Long-term safety of dietary salt: A 5-year ProspEctive rAndomized bliNded and controlled stUdy in healThy aged cats (PEANUT study).

BACKGROUND: High-salt diets promote urine dilution and decrease urolithiasis risk. OBJECTIVE: Prospectively evaluate the safety of chronic high dietary salt intake (randomized controlled trial). ANIMALS: Twenty research colony neutered, healthy aged cats (11.5 years [10.0-11.6], median [interquartile range]). METHODS: Healthy cats were randomized to control or high-salt dry diets (sodium: 1.02 ± 0.16 [mean, SD] and 3.26 ± 0.30 g/Mcal metabolizable energy [ME], respectively; chloride: 2.26 ± 0.33 and 5.71 ± 0.28 g/Mcal ME, respectively), fed for up to 60 months. Assessments included CBC, plasma biochemistry, urinalysis, glomerular filtration rate (GFR), blood pressure, renal and cardiac (conventional Doppler and 2-dimensional color tissue Doppler) imaging, annually. Cats that died or were euthanized underwent necropsy. Diet effects over time were evaluated with linear mixed models. RESULTS: Follow-up duration (median [Interquartile range]) was similar between the control (38.7 months [28.6-48.2]) and high-salt group (51.4 months [45.7-59.0]). Diet had no significant effect on changes in GFR, blood pressure, plasma creatinine concentration, end-diastolic left ventricular (LV) wall thicknesses, LV internal diameters, LV systolic function, left atrial size, or systolic and diastolic Doppler variables. One control cat developed hypertension. One high-salt group cat developed persistent azotemia. Serial plasma biochemistry and urine specific gravity suggested early chronic kidney disease in 4 nonazotemic cats (2 per group), consistent with necropsy findings. CONCLUSIONS AND CLINICAL IMPORTANCE: In healthy aged cats, a commercial veterinary diet containing 3.26 ± 0.30 g/Mcal ME sodium was safe with regard to renal and cardiac function for up to 5 years.

Evaluation of in vitro intrinsic radiosensitivity and characterization of five canine high-grade glioma cell lines.

Glioma is the most common primary brain tumor in dogs and predominantly affects brachycephalic breeds. Diagnosis relies on CT or MRI imaging, and the proposed treatments include surgical resection, chemotherapy, and radiotherapy depending on the tumor's location. Canine glioma from domestic dogs could be used as a more powerful model to study radiotherapy for human glioma than the murine model. Indeed, (i) contrary to mice, immunocompetent dogs develop spontaneous glioma, (ii) the canine brain structure is closer to human than mice, and (iii) domestic dogs are exposed to the same environmental factors than humans. Moreover, imaging techniques and radiation therapy used in human medicine can be applied to dogs, facilitating the direct transposition of results. The objective of this study is to fully characterize 5 canine glioma cell lines and to evaluate their intrinsic radiosensitivity. Canine cell lines present numerous analogies between the data obtained during this study on different glioma cell lines in dogs. Cell morphology is identical, such as doubling time, clonality test and karyotype. Immunohistochemical study of surface proteins, directly on cell lines and after stereotaxic injection in mice also reveals close similarity. Radiosensitivity profile of canine glial cells present high profile of radioresistance.

Micromolding-based encapsulation of mesenchymal stromal cells in alginate for intraarticular injection in osteoarthritis.

Osteoarthritis (OA) is an inflammatory joint disease that affects cartilage, subchondral bone, and joint tissues. Undifferentiated Mesenchymal Stromal Cells are a promising therapeutic option for OA due to their ability to release anti-inflammatory, immuno-modulatory, and pro-regenerative factors. They can be embedded in hydrogels to prevent their tissue engraftment and subsequent differentiation. In this study, human adipose stromal cells are successfully encapsulated in alginate microgels via a micromolding method. Microencapsulated cells retain their in vitro metabolic activity and bioactivity and can sense and respond to inflammatory stimuli, including synovial fluids from OA patients. After intra-articular injection in a rabbit model of post-traumatic OA, a single dose of microencapsulated human cells exhibit properties matching those of non-encapsulated cells. At 6 and 12 weeks post-injection, we evidenced a tendency toward a decreased OA severity, an increased expression of aggrecan, and a reduced expression of aggrecanase-generated catabolic neoepitope. Thus, these findings establish the feasibility, safety, and efficacy of injecting cells encapsulated in microgels, opening the door to a long-term follow-up in canine OA patients.

