Diagnostic performance of the urinary canine calgranulins in dogs with lower urinary or urogenital tract carcinoma.

BACKGROUND: Onset of canine transitional cell carcinoma (TCC) and prostatic carcinoma (PCA) is usually insidious with dogs presenting at an advanced stage of the disease. A biomarker that can facilitate early detection of TCC/PCA and improve patient survival would be useful. S100A8/A9 (calgranulin A/B or calprotectin) and S100A12 (calgranulin C) are expressed by cells of the innate immune system and are associated with several inflammatory disorders. S100A8/A9 is also expressed by epithelial cells after malignant transformation and is involved in the regulation of cell proliferation and metastasis. S100A8/A9 is up-regulated in human PCA and TCC, whereas the results for S100A12 have been ambiguous. Also, the urine S100A8/A9-to-S100A12 ratio (uCalR) may have potential as a marker for canine TCC/PCA. Aim of the study was to evaluate the diagnostic accuracy of the urinary S100/calgranulins to detect TCC/PCA in dogs by using data and urine samples from 164 dogs with TCC/PCA, non-neoplastic urinary tract disease, other neoplasms, or urinary tract infections, and 75 healthy controls (nested case-control study). Urine S100A8/A9 and S100A12 (measured by species-specific radioimmunoassays and normalized against urine specific gravity [S100A8/A9USG; S100A12USG], urine creatinine concentration, and urine protein concentration and the uCalR were compared among the groups of dogs. RESULTS: S100A8/A9USG had the highest sensitivity (96%) and specificity (66%) to detect TCC/PCA, with specificity reaching 75% after excluding dogs with a urinary tract infection. The uCalR best distinguished dogs with TCC/PCA from dogs with a urinary tract infection (sensitivity: 91%, specificity: 60%). Using a S100A8/A9USG ≥ 109.9 to screen dogs ≥6 years of age for TCC/PCA yielded a negative predictive value of 100%. CONCLUSIONS: S100A8/A9USG and uCalR may have utility for diagnosing TCC/PCA in dogs, and S100A8/A9USG may be a good screening test for canine TCC/PCA.

TOLERABILITY AND TUMOR RESPONSE OF A NOVEL LOW-DOSE PALLIATIVE RADIATION THERAPY PROTOCOL IN DOGS WITH TRANSITIONAL CELL CARCINOMA OF THE BLADDER AND URETHRA.

Previously reported radiation protocols for transitional cell carcinoma of the canine lower urinary tract have been ineffective or associated with increased side effects. Objectives of this retrospective, cross-sectional study were to describe safety of and tumor responses for a novel palliative radiation protocol for transitional cell carcinoma in dogs. Included dogs had cytologically or histologically confirmed transitional cell carcinoma of the bladder or urethra, and were treated with 10 once-daily fractions (Monday-Friday) of 2.7 Gy. Thirteen dogs were sampled, with six treated using radiation as first-line (induction) therapy and seven treated using radiation as rescue therapy after failing previous chemotherapy. Within 6 weeks of radiation, 7.6% (1/13) dogs had a complete response, 53.8% (7/13) partial response, 38.5% (5/13) stable disease, and none had progressive disease. Three patients presenting with urethral obstruction had spontaneous micturition restored during the treatment protocol. A single patient with unilateral ureteral obstruction was patent at recheck examination. Median survival time from time of initial diagnosis was 179 days. Median survival time from start of radiation was 150 days. Acute radiation side effects occurred in 31% (4/13) patients and were classified as grade 1 or 2. No significant late side radiation side effects were reported. No variables examined were identified as prognostic factors. Findings indicated that the reported radiation protocol was safe in this sample of dogs with bladder and urethral transitional cell carcinoma. Future prospective studies are needed to determine utility of this treatment as a rescue therapy in patients with complete urinary tract obstruction.

Evidence for a genetic basis of urogenital carcinoma in the wild California sea lion.

