Mammary carcinoma: Comparative oncology between small animals and humans-New therapeutic tools.

The poor outcomes associated with mammary carcinomas (MCs) in dogs and cats in terms of locoregional recurrence, distant metastasis and survival, highlight the need for better management of mammary cancers in small animals. By contrast, the outcomes of women with breast cancer (BC) have dramatically improved during the last 10 years, notably thanks to new therapeutic strategies. The aim of this article was to imagine what could be the future of therapy for dogs and cats with MCs if it became inspired from current practices in human BC. This article focuses on the importance of taking into account cancer stage and cancer subtypes in therapeutic plans, on locoregional treatments (surgery, radiation therapy), new developments in endocrine therapy, chemotherapy, PARP inhibitors and immunotherapy. Ideally, multimodal treatment regimens would be chosen according to cancer stage and cancer subtypes, and according to predictive factors that are still to be defined.

Prevalence of Reproductive Disorders including Mammary Tumors and Associated Mortality in Female Dogs.

Female dogs, especially intact or neutered lately, are at increased risk for reproductive disorders including mammary tumors (MTs). This retrospective study evaluated the prevalence of reproductive pathology and associated mortality in a cohort of female dogs presented at a single veterinary clinic. The medical records of female dogs born in 2000-2003 were reviewed. The study included 599 cases, of which 293 were followed up until death. Causes of death were analyzed according to the spaying status. Among the 599 female dogs, 306 were intact (51%), 50 (8%) had been spayed before 2 years of age (ES, early spaying), and 243 (41%) after 2 years (LS, late spaying). During their lifetime, 79 dogs (13.2%) developed pyometra, and 160 (26.7%) a mammary tumor. Among the 293 dogs with complete follow-up, 103 (35.1%) had at least one MT during their lifetime, of which 53 (51.5%) died of their mammary cancer. Spayed (ES + LS) female dogs had a 4-fold decreased risk of dying from mammary cancer (OR = 0.23, 95% CI 0.11-0.47, p < 0.0001) compared to intact females. In this low-sterilization rate population, MTs developed in 35.1% of female dogs over their lifetime and was the cause of death in half of them.

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.

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.

STING-dependent paracriny shapes apoptotic priming of breast tumors in response to anti-mitotic treatment.

A fascinating but uncharacterized action of antimitotic chemotherapy is to collectively prime cancer cells to apoptotic mitochondrial outer membrane permeabilization (MOMP), while impacting only on cycling cell subsets. Here, we show that a proapoptotic secretory phenotype is induced by activation of cGAS/STING in cancer cells that are hit by antimitotic treatment, accumulate micronuclei and maintain mitochondrial integrity despite intrinsic apoptotic pressure. Organotypic cultures of primary human breast tumors and patient-derived xenografts sensitive to paclitaxel exhibit gene expression signatures typical of type I IFN and TNFα exposure. These cytokines induced by cGAS/STING activation trigger NOXA expression in neighboring cells and render them acutely sensitive to BCL-xL inhibition. cGAS/STING-dependent apoptotic effects are required for paclitaxel response in vivo, and they are amplified by sequential, but not synchronous, administration of BH3 mimetics. Thus anti-mitotic agents propagate apoptotic priming across heterogeneously sensitive cancer cells through cytosolic DNA sensing pathway-dependent extracellular signals, exploitable by delayed MOMP targeting.

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.

Proposal for a Histological Staging System of Mammary Carcinomas in Dogs and Cats. Part 2: Feline Mammary Carcinomas.