A Polymer Prodrug Strategy to Switch from Intravenous to Subcutaneous Cancer Therapy for Irritant/Vesicant Drugs.

Chemotherapy is almost exclusively administered via the intravenous (IV) route, which has serious limitations (e.g., patient discomfort, long hospital stays, need for trained staff, high cost, catheter failures, infections). Therefore, the development of effective and less costly chemotherapy that is more comfortable for the patient would revolutionize cancer therapy. While subcutaneous (SC) administration has the potential to meet these criteria, it is extremely restrictive as it cannot be applied to most anticancer drugs, such as irritant or vesicant ones, for local toxicity reasons. Herein, we report a facile, general, and scalable approach for the SC administration of anticancer drugs through the design of well-defined hydrophilic polymer prodrugs. This was applied to the anticancer drug paclitaxel (Ptx) as a worst-case scenario due to its high hydrophobicity and vesicant properties (two factors promoting necrosis at the injection site). After a preliminary screening of well-established polymers used in nanomedicine, polyacrylamide (PAAm) was chosen as a hydrophilic polymer owing to its greater physicochemical, pharmacokinetic, and tumor accumulation properties. A small library of Ptx-based polymer prodrugs was designed by adjusting the nature of the linker (ester, diglycolate, and carbonate) and then evaluated in terms of rheological/viscosity properties in aqueous solutions, drug release kinetics in PBS and in murine plasma, cytotoxicity on two different cancer cell lines, acute local and systemic toxicity, pharmacokinetics and biodistribution, and finally their anticancer efficacy. We demonstrated that Ptx-PAAm polymer prodrugs could be safely injected subcutaneously without inducing local toxicity while outperforming Taxol, the commercial formulation of Ptx, thus opening the door to the safe transposition from IV to SC chemotherapy.

Comparison of MRI T1, T2, and T2* mapping with histology for assessment of intervertebral disc degeneration in an ovine model.

An easy, reliable, and time-efficient standardized approach for assessing lumbar intervertebral disc (IVD) degeneration with relaxation times measurements in pre-clinical and clinical studies is lacking. This prospective study aims to determine the most appropriate method for lumbar IVD degeneration (IDD) assessment in sheep by comparing three quantitative MRI sequences (variable-flip-angle T1 mapping, and multi-echo T2 and T2* mapping), correlating them with Pfirrmann grading and histology. Strong intra- and interrater agreements were found for Nucleus pulposus (NP) regions-of-interest (ROI). T1, T2, and T2* mapping correlated with Pfirrmann grading and histological scoring (p < 0.05) except for the most ventral rectangular ROI on T2 maps. Correlations were excellent for all of the T1 ROIs and the T2* NP ROIs. Highly significant differences in T1 values were found between all Pfirrmann grades except between grades I/II and between grades III/IV. Significant differences were identified in the T2 and the T2* values between all grades except between grades I/III. T1, T2, and T2* relaxation times measurements of the NP are an accurate and time-efficient tool to assess lumbar IDD in sheep. Variable-flip-angle T1 mapping may be further considered as a valuable method to investigate IDD and to assess the efficacy of regenerative treatments in longitudinal studies.

Multiple follicular abnormalities in a 1-year old cat consistent with basaloid follicular hamartomas.