Although neoplasia is a major cause of mortality in humans and domestic animals, it has rarely been described in wildlife species. One of the few examples is a highly prevalent urogenital carcinoma in California sea lions (CSLs). Although the aetiology of this carcinoma is clearly multifactorial, inbreeding depression, as estimated using levels of microsatellite multilocus heterozygosity, is identified as predictive for this neoplasia. On further analysis, this relationship appears to be largely driven by one marker, suggesting that a single locus might be associated with the occurrence of this disease in CSLs. In a case-control study, carcinoma was significantly associated with homozygosity at the Pv11 microsatellite locus. Pv11 was mapped to intron 9 of the heparanase 2 gene (HPSE2) locus, a very large gene encoding heparanase 2, which in humans is associated with multiple carcinomas. Correspondingly, immunohistochemical labelling in tissues was present in carcinoma cases within a single homozygous Pv11 genotype. To our knowledge, this is the first report of an individual locus being associated with cancer in any wildlife species. This adds emphasis to the study of HPSE2 in other species, including humans and will guide future studies on this sentinel species that shares much of its diet and environment with humans.

Cancer selection.

Cancers are often thought to be selectively neutral. This is because most of the individuals that they kill are post-reproductive. Some cancers, however, kill the young and so select for anticancer adaptations that reduce the chance of death. These adaptations could reduce the somatic mutation rate or the selective value of a mutant clone of cells, or increase the number of stages required for neoplasia. New theory predicts that cancer selection--selection to prevent or postpone deaths due to cancer--should be especially important as animals evolve new morphologies or larger, longer-lived bodies, and might account for some of the differences in the causes of cancer between mice and men.

Tumor morphology and immunohistochemical expression of estrogen receptor, progesterone receptor, p53, and Ki67 in urogenital carcinomas of California sea lions (Zalophus californianus).

Metastatic carcinoma of urogenital origin is a common cause of mortality in free-ranging California sea lions (Zalophus californianus). The etiology of this cancer is likely multifactorial, with viral infection, genetic factors, and exposure to environmental organochlorine contaminants possible contributing factors. In this study, expression of estrogen receptor alpha (ER alpha), progesterone receptor (PR), p53, and Ki67 were evaluated by immunohistochemistry in 12 sea lions with metastatic carcinoma, genital epithelial dysplasia, and intraepithelial neoplasia; 4 with genital epithelial dysplasia and intraepithelial neoplasia without metastases; and 6 control animals. Dysplastic and neoplastic lesions were identified in multiple areas of the cervix, vagina, penis, prepuce, and urethra in affected animals, suggesting multicentric development. Lesions were graded according to degree of epithelial dysplasia and infiltration and lesions of different grades were evaluated separately. Estrogen receptor expression was lower in intraepithelial lesions compared with normal genital epithelium, and expression in metastatic lesions was completely absent. There was progesterone receptor expression in neoplastic cells in intraepithelial lesions of all grades and in metastases, with no significant difference between lesion grades or between control and affected epithelium. Ki67 index and p53 expression increased with lesion grade and were higher in lesions than normal epithelium. Metastatic tumors exhibited highly variable morphology; however, proliferation index, ER alpha, PR, and p53 expression were similar in tumors with different patterns of growth. These results suggest that endogenous hormones, environmental contaminants that interact with steroid hormone receptors, and alterations in p53 may play a role in urogenital carcinogenesis in California sea lions.

Transcriptional Profiles of California Sea Lion Peripheral NK and CD+8 T Cells Reflect Ecological Regionalization and Infection by Oncogenic Viruses.

