Gastrointestinal colonization by Diutina (Candida) rugosa in a 6-year-old Siberian Husky.

A 6-year-old 21.5 kg castrated male Siberian Husky was presented for acute onset of lethargy, vomiting, hemorrhagic diarrhea, and inappetence. Physical examination revealed marked discomfort upon abdominal palpation and 5%-7% dehydration. The CBC and biochemical profile revealed changes consistent with mild to moderate inflammation, dehydration, and gastrointestinal (GI) disease. Despite aggressive gastrointestinal support, anorexia persisted, and an upper GI endoscopy was performed in conjunction with esophagostomy tube placement. Endoscopy revealed abnormal gastric mucosa characterized by moderately well-demarcated areas of blue-black discoloration. Impression smears of a gastric biopsy revealed abundant extracellular yeasts with morphology most consistent with Candida spp. and frequent extracellular cocci. Similar yeast and bacteria, in lower numbers, were observed on cytologic analysis of a direct smear of the rectal mucosa. A rectal swab submitted for fungal culture yielded pure growth of fungal yeasts identified as Diutina (formerly Candida) rugosa by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. The dog's clinical signs improved with fluconazole, and he was discharged. Follow-up fungal culture of a rectal swab showed no growth of D. rugosa. To the authors' knowledge, this is the first case report that describes the clinical, hematologic, cytologic, and gross findings of enteric colonization by D. rugosa in a dog.

Host-derived growth factors drive ERK phosphorylation and MCL1 expression to promote osteosarcoma cell survival during metastatic lung colonization.

PURPOSE: For patients with osteosarcoma, disease-related mortality most often results from lung metastasis-a phenomenon shared with many solid tumors. While established metastatic lesions behave aggressively, very few of the tumor cells that reach the lung will survive. By identifying mechanisms that facilitate survival of disseminated tumor cells, we can develop therapeutic strategies that prevent and treat metastasis. METHODS: We analyzed single cell RNA-sequencing (scRNAseq) data from murine metastasis-bearing lungs to interrogate changes in both host and tumor cells during colonization. We used these data to elucidate pathways that become activated in cells that survive dissemination and identify candidate host-derived signals that drive activation. We validated these findings through live cell reporter systems, immunocytochemistry, and fluorescent immunohistochemistry. We then validated the functional relevance of key candidates using pharmacologic inhibition in models of metastatic osteosarcoma. RESULTS: Expression patterns suggest that the MAPK pathway is significantly elevated in early and established metastases. MAPK activity correlates with expression of anti-apoptotic genes, especially MCL1. Niche cells produce growth factors that increase ERK phosphorylation and MCL1 expression in tumor cells. Both early and established metastases are vulnerable to MCL1 inhibition, but not MEK inhibition in vivo. Combining MCL1 inhibition with chemotherapy both prevented colonization and eliminated established metastases in murine models of osteosarcoma. CONCLUSION: Niche-derived growth factors drive MAPK activity and MCL1 expression in osteosarcoma, promoting metastatic colonization. Although later metastases produce less MCL1, they remain dependent on it. MCL1 is a promising target for clinical trials in both human and canine patients.

Osteosarcoma tumors maintain intra-tumoral transcriptional heterogeneity during bone and lung colonization.

BACKGROUND: Tumors are complex tissues containing collections of phenotypically diverse malignant and nonmalignant cells. We know little of the mechanisms that govern heterogeneity of tumor cells nor of the role heterogeneity plays in overcoming stresses, such as adaptation to different microenvironments. Osteosarcoma is an ideal model for studying these mechanisms-it exhibits widespread inter- and intra-tumoral heterogeneity, predictable patterns of metastasis, and a lack of clear targetable driver mutations. Understanding the processes that facilitate adaptation to primary and metastatic microenvironments could inform the development of therapeutic targeting strategies. RESULTS: We investigated single-cell RNA-sequencing profiles of 47,977 cells obtained from cell line and patient-derived xenograft models as cells adapted to growth within primary bone and metastatic lung environments. Tumor cells maintained phenotypic heterogeneity as they responded to the selective pressures imposed during bone and lung colonization. Heterogenous subsets of cells defined by distinct transcriptional profiles were maintained within bone- and lung-colonizing tumors, despite high-level selection. One prominent heterogenous feature involving glucose metabolism was clearly validated using immunofluorescence staining. Finally, using concurrent lineage tracing and single-cell transcriptomics, we found that lung colonization enriches for multiple clones with distinct transcriptional profiles that are preserved across cellular generations. CONCLUSIONS: Response to environmental stressors occurs through complex and dynamic phenotypic adaptations. Heterogeneity is maintained, even in conditions that enforce clonal selection. These findings likely reflect the influences of developmental processes promoting diversification of tumor cell subpopulations, which are retained, even in the face of selective pressures.