BACKGROUND: In humans, basaloid follicular hamartomas are benign follicular tumours, that can be solitary or multiple, in which case they show autosomal dominant inheritance. HYPOTHESIS/OBJECTIVES: This study describes clinical and histopathological findings observed in a young cat, which could be consistent with basaloid follicular hamartomas. CASE DESCRIPTION: Multiple follicular abnormalities, consistent with cutaneous diffuse basaloid follicular hamartomas, were observed in skin samples from a one-year old neutered domestic short hair cat. Clinical signs were diffuse symmetrical alopecia with exaggerated skin markings (ventral abdomen, thorax and medial aspects of the limbs) and intense follicular-centred thickening (face and feet). Microscopic lesions were characterised by multiple proliferative follicular abnormalities in all samples. The epidermis showed a very irregular surface with the follicles filled with variably pigmented keratin. The epithelial walls of the follicles had multiple small hyperplastic basaloid cells foci. In the superficial dermis under the epidermis and around the follicles, fibroblastic spindle-shaped mesenchymal cells with a homogeneous moderate density were present in the collagenous connective tissue. The interfollicular epidermis was also abnormal with multiple small proliferating trichoblastic foci originating from the basal layer. RNAscope testing for feline papillomavirus was negative. CONCLUSIONS AND CLINICAL RELEVANCE: This case report provides the first evidence of clinical and histopathological findings of multiple follicular abnormalities, consistent with cutaneous diffuse basaloid follicular hamartomas in a cat.

De multiples anomalies folliculaires, compatibles avec des hamartomes folliculaires basaloïdes diffus cutanés, ont été observées dans des échantillons de peau d'un chat domestique à poils courts castré âgé d'un an. Les signes cliniques étaient une alopécie diffuse symétrique avec des marques cutanées exagérées (abdomen ventral, thorax et face médiale des membres) et un épaississement folliculaire intense (face et pieds).

Múltiplas anormalidades foliculares, consistentes com hamartomas cutâneos foliculares basaloides difusos, foram observadas em amostras de pele de um gato doméstico de pelo curto castrado de um ano de idade. Os sinais clínicos foram alopecia simétrica difusa com marcações cutâneas exuberantes (abdômen, tórax e aspecto medial dos membros) e espessamento folicular central intenso (face e patas).

Se observaron múltiples anomalías foliculares, consistentes con hamartomas foliculares basaloides difusos cutáneos, en muestras de piel de un gato doméstico de pelo corto castrado de 1 año. Los signos clínicos fueron alopecia simétrica difusa con marcas cutáneas exageradas (abdomen ventral, tórax y cara medial de las extremidades) e intenso engrosamiento de la piel centrado en los folículos (cara y pies).

Canine Oral Melanoma Genomic and Transcriptomic Study Defines Two Molecular Subgroups with Different Therapeutical Targets.

Mucosal melanoma (MM) is a rare, aggressive clinical cancer. Despite recent advances in genetics and treatment, the prognosis of MM remains poor. Canine MM offers a relevant spontaneous and immunocompetent model to decipher the genetic bases and explore treatments for MM. We performed an integrative genomic and transcriptomic analysis of 32 canine MM samples, which identified two molecular subgroups with a different microenvironment and structural variant (SV) content. The overexpression of genes related to the microenvironment and T-cell response was associated with tumors harboring a lower content of SVs, whereas the overexpression of pigmentation-related pathways and oncogenes, such as TERT, was associated with a high SV burden. Using whole-genome sequencing, we showed that focal amplifications characterized complex chromosomal rearrangements targeting oncogenes, such as MDM2 or CDK4, and a recurrently amplified region on canine chromosome 30. We also demonstrated that the genes TRPM7, GABPB1, and SPPL2A, located in this CFA30 region, play a role in cell proliferation, and thus, may be considered as new candidate oncogenes for human MM. Our findings suggest the existence of two MM molecular subgroups that may benefit from dedicated therapies, such as immune checkpoint inhibitors or targeted therapies, for both human and veterinary medicine.

Evaluation of the Chemotherapy Drug Response Using Organotypic Cultures of Osteosarcoma Tumours from Mice Models and Canine Patients.