The California sea lion is one of the few wild mammals prone to develop cancer, particularly urogenital carcinoma (UGC), whose prevalence is currently estimated at 25% of dead adult sea lions stranded along the California coastline. Genetic factors, viruses and organochlorines have been identified as factors that increase the risk of occurrence of this pathology. Given that no cases of UGC have as yet been reported for the species along its distribution in Mexican waters, the potential relevance of contaminants for the development of urogenital carcinoma is highlighted even more as blubber levels of organochlorines are more than two orders of magnitude lower in the Gulf of California and Mexican Pacific than in California. In vitro studies have shown that organochlorines can modulate anti-viral and tumor-surveillance activities of NK and cytotoxic T-cells of marine mammals, but little is known about the activity of these effectors in live, free-living sea lions. Here, we examine leukocyte transcriptional profiles of free-ranging adult California sea lions for eight genes (Eomes, Granzyme B, Perforin, Ly49, STAT1, Tbx21, GATA3, and FoxP3) selected for their key role in anti-viral and tumor-surveillance, and investigate patterns of transcription that could be indicative of differences in ecological variables and exposure to two oncogenic viruses: sea lion type one gammaherpesvirus (OtHV-1) and sea lion papillomavirus type 1 (ZcPV-1) and systemic inflammation. We observed regional differences in the expression of genes related to Th1 responses and immune modulation, and detected clear patterns of differential regulation of gene expression in sea lions infected by genital papillomavirus compared to those infected by genital gammaherpesvirus or for simultaneous infections, similar to what is known about herpesvirus and papillomavirus infections in humans. Our study is a first approach to profile the transcriptional patterns of key immune effectors of free-ranging California sea lions and their association with ecological regions and oncogenic viruses. The observed results add insight to our understanding of immune competence of marine mammals, and may help elucidate the marked difference in the number of cases of urogenital carcinoma in sea lions from US waters and other areas of their distribution.

Prevalence of Urogenital Carcinoma in Stranded California Sea Lions ( Zalophus californianus) from 2005-15.

Urogenital carcinoma is common in wild California sea lions ( Zalophus californianus) along the west coast of the US. From 1979 to 1994, this cancer was observed in 18% (66/370) of necropsied subadult and adult sea lions at The Marine Mammal Center in Sausalito, California. A retrospective review of records from 1 January 2005 to 31 December 2015 was performed to characterize prevalence and characteristics of cancer over this decade. Fourteen percent (263/1917) of necropsied sea lions had cancer, of which 90% (237/263) were urogenital carcinoma. The prevalence of urogenital carcinoma was significantly higher in adults compared to juveniles and subadults. Advanced-stage disease with metastases was identified histologically in 78% (182/232) of cases and was the cause of death in 95% (172/182) of these cases. Metastases were most common in lung and lymph nodes, and hydronephrosis, secondary to ureter obstruction by metastases, was identified in 62% (114/185) of animals with advanced disease. No significant temporal change in prevalence was detected over the decade, and this highly aggressive, fatal cancer remains common in stranded California sea lions.

Aerobic bacterial flora of the vagina and prepuce of California sea lions (Zalophus californianus) and investigation of associations with urogenital carcinoma.

To investigate the association between genital bacterial infection and urogenital carcinoma in California sea lions (Zalophus californianus), vaginal and preputial swabs for bacterial isolation were taken from 148 free-ranging and 51 stranded California sea lions including 16 animals with urogenital carcinoma. Cytological examination of vaginal or preputial smears showed a majority (65.5%, 57/87) of animals examined had mild or no inflammation. Aerobic bacteria were isolated from 116 (78.4%) wild sea lions and 100% of stranded animals. A total of 403 isolates were identified representing 51 unique bacterial species. The median number of isolates per animal increased with age in the wild group, but there was no difference in the number of isolates per animal between wild and stranded adults. The most common bacteria isolated from the wild sea lions were Psychrobacter phenylpyruvicus (39 isolates), non-hemolytic Streptococcus (35 isolates), Corynebacterium spp. (30 isolates), and Escherichia coli (20 isolates). More bacterial species were isolated from stranded animals than wild animals (33 versus 26) and there was significantly less growth of P. phenylpyruvicus, Corynebacterium spp., and Moraxella-like spp. in the stranded animals. Beta-hemolytic Streptococcus was the only bacterium significantly associated with urogenital carcinomas in California sea lions, but only in females.

Otarine Herpesvirus-1, not papillomavirus, is associated with endemic tumours in California sea lions (Zalophus californianus).