Comparison of pathologist review protocols for cytologic detection of prostatic and urothelial carcinomas in canines: A bi-institutional retrospective study of 298 cases.

Urothelial carcinoma, also known as transitional cell carcinoma, is the most common primary bladder tumour in dogs, and can also involve the prostate gland. Cytology is a common diagnostic tool utilized for dogs with bladder or prostate gland lesions. The objectives of this retrospective study were to compare the sensitivity and specificity of cytologic evaluation for urothelial or prostatic carcinoma between two institutions with different cytology review protocols as well as determine if certain collection methods resulted in higher cytologic accuracy. A total of 298 cases met inclusion criteria. The overall sensitivity and specificity for institution 1 were 91.8% and 50%, respectively, compared to 31.1% and 97.4%, respectively, for institution 2. When the urine sample review protocol at institution 2 was matched to that of institution 1, sensitivity and specificity were more similar to institution 1 (71.2% and 88.9%, respectively). Our results show that the sensitivity and specificity of cytology are affected by screening and review protocols implemented by different institutions. The data also demonstrate that sensitivity and specificity vary by collection method. Diagnostic catheterization had the highest performance: of the 11 cases between two institutions, it had 100% sensitivity and specificity. In contrast, examination of urine sediment not collected via diagnostic catheterization had low sensitivity and specificity that varied greatly by institution. In summary, cytologic interpretation should be undertaken with consideration given to both processing and collection protocols.

Ectodomain shedding by ADAM17 (a disintegrin and metalloproteinase 17) in canine neutrophils.

ADAM17 is a transmembrane protease expressed by most cells in humans and mice that cleaves cell surface substrates primarily in a cis manner, a process referred to as ectodomain shedding. ADAM17 has numerous substrates and plays a broad role in various physiological processes, including as a key regulator of inflammation. At this time, little is known about ADAM17 expression and function in dogs. A well-established ADAM17 substrate is the leukocyte adhesion protein CD62L (L-selectin). We show that a selective inhibitor of ADAM17, but not an inhibitor of its most closely related family member ADAM10, blocks CD62L shedding upon canine neutrophil activation. We also tested several anti-human ADAM17 monoclonal antibodies (mAbs) for staining canine neutrophils. Although most did not recognize canine neutrophils, the mAbs MEDI3622 and D1(A12) did. They also blocked the downregulation of CD62L upon neutrophil activation. MEDI3622 is a human IgG antibody and we found that a canine chimeric version of this mAb also blocked CD62L shedding by canine leukocytes. Taken together, our findings provide the first direct evidence of ADAM17 expression and sheddase activity in dogs, establishing a potential therapeutic target for various inflammatory disorders.

Evaluation of the diagnostic utility of cytologic examination of renal fine-needle aspirates from dogs and the use of ultrasonographic features to inform cytologic diagnosis.

OBJECTIVE To describe cytologic characteristics of renal fine-needle aspirate (FNA) samples from dogs, evaluate proportions of cytologic specimens deemed adequate for interpretation (diagnostic yield), assess diagnostic utility of cytologic examination for neoplastic and nonneoplastic diseases, and characterize ultrasonographic features of evaluated kidneys to determine whether the imaging characteristics could be used to inform cytologic interpretations. DESIGN Retrospective, observational study. SAMPLE 102 cytologic specimens and 97 ultrasonographic studies from 100 dogs. PROCEDURES Medical records were reviewed to identify dogs that underwent ultrasound-guided renal FNA. Slides were categorized as adequate or inadequate for interpretation; adequate slides were used for retrospective cytologic diagnosis. Sensitivity, specificity, and predictive values of cytologic examination for detection of neoplastic and nonneoplastic conditions were calculated by comparison with histologic or lymphoid cell clonality assay results. Ultrasonographic characteristics of neoplastic and nonneoplastic renal lesions were described. RESULTS 74 of 102 (72%) specimens had slides adequate for interpretation; 26 were included in the diagnostic accuracy analysis. Sensitivity of cytologic examination was 78% and 50% for detection of neoplastic and nonneoplastic conditions, respectively, with specificities of 50% and 77%, respectively; sensitivity for detection of lymphoma was 100%. Ultrasonographic appearance of kidneys with confirmed neoplasia varied; masses were most commonly found in kidneys with carcinoma (5/5), lymphoma (5/7), or other neoplasia (3/4) and absent in kidneys with nonneoplastic conditions (n = 5). CONCLUSIONS AND CLINICAL RELEVANCE Renal FNA specimens were adequate for interpretation at rates comparable with those reported for other organs and were considered clinically useful for diagnosis of neoplasia. Imaging characteristics may potentially aid differentiation between neoplastic and nonneoplastic lesions; however, further investigation is needed.