Improvements in the clinical outcome of osteosarcoma have plateaued in recent decades with poor translation between preclinical testing and clinical efficacy. Organotypic cultures retain key features of patient tumours, such as a myriad of cell types organized within an extracellular matrix, thereby presenting a more realistic and personalised screening of chemotherapeutic agents ex vivo. To test this concept for the first time in osteosarcoma, murine and canine osteosarcoma organotypic models were maintained for up to 21 days and in-depth analysis identified proportions of immune and stromal cells present at levels comparable to that reported in vivo in the literature. Cytotoxicity testing of a range of chemotherapeutic drugs (mafosfamide, cisplatin, methotrexate, etoposide, and doxorubicin) on murine organotypic culture ex vivo found limited response to treatment, with immune and stromal cells demonstrating enhanced survival over the global tumour cell population. Furthermore, significantly decreased sensitivity to a range of chemotherapeutics in 3D organotypic culture relative to 2D monolayer was observed, with subsequent investigation confirming reduced sensitivity in 3D than in 2D, even at equivalent levels of drug uptake. Finally, as proof of concept for the application of this model to personalised drug screening, chemotherapy testing with doxorubicin was performed on biopsies obtained from canine osteosarcoma patients. Together, this study highlights the importance of recapitulating the 3D tumour multicellular microenvironment to better predict drug response and provides evidence for the utility and possibilities of organotypic culture for enhanced preclinical selection and evaluation of chemotherapeutics targeting osteosarcoma.

Identification of common predisposing loci to hematopoietic cancers in four dog breeds.

Histiocytic sarcoma (HS) is a rare but aggressive cancer in both humans and dogs. The spontaneous canine model, which has clinical, epidemiological, and histological similarities with human HS and specific breed predispositions, provides a unique opportunity to unravel the genetic basis of this cancer. In this study, we aimed to identify germline risk factors associated with the development of HS in canine-predisposed breeds. We used a methodology that combined several genome-wide association studies in a multi-breed and multi-cancer approach as well as targeted next-generation sequencing, and imputation We combined several dog breeds (Bernese mountain dogs, Rottweilers, flat-coated retrievers, and golden retrievers), and three hematopoietic cancers (HS, lymphoma, and mast cell tumor). Results showed that we not only refined the previously identified HS risk CDKN2A locus, but also identified new loci on canine chromosomes 2, 5, 14, and 20. Capture and targeted sequencing of specific loci suggested the existence of regulatory variants in non-coding regions and methylation mechanisms linked to risk haplotypes, which lead to strong cancer predisposition in specific dog breeds. We also showed that these canine cancer predisposing loci appeared to be due to the additive effect of several risk haplotypes involved in other hematopoietic cancers such as lymphoma or mast cell tumors as well. This illustrates the pleiotropic nature of these canine cancer loci as observed in human oncology, thereby reinforcing the interest of predisposed dog breeds to study cancer initiation and progression.

Nonhypoalbuminemic Inflammatory Bowel Disease in Dogs as Disease Model.

BACKGROUND: The incidence of inflammatory bowel disease (IBD) is increasing worldwide, emphasizing the need of relevant models, as dogs spontaneously affected by IBD may be, for better knowledge of the disease's physiopathology. METHODS: We studied 22 client-owned dogs suffering from IBD without protein loss and 14 control dogs. Biopsies were obtained from the duodenum, ileum, and colon. Inflammatory grade was assessed by histopathology, immunohistochemistry, and chemokine analysis. The expression of Toll-like receptors (TLR) in mucosa was immunohistochemically evaluated. Antibody levels against bacterial ligands (lipopolysaccharide [LPS] and flagellin) were measured in sera using enzyme-linked immunoassay. RESULTS: Dogs with IBD showed low to severe clinical disease. Histopathologically, the gut of dogs with IBD did not exhibit significant alterations compared with controls except in the colon. The number of CD3+ T lymphocytes was decreased in the ileum and colon of dogs with IBD compared with controls, whereas the numbers of Foxp3+, CD20+, and CD204+ cells were similar in the 2 groups. Three chemokines, but no cytokines, were detected at the protein level in the mucosa, and the disease poorly affected their tissue concentrations. Dogs with IBD exhibited higher serum reactivity against LPS and flagellin than controls but similar immunoreactivity against the receptors TLR4 and TLR5. In addition, TLR2 and TLR9 showed similar expression patterns in both groups of dogs. CONCLUSIONS: Our data described dysregulated immune responses in dogs affected by IBD without protein loss. Despite fairly homogeneous dog cohorts, we were still faced with interindividual variability, and new studies with larger cohorts are needed to validate the dog as a model.