The purpose of this study was to determine if Otarine Herpesvirus-1 (OtHV-1) is associated with the presence of urogenital carcinomas in California sea lions. Polymerase chain reaction (PCR) analysis with primers specific for OtHV-1 was used to compare the prevalence of OtHV-1 infection in 15 sea lions affected by urogenital carcinoma with that of age-matched and juvenile tumour-free animals, and animals with tumours of non-urogenital origin. The herpesvirus was more prevalent (100%) and more widespread in the 15 animals with urogenital carcinoma than in 25 control animals, and was most often found in the urogenital tissue (vagina and prostate) and in the draining lymph nodes. Moreover, OtHV-1 DNA was not found in any juvenile animal, or in the neoplastic tissues of animals with non-urogenital tumours. Papillomavirus-specific PCR analysis of urogenital carcinoma tissues detected papillomavirus sequences in only one carcinomatous tissue. Further studies are needed to determine if OtHV-1 contributes to oncogenesis in the California sea lion; these data show, however, that OtHV-1 is associated with urogenital carcinomas, is preferentially present in urogenital tissues, and may be sexually transmitted. Papillomaviruses, which are known to contribute to urogenital tumours in other species, did not appear to be associated with the sea lion carcinomas.

Incidence patterns of spontaneous tumors in BN/Bi rats.

The occurrence of tumors in 236 female and 74 male BN/Bi rats that were allowed to live out their normal lifespans was described. For most neoplasms, the risk of a rat dying with a specific tumor increased with age. Some tumors, however, had a peak incidence; therefore, animals surviving these periods were at less risk than were their younger cohorts. The number dying with metastatic cancers was greatest in females over 30 months of age. Unexpectedly, males had a peak risk period at 25-30 months, so that males that died in older age groups had less risk of dying with a metastatic tumor. Lesions were often multiple, even in rats dying at a young age. Surprisingly, the percent dead with multiple tumors did not increase significantly with age. Some animals in every age group died without a tumor, and the percentage without neoplasms did not decrease, even in the older age groups.

Nature and rate of neoplasia found in captive wild mammals, birds, and reptiles at necropsy.

The nature and rate of neoplasia found at necropsy of captive wild animals of the Zoological Society of San Diego collection were studied. Neoplasia was present at necropsy in 2.75% of 3,127 mammals, 1.89% of 5,957 birds, and 2.19% of 1,233 reptiles. Neoplasms were not detected during 198 necropsies of amphibians. Gross and histologic examinations were performed on the 92 mammalian, 111 avian, and 28 reptilian neoplasms. The lesions were diagnosed. The findings findings included a high frequency of lymphosarcomas in birds and reptiles, multiple lung adenomas in mammals, multiple endocrine tumors in 2 European mouflons (Ovis musimon), and proliferative lesions of the biliary and pancreatic ductal systems in several species.

Tumor-blocking and -inhibitory serum factors in the clinical course of canine venereal tumor.

An in vitro method for growing colonies of canine transplantable venereal tumor cells in a semisolid agar medium is described. Using autologous or pooled homologous normal dog sera as a feeder layer, 49.3 +/- 3.5 and 47.5 +/- 4.5 tumor colonies were obtained, respectively, when 2 X 10(4) tumor cells were plated. With this assay system, assessment of colony counts provided an accurate and rapid technique for monitoring serum factors in tumor-bearing dogs that inhibited colony formation, or blocked the inhibition of colony formation. In 52 dogs given tumor transplants, a direct correlation was demonstrated between serum activity tested in vitro and the in vivo growth characteristics of the tumor. Tumor cells preincubated with serum from dogs with active tumor growth consistently showed normal colony growth when cultured in agar containing colony-inhibitory sera (blocking effect). In vivo regression was characterized by loss of serum blocking and the development of serum colony-inhibitory activity in culture. In metastatic disease, only blocking activity could be identified, while persistent local invasive disease was characterized by low levels of both blocking and inhibitory serum activity. The sensitivity of this technique coupled with its in vivo predictive capabilities provides a model for monitoring serological responses to a naturally occurring neoplasm in a large, randomly bred animal.

A NOVEL GAMMAHERPESVIRUS IN NORTHERN FUR SEALS (CALLORHINUS URSINUS) IS CLOSELY RELATED TO THE CALIFORNIA SEA LION (ZALOPHUS CALIFORNIANUS) CARCINOMA-ASSOCIATED OTARINE HERPESVIRUS-1.

