables of potential prognostic significance with progression-free survival (PFS) were tested using Cox proportional hazards modeling. In the final multivariable model, only a complete (rather than partial) remission to chemotherapy (Hazard ratio [HR] 0.02; 95% confidence intervals [CI] 0.01-0.07; P < 0.001) and serum CRP concentration > 1.0 mg/dL (HR, 1.73; 95% CI, 1.02-2.92; P = 0.042) were significantly associated with PFS. The median PFS for dogs with CRP concentration ≤ 1.0 mg/dL (within the test reference interval) was 315 days, while that for dogs with CRP concentration > 1.0 mg/dL was 232 days (P = 0.06). These results suggest that baseline serum CRP concentration is independently associated with progression-free survival in dogs with DLBCL, making it a potentially useful prognostic biomarker for dogs with this cancer.

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.

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.

Feline Pituitary Adenomas: Correlation of Histologic and Immunohistochemical Characteristics With Clinical Findings and Case Outcome.

Pituitary glands from 141 feline autopsy cases were reviewed histologically. Adenoma and hyperplasia were the most common lesions at 13 cases each. Pituitary adenoma was more likely than hyperplasia to be associated with clinical evidence of endocrinopathy or an intracranial mass (P < .001). A histochemical and immunohistochemical panel was applied to 44 autopsy- or hypophysectomy-derived pituitary adenomas in 43 cats from 2 diagnostic laboratories. Adenomas were differentiated from hyperplasia by the presence of disrupted reticulin fibers. One cat had a double (somatotroph and melanotroph) adenoma. Twenty somatotroph adenomas consisted of periodic acid-Schiff (PAS)-negative acidophils that expressed growth hormone; 16/20 had hypersomatotropism; 17/20 had diabetes mellitus. Eleven melanotroph adenomas consisted of PAS-positive basophils or chromophobes that expressed melanocyte-stimulating and adrenocorticotrophic hormones; 5/11 had hypercortisolism; 6/11 had diabetes mellitus. Eleven gonadotroph adenomas consisted of PAS-negative chromophobes that expressed follicle-stimulating and/or luteinizing hormones. Two thyrotroph adenomas consisted of PAS-negative basophils or chromophobes that expressed thyroid-stimulating hormone. Pituitary-dependent disease was not recognized in cats with gonadotroph or thyrotroph adenomas. The Ki-67 proliferation index in hypophysectomy specimens was lower in somatotroph than in melanotroph adenomas. Fourteen cats with hypophysectomy-treated somatotroph or melanotroph adenoma had an 899-day median survival time versus 173 days in 17 nonsurgical cases. After adjusting for age, adenoma size and type, hypophysectomized cats had an overall better survival time than nonsurgical cases (P = .029). The study results underscore the value of hypophysectomy and trophic hormone immunohistochemistry in the treatment and classification of feline pituitary adenomas.

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.

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.

Ultrasonographic and computed tomographic features of rice bodies in an Arabian horse with atlantal bursitis.

A 19-year-old castrated Arabian male horse presented for evaluation of a firm mass at the dorsal cervical region. Ultrasonography and computed tomography revealed multiple well defined fusiform structures within the atlantal bursa. Multiple glossy smooth, white to yellowish, flattened fusiform structures were removed surgically. These structures were composed of dense fibrin with some leukocytes and red blood cells. The imaging and histopathological features of these structures were similar to chronic 'rice bodies' reported in humans with bursitis or tenosynovitis. This is the first veterinary report describing the imaging features of 'rice bodies' in a horse with atlantal bursitis.

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.

Quantification and prognostic value of programmed cell death ligand-1 expression in dogs with diffuse large B-cell lymphoma.

OBJECTIVE To measure programmed cell death ligand-1 (PD-L1) mRNA expression in archived primary nodal diffuse large B-cell lymphoma (DLBCL) specimens of dogs and determine whether that expression was associated with progression-free survival time (PFST). SAMPLE Archived tumoral lymph node specimens from 42 dogs with DLBCL and lymph node specimens from 10 healthy dogs (controls). PROCEDURES Archived tumoral and control lymph node specimens underwent multiplex qPCR analysis with probes and primers against canine PD-L1 and glyceraldehyde 3-phosphate dehydrogenase (housekeeping gene) to determine PD-L1 mRNA expression. The 2-ΔΔCt method was used to calculate the fold change in PD-L1 expression in DLBCL specimens relative to that in control lymph nodes. Kaplan-Meier and Cox proportional hazard analyses were used to evaluate the association of various tumoral and clinical factors with PFST. RESULTS The fold change in PD-L1 mRNA expression in DLBCL specimens relative to control specimens ranged from 0.21 to 7.44. Twenty-one of 42 (50%) DLBCL specimens had a PD-L1-fold change > 1, which suggested PD-L1 was overexpressed in those specimens. Median PFST was 249 days for dogs with DLBCL. The PFST was not associated with PD-L1 mRNA expression but was associated with thrombocytopenia at the time of diagnosis (hazard ratio, 2.56; 95% confidence interval, 1.28 to 5.15). CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that tumoral PD-L1 mRNA expression varied among dogs with DLBCL and that PD-L1 MRNA was overexpressed in half the study population. Therefore, anti-PD-L1 therapies may be clinically beneficial for some dogs with DLBCL.