Circulating tumor DNA is detectable in canine histiocytic sarcoma, oral malignant melanoma, and multicentric lymphoma.

Circulating tumor DNA (ctDNA) has become an attractive biomarker in human oncology, and its use may be informative in canine cancer. Thus, we used droplet digital PCR or PCR for antigen receptor rearrangement, to explore tumor-specific point mutations, copy number alterations, and chromosomal rearrangements in the plasma of cancer-affected dogs. We detected ctDNA in 21/23 (91.3%) of histiocytic sarcoma (HS), 2/8 (25%) of oral melanoma, and 12/13 (92.3%) of lymphoma cases. The utility of ctDNA in diagnosing HS was explored in 133 dogs, including 49 with HS, and the screening of recurrent PTPN11 mutations in plasma had a specificity of 98.8% and a sensitivity between 42.8 and 77% according to the clinical presentation of HS. Sensitivity was greater in visceral forms and especially related to pulmonary location. Follow-up of four dogs by targeting lymphoma-specific antigen receptor rearrangement in plasma showed that minimal residual disease detection was concordant with clinical evaluation and treatment response. Thus, our study shows that ctDNA is detectable in the plasma of cancer-affected dogs and is a promising biomarker for diagnosis and clinical follow-up. ctDNA detection appears to be useful in comparative oncology research due to growing interest in the study of natural canine tumors and exploration of new therapies.

One-year conditional survival of dogs and cats with invasive mammary carcinomas: A concept inspired from human breast cancer.

Numerous studies have described the prognostic factors of canine and feline mammary carcinomas (MCs), that is, variables that predict patient survival after diagnosis. But how does survival estimation evolve in patients that escaped early death from their cancer? In human oncology, conditional survival (CS), the probability of surviving X further years when cancer patients have already survived Y years, is used to analyse cancer outcomes in a long-term perspective. In this cohort of 344 dogs and 342 cats with surgically removed stage I to III invasive MCs, with a minimal follow-up of 2 years, we calculated the 1-year CS, that is, the probability for patients that have survived 1 year, to survive or to die from cancer during the subsequent year. The 1-year conditional specific survival probabilities were 59% and 48% at diagnosis of invasive MC respectively in dogs and cats, and 80% and 52% in 1-year surviving dogs and cats respectively, suggesting that 1-year surviving dogs were relatively protected from cancer-related death, whereas feline MCs remained life-threatening cancers for longer periods of time. Among the most significant parameters associated with CS in surviving dogs and cats were the nodal stage and lymphovascular invasion, as well as patient age, cancer stage and margin status in surviving dogs. By comparison, tumour size and the histological grade did not significantly alter CS probabilities in surviving dogs and cats. Conditional survival may be considered a very interesting tool for veterinary practitioners to estimate the likely outcome of cancer survivors.

SPECT-CT Imaging of Dog Spontaneous Diffuse Large B-Cell Lymphoma Targeting CD22 for the Implementation of a Relevant Preclinical Model for Human.

Antibodies directed against CD22 have been used in radioimmunotherapy (RIT) clinical trials to treat patients with diffuse large B-cell lymphoma (DLBCL) with promising results. However, relevant preclinical models are needed to facilitate the evaluation and optimization of new protocols. Spontaneous DLBCL in dogs is a tumor model that may help accelerate the development of new methodologies and therapeutic strategies for RIT targeting CD22. Seven murine monoclonal antibodies specific for canine CD22 were produced by the hybridoma method and characterized. The antibodies' affinity and epitopic maps, their internalization capability and usefulness for diagnosis in immunohistochemistry were determined. Biodistribution and PET imaging on a mouse xenogeneic model of dog DLBCL was used to choose the most promising antibody for our purposes. PET-CT results confirmed biodistribution study observations and allowed tumor localization. The selected antibody, 10C6, was successfully used on a dog with spontaneous DLBCL for SPECT-CT imaging in the context of disease staging, validating its efficacy for diagnosis and the feasibility of future RIT assays. This first attempt at phenotypic imaging on dogs paves the way to implementing quantitative imaging methodologies that would be transposable to humans in a theranostic approach. Taking into account the feedback of existing human radioimmunotherapy clinical trials targeting CD22, animal trials are planned to investigate protocol improvements that are difficult to consider in humans due to ethical concerns.