Otarine herpesvirus 1 (OtHV1) is strongly associated with California sea lion (CSL, Zalophus californianus) urogenital carcinoma, the most common cancer documented in marine mammals. In addition to CSL, OtHV1 has also been found in association with carcinoma in South American fur seals (Arctocephalus australis), demonstrating it can infect related species. Northern fur seals (NFS, Callorhinus ursinus) are sympatric with CSL, and copulation between these species has been observed; yet, there are no reports of urogenital carcinoma in NFS. We describe a new Otarine herpesvirus found in vaginal swabs from NFS, herein called OtHV4. Partial sequencing of the polymerase gene and the glycoprotein B gene revealed OtHV4 is closely related to OtHV1, with 95% homology in the region of polymerase sequenced, and phylogenetic analyses demonstrate that they are sister taxa. An OtHV4-specific hydrolysis probe quantitative PCR was developed and validated, and its use on vaginal swabs revealed 16 of 50 (32%) wild adult female NFS were positive for OtHV4. The identification of a virus highly similar to the carcinoma-associated OtHV1 in a sympatric species without carcinoma suggests that comparative genomics of OtHV1 and OtHV4 may identify candidate viral oncogenes.

Common cancer in a wild animal: the California sea lion (Zalophus californianus) as an emerging model for carcinogenesis.

Naturally occurring cancers in non-laboratory species have great potential in helping to decipher the often complex causes of neoplasia. Wild animal models could add substantially to our understanding of carcinogenesis, particularly of genetic and environmental interactions, but they are currently underutilized. Studying neoplasia in wild animals is difficult and especially challenging in marine mammals owing to their inaccessibility, lack of exposure history, and ethical, logistical and legal limits on experimentation. Despite this, California sea lions (Zalophus californianus) offer an opportunity to investigate risk factors for neoplasia development that have implications for terrestrial mammals and humans who share much of their environment and diet. A relatively accessible California sea lion population on the west coast of the USA has a high prevalence of urogenital carcinoma and is regularly sampled during veterinary care in wildlife rehabilitation centres. Collaborative studies have revealed that genotype, persistent organic pollutants and a herpesvirus are all associated with this cancer. This paper reviews research to date on the epidemiology and pathogenesis of urogenital carcinoma in this species, and presents the California sea lion as an important and currently underexploited wild animal model of carcinogenesis.

Randomized phase III trial of piroxicam in combination with mitoxantrone or carboplatin for first-line treatment of urogenital tract transitional cell carcinoma in dogs.

BACKGROUND: Reported response rates of transitional cell carcinoma (TCC) in dogs to piroxicam in combination with either mitoxantrone or carboplatin are similar; however, it is unknown whether either drug might provide superior duration of response. HYPOTHESIS/OBJECTIVES: To determine if the progression-free interval (PFI) of dogs with TCC treated with mitoxantrone and piroxicam was different than that of dogs receiving carboplatin and piroxicam. The hypothesis was that the efficacy of mitoxantrone is no different from carboplatin. ANIMALS: Fifty dogs with TCC without azotemia. METHODS: Prospective open-label phase III randomized study. Either mitoxantrone or carboplatin was administered every 3 weeks concurrently with piroxicam with restaging at 6-week intervals. Twenty-four dogs received carboplatin and 26 received mitoxantrone. RESULTS: Response was not different between groups (P = .56). None of the dogs showed complete response. In the mitoxantrone group, there were 2 (8%) partial responses (PR) and 18 (69%) dogs with stable disease (SD). In the carboplatin group, there were 3 PR (13%) and 13 (54%) dogs with SD. The PFI was not significantly different between groups (mitoxantrone = 106 days; carboplatin = 73.5 days; P = .62; hazard ratio 0.86; 95% confidence interval 0.47-1.56). Dogs with prostatic involvement experienced a shorter survival (median, 109 days) compared to dogs with urethral, trigonal, or apically located tumors; this difference was significant (median 300, 190, and 645 days, respectively; P = .005). CONCLUSIONS AND CLINICAL IMPORTANCE: This study did not detect a different in outcome in dogs with TCC treated with either mitoxantrone or carboplatin in combination with piroxicam.