Naturally-occurring canine invasive urothelial carcinoma harbors luminal and basal transcriptional subtypes found in human muscle invasive bladder cancer.

There is growing evidence that molecular subtypes (e.g. luminal and basal subtypes) affect the prognosis and treatment response in patients with muscle invasive urinary bladder cancer (invasive urothelial carcinoma, iUC). Modeling these subtypes in pre-clinical animal studies is essential, but it is challenging to produce these subtypes, along with other critical host and tumor features, in experimentally-induced animal models. This study was conducted to determine if luminal and basal molecular subtypes are present in naturally-occurring canine iUC, a cancer that mimics the human condition in other key aspects. RNA sequencing was performed on 29 canine treatment naive iUC tissue samples and on four normal canine bladder mucosal samples. Data were aligned to CanFam 3.1, and differentially expressed genes were identified. Unsupervised hierarchical clustering of these genes revealed two distinct groups (n = 13, n = 16). When genes that distinguish basal and luminal subtypes in human cancer (n = 2015) were used to probe genes differentially expressed between normal canine bladder and iUC, 829 enriched signature genes were identified. Unsupervised hierarchical clustering of these genes revealed two distinct groups comprised of 18 luminal subtype tumors and 11 basal subtype tumors. The enriched genes included MMP9, SERPINE2, CAV1, KRT14, and RASA3 in basal tumors, and PPARG, LY6E, CTSE, CDK3, and TBX2 in luminal tumors. In supervised clustering, additional genes of importance in human iUC were identified in canine iUC associated with claudin-low and infiltrated tumors. A smaller panel of genes (n = 60) was identified that distinguished canine luminal and basal iUC with overall 93.1% accuracy. Immune signature patterns similar to those in human iUC were also identified with the greatest enrichment of immune genes being in the basal subtype tumors. These findings provide additional compelling evidence that naturally-occurring canine iUC is a highly relevant and much needed model of human iUC for translational research.

Immunohistochemical Evaluation of Canine Pituitary Adenomas Obtained by Transsphenoidal Hypophysectomy.

Hypophysectomy specimens from 16 dogs with pituitary adenoma were evaluated with periodic acid-Schiff (PAS), reticulin, and immunohistochemistry for adrenocorticotrophic hormone (ACTH), melanocyte stimulating hormone (MSH), growth hormone (GH), and Ki-67. The reticulin network was obliterated in all adenomas. One adenoma expressed ACTH and GH. Eight corticotroph adenomas were basophilic to chromophobic, and PAS- and ACTH-positive. Seven melanotroph adenomas were distinguished from corticotroph adenomas by expression of MSH. Pituitary-dependent hypercortisolism was diagnosed in 5 of 8 dogs with corticotroph and 4 of 7 with melanotroph adenoma. Pituitary height/brain area (P/B) ratio was elevated in all dogs. Previous canine hypophysectomy studies suggested that melanotroph adenomas were larger and carried a worse prognosis than corticotroph adenomas; however, in this study, corticotroph adenomas in comparison to melanotroph adenomas were larger (median P/B ratio: 1.06 versus 0.76), more proliferative (median Ki-67 index: 9.47% versus 1.99%), and associated with shorter survival (median: 300 versus 793 days). Recommended immunohistochemistry for PAS-positive pituitary adenomas includes ACTH and MSH to distinguish corticotrophs from melanotrophs and Ki-67 for proliferation index.

Histopathologic Findings in Canine Pituitary Glands.