PTPN11 mutations in canine and human disseminated histiocytic sarcoma.

In humans, histiocytic sarcoma (HS) is an aggressive cancer involving histiocytes. Its rarity and heterogeneity explain that treatment remains a challenge. Sharing high clinical and histopathological similarities with human HS, the canine HS is conversely frequent in specific breeds and thus constitutes a unique spontaneous model for human HS to decipher the genetic bases and to explore therapeutic options. We identified sequence alterations in the MAPK pathway in at least 63.9% (71/111) of HS cases with mutually exclusive BRAF (0.9%; 1/111), KRAS (7.2%; 8/111) and PTPN11 (56.75%; 63/111) mutations concentrated at hotspots common to human cancers. Recurrent PTPN11 mutations are associated to visceral disseminated HS subtype in dogs, the most aggressive clinical presentation. We then identified PTPN11 mutations in 3/19 (15.7%) human HS patients. Thus, we propose PTPN11 mutations as key events for a specific subset of human and canine HS: the visceral disseminated form. Finally, by testing drugs targeting the MAPK pathway in eight canine HS cell lines, we identified a better anti-proliferation activity of MEK inhibitors than PTPN11 inhibitors in canine HS neoplastic cells. In combination, these results illustrate the relevance of naturally affected dogs in deciphering genetic mechanisms and selecting efficient targeted therapies for such rare and aggressive cancers in humans.

Identification of an immune-suppressed subtype of feline triple-negative basal-like invasive mammary carcinomas, spontaneous models of breast cancer.

Feline invasive mammary carcinomas are characterized by their high clinical aggressiveness, rare expression of hormone receptors, and pathological resemblance to human breast cancer, especially triple-negative breast cancer (negative to estrogen receptor, progesterone receptor, and epidermal growth factor receptor type 2). Recent gene expression studies of triple-negative breast cancers have highlighted their heterogeneity and the importance of immune responses in their biology and prognostic assessment. Indeed, regulatory T cells may play a crucial role in producing an immune-suppressed microenvironment, notably in triple-negative breast cancers. Feline invasive mammary carcinomas arise spontaneously in immune-competent animals, in which we hypothesized that the immune tumor microenvironment also plays a role. The aims of this study were to determine the quantity and prognostic value of forkhead box protein P3-positive peritumoral and intratumoral regulatory T cells in feline invasive mammary carcinomas, and to identify an immune-suppressed subgroup of triple-negative basal-like feline invasive mammary carcinomas. One hundred and eighty female cats with feline invasive mammary carcinomas, treated by surgery only, with 2-year follow-up post-mastectomy, were included in this study. Forkhead box protein P3, estrogen receptor, progesterone receptor, Ki-67, epidermal growth factor receptor type 2, and cytokeratin 14 expression were assessed by automated immunohistochemistry. Peritumoral regulatory T cells were over 300 times more abundant than intratumoral regulatory T cells in feline invasive mammary carcinomas. Peritumoral and intratumoral regulatory T cells were associated with shorter disease-free interval and overall survival in both triple-negative (ER-, PR-, HER2-, N = 123 out of 180) and luminal (ER+ and/or PR+, N = 57) feline invasive mammary carcinomas. In feline triple-negative basal-like (CK14+) mammary carcinomas, a regulatory T-cell-enriched subgroup was associated with significantly poorer disease-free interval, overall survival, and cancer-specific survival than regulatory T-cell-poor triple-negative basal-like feline invasive mammary carcinomas. High regulatory T-cell numbers had strong and negative prognostic value in feline invasive mammary carcinomas, especially in the triple-negative basal-like subgroup, which might contain a "basal-like immune-suppressed" subtype, as described in triple-negative breast cancer. Cats with feline invasive mammary carcinomas may thus be interesting spontaneous animal models to investigate new strategies of cancer immunotherapy in an immune-suppressed tumor microenvironment.