Otarine herpesvirus-1: a novel gammaherpesvirus associated with urogenital carcinoma in California sea lions (Zalophus californianus).

The incidence of neoplasia in California sea lions (CSLs) is considered to be unusually high. Electron microscopic examination of some of these urogenital tumours revealed the presence of virions with typical herpes-like structure. While current attempts to cultivate this virus have not been successful, molecular studies employing DNA extracted from tumour tissues allowed both the classification of the agent and its identification in tumours and archived tissue samples. Two genome fragments generated using degenerate primers in PCR demonstrated highest identities with other mammalian gammaherpesviruses. Phylogenetic analysis showed that this novel virus, tentatively designated Otarine herpesvirus-1 (OtHV-1), grouped with members of the gammaherpesvirus subfamily and was distinct from PHV-2, a previously described pinniped gammaherpesvirus. An OtHV-1 specific PCR was established and used to investigate the presence of this virus in CSL tissues. PCR of DNA isolated from animals with these tumours, demonstrated that this virus was present in 100% (16/16) of tumours. Furthermore, DNA extracted from archived brain and muscle tissues was also positive in 29% (4/14) and 50% (7/14) of cases examined. This preliminary study provides evidence to support the hypothesis that the presence of this novel gammaherpesvirus is a factor in the development of urogenital carcinoma in CSLs.

Immunophenotype of the canine transmissible venereal tumour.

The canine transmissible venereal tumour is a naturally occurring contagious round-cell neoplasia which is primarily located in the mucous membrane of the external genitalia in dogs of either sex. In order to specify the controversial cytogenetic origin of this round-cell tumour, 14 cases of canine transmissible venereal tumour, formalin- or Bouin-fixed and paraffin-embedded, were subjected to extensive immunophenotypic analysis using reagents specific to a variety of cytoplasmic or surface antigens: lysozyme, ACM1 antigen, vimentin, neuron-specific enolase, glial fibrillary acidic protein, desmin, alpha smooth muscle actin, CD3, IgG, kappa and lambda light chains, and keratin. Lysozyme immunoreactivity was detected in all cases, ACM1 antigen in 11 of 14, neuron-specific enolase in 11 of 14, vimentin in 10 of 14, glial fibrillary acidic protein in 4 of 14 and desmin in 1 of 14. All the sections were negative to keratins, alpha smooth muscle actin and CD3, whereas in five cases, perivascular tumour cells contained Ig G, kappa and lambda light chains. The immunoreactivity to lysozyme and ACM1 antigen supports the hypothesis of a histiocytic immunophenotype for the canine transmissible venereal tumour.

Tumors in domestic animals examined during a ten-year period (1980 to 1989) at Miyazaki University.

During the ten years from 1980 to 1989 inclusive, a total of 468 (16.1%) tumors were found in 2,907 pathological samples from domestic animals, collected from Southern Kyushu, around Miyazaki City. In this study, canine tumors were collected most commonly (340/468 cases, 73%). In small animals, the skin and mammary gland were associated particularly with tumorigenetic hazards. In cattle, high incidence of leukemia and mesothelioma was found. These tendencies were almost the same as those we reported for the preceding ten-year period, although the number of cases of tumor was higher in this study.

Ultrastructural characteristics of canine transmissible venereal tumor at various stages of growth and regression.

Ultrastructural study of canine transmissible tumors in developing, mature, and regressing stages from 6 dogs revealed the presence of healthy and degenerating tumor cells in all neoplasms. The total number of neoplastic cells seemed to decrease, and the number of degenerating neoplastic cells seemed to increase in mature tumors. Macrophages, lymphocytes, and plasma cells infiltrated mature and regressing tumors. Alteratons in degenerating tumor cells consisted mainly of cytoplasmic changes in early stages and of both nuclear and cytoplasmic changes in cells in which degeneration was more advanced. Amounts of endoplasmic reticulum and ribosomes were decreased. There were swelling and vacuolation of mitochondria. Nuclear chromatin was clumped along the nuclear envelope, and the perinuclear space was widened. Degenerating cells often contained membrane-bound granules and clusters. Lamellar complexes were observed in tumor cells from 2 dogs. Virus particles were not seen.