Diagnostic utility of renal fine-needle aspirate cytology and ultrasound in the cat.

Objectives The primary objective of this study was to retrospectively assess the diagnostic utility of feline renal fine-needle aspiration cytology by assessing diagnostic yield, cytologic characteristics and diagnostic accuracy. The secondary objective was to characterize ultrasonographic features of sampled kidneys to determine if they influenced diagnostic yield. Methods Slides, images and patient data were collected from the University of Minnesota Veterinary Medical Center database. Slides were designated as diagnostic or non-diagnostic. Non-diagnostic slides were used in calculating diagnostic yield and excluded from other analysis. Slides were evaluated for cytologic characteristics and assigned a single primary diagnosis. Ultrasound still images were evaluated for descriptive characteristics and characteristics of specific lesions were described. Cases with confirmatory testing were used to determine diagnostic sensitivity, specificity and positive and negative predictive values for detecting neoplasia. Results Of 96 cytologic submissions available for review, diagnostic yield was 68%; 48% of samples were at least moderately cellular. Of 87 cases with ultrasound data, kidneys showing subcapsular renal infiltrate, diffuse renal enlargement without pelvic dilation and infiltrative/nodular change were more likely to yield diagnostic samples. Of 12 confirmed cases, cytology was 100% sensitive and specific for the detection of neoplasia (four round-cell tumors and two carcinomas). Three cases with non-neoplastic histologic diagnoses were considered cytologically normal, two incorrectly diagnosed the pathology present, and one correctly diagnosed the pathology. While some imaging characteristics were more commonly seen in neoplastic vs non-neoplastic lesions, the sample size was insufficient for definitive correlation. Conclusions and relevance This is the first major analysis of feline ultrasound-guided renal fine-needle aspiration cytology. This technique generates adequate samples for interpretation at rates comparable to other soft tissues and is most useful in the diagnosis of neoplasia. Some imaging characteristics are indicative of the likelihood of obtaining an adequate sample for cytologic interpretation.

Polymorphisms within the Telomerase Reverse Transcriptase gene (TERT) in four breeds of dogs selected for difference in lifespan and cancer susceptibility.

BACKGROUND: Enzymatic activity of Telomerase Reverse Transcriptase (TERT) is important in maintaining the telomere length and has been implicated in cancer and aging related pathology. Since cancer susceptibility as well as longevity of dogs vary between breeds, this study involved sequencing the entire TERT gene of Canis familiaris from DNA samples obtained from forty dogs, with ten dogs each of four breeds: Shih Tzu, Dachshund, Irish Wolfhound, and Newfoundland, each with different life expectancies and susceptibility to cancer. RESULTS: We compared the sequences of all forty individuals amongst one another and with the published sequence of canine TERT, and analyzed relationships between members of the same or different breeds. Two separate phylogenetic trees were generated and analyzed from these individuals. Polymorphisms were found most frequently in intronic regions of the gene, although exonic polymorphisms also were observed. In many locations genotypes were observed that were either homozygous for the reference sequence or heterozygous, but the variant homozygous genotype was not observed. CONCLUSIONS: We propose that these homozygous variants are likely to have adverse effects in dogs. It was also found that the polymorphisms did not segregate by breed. Because the four breeds chosen come from geographically and physiologically distinct backgrounds, it can be inferred that the polymorphic diversification of TERT preceded breed derivation.