To optimize the histologic evaluation of hypophysectomy specimens, sections of 207 canine pituitary glands (196 postmortem, 11 hypophysectomy specimens) were reviewed. Adenohypophyseal proliferation was the most common (n = 79) lesion. Proliferative lesions were sparsely to densely granulated; the granules were usually basophilic to chromophobic and periodic acid-Schiff-positive. Adenohypophyseal proliferation was classified as hyperplasia (n = 40) if ≤2 mm diameter with intact reticulin network, as microadenoma (n = 22) for 1-5 mm homogeneous nodules with lost reticulin network, or as macroadenoma (n = 17) for larger tumors. Craniopharyngeal duct cysts were common incidental lesions and the only lesion in 15 dogs. Uncommon diagnoses included lymphoma (n = 4), hemorrhagic necrosis (n = 4), metastatic carcinoma (n = 3), hypophysitis (n = 3), ependymoma (n = 2), craniopharyngioma (n = 2), and 1 case each of metastatic melanoma, pituicytoma, gliomatosis, germ cell tumor, meningioma, and atrophy. The pituitary histologic diagnosis was associated with hyperadrenocorticism (HAC; P < .001) and adrenocortical histologic diagnosis ( P = .025). Both HAC and adrenocortical hyperplasia showed a positive trend with the degree of adenohypophyseal proliferation. The association of adrenocortical hyperplasia with HAC was not significant ( P = .077). Dogs with adenohypophyseal proliferations were older than dogs with normal pituitary glands ( P < .05). Brachycephalic breeds were overrepresented among dogs with pituitary macroadenoma or craniopharyngeal duct cysts, but the association was not statistically significant ( P = .076). Adenohypophyseal hyperplasia was more common than adenoma among postmortem specimens, but was unexpected in >80% of cases. Pituitary macroadenoma was the most common diagnosis in hypophysectomy specimens.

Immunohistochemical Expression of CD31 (PECAM-1) in Nonendothelial Tumors of Dogs.

CD31 immunoreactivity has been reported in human nonendothelial tumors of both epithelial and mesenchymal origin. This study examined CD31 immunoreactivity of 347 formalin-fixed, paraffin-embedded normal, nonneoplastic, and neoplastic canine tissues. CD31 expression was considered positive if at least 10% of the cell population had membranous reactivity. Labeling with the CD31 antibody (clone JC/70A) was observed in 16 samples of normal organs (liver, kidney, lymph node), 6 of 6 specimens of hepatic nodular hyperplasia, 3 of 3 hepatic regenerative nodules, 1 of 4 anal sac carcinomas, 6 of 6 hemangiosarcomas, 18 of 20 hepatocellular carcinomas, 1 of 6 mammary carcinomas, 3 of 5 plasmacytomas, 18 of 53 renal cell carcinomas, and 1 of 5 cutaneous histiocytomas. CD31 expression did not correlate with case outcome in hepatocellular or renal cell carcinomas. Although distinguishing hemangiosarcoma from other neoplasms is typically straightforward, pathologists should be aware of potential cross-reactivity when relying on CD31 immunohistochemistry for diagnosis, particularly in small biopsy samples or when faced with an epithelioid or poorly differentiated vascular neoplasm.

Phase I/II clinical trial of the targeted chemotherapeutic drug, folate-tubulysin, in dogs with naturally-occurring invasive urothelial carcinoma.

PURPOSE: The purpose was to determine the safety and antitumor activity of a folate-tubulysin conjugate (EC0531) in a relevant preclinical animal model, dogs with naturally-occurring invasive urothelial carcinoma (iUC). Canine iUC is an aggressive cancer with high folate receptor (FR) expression similar to that in certain forms of human cancer. EXPERIMENTAL DESIGN: A 3+3 dose escalation study of EC0531 (starting dose 0.2 mg/kg given intravenously at two-week intervals) was performed in dogs with iUC expressing high levels of FRs (>50% positive tumor cells). Pharmacokinetic (PK) analysis was performed, and the maximum tolerated dose (MTD) was determined. The dose cohort at the MTD was expanded to determine antitumor activity. RESULTS: The MTD of EC0531 was 0.26 mg/kg every two weeks, with grade 3-4 neutropenia and gastrointestinal toxicity observed at higher doses. Treatment at the MTD was well tolerated. Clinical benefit was found in 20 of 28 dogs (71%), including three dogs with partial remission and 17 dogs with stable disease. Plasma EC0531 concentrations in the dogs far exceeded those required to inhibit proliferation of FR-expressing cell in vitro. Unlike human neutrophils, canine neutrophils were found to express FRs, which contributes to the neutropenia at higher doses of EC0531 in dogs. CONCLUSION: EC0531 was well tolerated and had good antitumor activity in dogs with iUC. It is likely that humans will tolerate higher, potentially more effective doses of folate-tubulysin without myelotoxicity because of the absence of FRs on human neutrophils. The results clearly justify the evaluation of folate-tubulysin in human clinical trials.