Unfavorable Prognostic Effects of the Stem Cell Pluripotency Factor Sox2 in Feline Invasive Mammary Carcinomas.

Background: Sex-determining Region Y (SRY)-box transcription factor-2 (Sox2) belongs to the "Yamanaka's factors," necessary and sufficient to convert somatic cells into pluripotent stem cells. In breast cancers, Sox2 expression has been associated with poor prognosis, and resistance to therapy. The aims of this study were to determine the frequency of Sox2 positivity in feline invasive mammary carcinomas (FMCs), its relationships with other clinical-pathologic variables, and with patient outcomes. Materials and Methods: This study relies on a previously described retrospective cohort of 180 FMCs, diagnosed in female cats treated by mastectomy alone, with 2-year follow-up. Sox2 (clone SP76), Estrogen Receptor alpha (ER), Progesterone Receptor (PR), Ki-67, Human Epidermal growth factor Receptor 2 (HER2), Androgen Receptor (AR), Bcl-2, Forkhead box protein A1 (FOXA1), basal markers and FoxP3-positive regulatory T cells (Tregs) were detected by automated immunohistochemistry. Sox2 expression was quantitated as an index (percentage of neoplastic cells demonstrating a positive nuclear signal). The FMCs were considered Sox2-positive at threshold >42%. Results: Sox2 was not expressed in the normal mammary gland or in mammary hyperplasia without atypia, but was occasionally detected in atypical hyperplasia. In FMCs, the mean Sox2 index was 38 ± 30%, and 79/180 FMCs (44%) were Sox2-positive. Sox2 expression was associated with older age at diagnosis, lymphovascular invasion, high Ki-67 proliferation indexes, low PR and FOXA1 expression, and increased numbers of tumor-associated Tregs, but was not significantly associated with the clinical stage, histological types, and histological grade. By multivariate survival analysis, Sox2 was associated with poor cancer-specific survival (Hazard Ratio = 1.48, 95% confidence interval 1.04-2.11, p = 0.0292), independently of the pathologic tumor size, pathologic nodal stage, distant metastasis, and AR expression. A rare subgroup of FMCs characterized by an AR+Sox2-phenotype (19/180 cases, 11%) was associated with very favorable outcomes. Conclusion: Sox2 expression was associated with poor cancer-specific survival of female cats with invasive mammary carcinomas, as previously reported in human breast cancer, but was more commonly expressed in cats than reported in breast cancers. Sox2 showed complementarity with AR in FMC prognostication.

Prognostic value of somatic focal amplifications on chromosome 30 in canine oral melanoma.

Canine oral melanoma is the first malignancy of the oral cavity in dogs and is characterized by a local invasiveness and a high metastatic propensity. A better knowledge of genetic alterations is expected to improve management of this tumour. Copy number alterations are known characteristics of mucosal melanomas both in dogs and humans. The goal of this study was to explore the prognostic value of somatic focal amplifications on chromosomes (Canis Familiaris [CFA]) 10 and 30 in canine oral melanoma. The cohort included 73 dogs with oral melanoma confirmed by histology, removed surgically without adjuvant therapy and with a minimal follow-up of 6 months. Epidemiological, clinical and histological data were collected and quantitative-PCR were performed on formalin-fixed paraffin-embedded (FFPE) samples to identify specific focal amplifications. The 73 dogs included in the study had a median survival time of 220 days. Focal amplifications on CFA 10 and 30 were recurrent (49.3% and 50.7% of cases, respectively) and CFA 30 amplification was significantly associated with the amelanotic phenotype (P = .046) and high mitotic index (MI; P = .0039). CFA 30 amplification was also linked to poor prognosis (P = .0005). Other negative prognostic factors included gingiva location (P = .003), lymphadenomegaly (P = .026), tumour ulceration at diagnosis (P = .003), MI superior to 6 mitoses over 10 fields (P = .001) and amelanotic tumour (P = .029). In multivariate analyses using Cox proportional hazards regression, CFA 30 amplification (Hazard ratio [HR] = 2.08; P = .011), tumour location (HR = 2.20; P = .005) and histological pigmentation (HR = 1.87; P = .036) were significantly associated with shorter survival time. Focal amplification of CFA 30 is linked to an aggressive subset and constitutes a new prognostic factor.

Proposal for a Histological Staging System of Mammary Carcinomas in Dogs and Cats. Part 1: Canine Mammary Carcinomas.

Background: Staging of mammary carcinomas of dogs and cats is not only important for prognostic purposes, but also to guide therapy, in particular regarding adjuvant chemotherapy. The classical staging system relies on T, the clinical tumor size, N, the clinical nodal stage, and M, distant metastasis, evaluated by the clinician. However, a more precise and reliable staging system is applied to human stage I-III breast cancer, i.e., without distant metastasis, in which T is replaced by the pathologic tumor size (pT), and N is replaced by the pathologic nodal stage (pN), both evaluated by the pathologist. This staging system is strongly associated with patient outcomes, and is used to select treatment options. The purpose of this study was to design a histologic staging system for Canine Mammary Carcinomas (CMCs, part 1 of this article), and Feline Mammary Carcinomas (part 2), inspired from human oncology, and to assess its association with patient outcomes. Materials and Methods: This retrospective study included 433 female dogs with a surgically removed CMC. Patient outcomes were recorded over a 2-years follow up period. CMCs were staged according to pT (greatest diameter in millimeters on histological slides), lymphovascular invasion (LVI), and pN (confirmed by cytokeratin AE1/AE3 immunohistochemistry). The histological stages were defined as: Stage 0 (CMCs in situ, surrounded by a continuous layer of p63+ myoepithelial cells), Stage I (pT1 ≤ 20 mm, LVI-, pN0-pNX, where pNX refers to the absence of lymph node sample), Stage II (pT2 > 20 mm, LVI-, pN0-pNX), Stage IIIA (pT1, LVI+, and/or pN+), and Stage IIIB (pT2, LVI+, and/or pN+). Results: Disease-free-interval, overall survival and specific survival significantly differed by histological stage. For specific survival, median survival times and hazard ratios (HR) by Cox proportional hazards regression (p < 0.0001) were: Stage 0 (median survival not reached; HR = 1.00; N = 89; 21% of the dogs), Stage I (1,720 days; HR = 3.05; p = 0.0018; N = 81; 19%), Stage II (1,181 days; HR = 4.39; p < 0.0001; N = 79; 18%), Stage IIIA (348 days; HR = 10.59; p < 0.0001; N = 79; 18%), and Stage IIIB (163 days; HR = 16.59; p < 0.0001; N = 105; 24%). Conclusion: The proposed histological staging system (invasiveness, pT, LVI, pN) is a very strong prognostic factor for CMCs.

Plasma cell leukemia with plasmablastic morphology in a dog.

A 5-y-old female Golden Retriever was presented with a 2-wk history of hyporexia, vomiting, diarrhea, lethargy, weight loss, polyuria, and polydipsia. Clinical examination and ultrasonography revealed multiple organ enlargement with gallbladder and kidney nodules suggestive of disseminated neoplasia. Hematologic and biochemical analyses revealed pancytopenia, hypercalcemia, and monoclonal IgA gammopathy suspicious for a plasma cell neoplasm. Bone marrow and blood smear examination revealed neoplastic atypical cells highly suggestive of lymphoid origin. Autopsy confirmed the presence of homogeneous white masses and multifocal pale infiltrates in the spleen, kidney, small intestine, gallbladder, and urinary tract. Histologic features were consistent with a multicentric atypical plasma cell tumor. Tumor cells were negative for CD204, IBA-1, E-cadherin, CD3, CD5, CD79a, CD20, and PAX5, and positive for MUM1, consistent with plasma cell origin. The presence of > 20% of circulating blastic plasma cells was consistent with primary plasma cell leukemia with plasmablastic morphology, a disease rarely described in veterinary medicine.