Background: Feline mammary carcinomas (FMCs) are characterized by a high frequency of metastatic spread. The clinical TNM (Tumor, Node, Metastasis) system is used to describe local, regional, and distant tumor extent within the patient, but few publications confirmed its association with survival in cats with FMC. The purpose of this study was to determine if the histological staging system proposed for dogs in part 1 of this article had significant association with prognosis in cats. Materials and Methods: This retrospective study included 395 female cats with a surgically removed mammary carcinoma, with a 2-year follow-up. Invasiveness (distinction between in situ and invasive FMCs), the pathologic tumor size (pT), lymphovascular invasion (LVI), and the pathologic nodal stage (pN) defined a 5-stage system: Stage 0 (FMCs in situ), Stage I (pT1, LVI-, pN0-pNX), Stage II (pT2, LVI-, pN0-pNX), Stage IIIA (pT1, LVI+ and/or pN+), and Stage IIIB (pT2, LVI+ and/or pN+), where pT1 was ≤20 mm, pT2 was >20 mm, and pNX corresponded to unsampled draining lymph node. Results: Higher histological stages were associated with reduced disease-free interval, overall survival, and specific survival. For cancer-specific survival, by univariate analysis (p < 0.0001), median survival times and 1-year specific survival rates (1ySSR) were: stage 0 (1484 days; 1ySSR = 85%; N = 55; 14% of the cats), stage I (808 days; 1ySSR = 76%; N = 103; 26%), stage II (377 days; 1ySSR = 51%; N = 56; 14%), stage IIIA (448 days; 1ySSR = 60%; N = 83; 21%), and stage IIIB (207 days; 1ySSR = 29%; N = 98; 25%). The histological stages were also associated with specific survival by multivariate analysis (Hazard Ratio (HR) = 2.72 for stage IIIB, HR = 1.76 for stage IIIA, HR = 1.50 for stage II compared with stage I), independently of Progesterone Receptor expression (HR = 0.34 for PR+ compared with PR- FMCs) and tumor-associated inflammation (HR = 1.33 when moderate to severe compared with absent to mild). Conclusion: A same histological staging system could be applied in dogs and cats with mammary carcinoma to refine prognosis assessment. In the near future, a preoperative complete tumor clinical staging and treatment based on the published standard of care should be performed in order to better validate the histological staging system here proposed.

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.

Feline Invasive Mammary Carcinomas: Prognostic Value of Histological Grading.

Feline mammary carcinomas are highly malignant tumors usually associated with poor outcome. Nevertheless, survival times can differ significantly according to various prognostic factors. The Elston and Ellis (EE) histologic grading system, originally developed for human breast cancer, is commonly used to grade feline mammary carcinomas, although it is not really adapted for this species, hence the need of a more relevant grading system. Although few veterinary studies attempted to validate previously published results in an independent cohort, the aim of our study was to evaluate the prognostic value of different histologic grading systems in feline invasive mammary carcinomas, including the EE grading system applicable to human breast cancers and the modified and newly designed histologic grading systems recently proposed by Mills et al. Survey data and histologic features of 342 feline invasive mammary carcinomas were analyzed with respect to overall and cancer-specific survival. The histological grading system with best prognostic value was the mitotic-modified Elston and Ellis (MMEE) grading system: grade III carcinomas (P = .04, hazard ratio [HR] = 1.46, 95% CI, 1.01-2.11), grade II (P = .03, HR = 1.39, 95% CI, 1.03-1.88), and grade I carcinomas (HR = 1.00, reference), with decreasing hazard ratios significantly were associated with a worse overall survival, independently from the pathologic tumor size (pT ≥ 20 mm: P = .002, HR = 1.45, 95% CI, 1.15-1.83) and positive nodal stage (P = .001, HR = 1.51, 95% CI, 1.18-1.94). This retrospective study validates Mills et al's proposal to adapt the thresholds for mitotic counts to better assess the histological grade of the highly proliferative mammary carcinomas encountered in the cat.

Bcl-2 expression and prognostic significance in feline invasive mammary carcinomas: a retrospective observational study.

BACKGROUND: Cats spontaneously develop invasive mammary carcinomas with high clinical aggressiveness, and are considered relevant animal models for human breast cancer. Bcl-2 is an anti-apoptotic pro-survival protein, whose expression is associated with a favorable outcome in human breast cancer. The aim of our study was to determine the frequency of Bcl-2 expression in feline invasive mammary carcinomas (FMCs), its relationship with other clinicopathologic variables, and its prognostic value. This retrospective study included 180 FMCs, diagnosed in female cats treated by surgery only, with a 2-year follow-up post-mastectomy. Bcl-2, ER, PR, Ki-67, HER2, and CK5/6 expression were determined by automated immunohistochemistry. A receiver-operating-characteristic curve was used to set the threshold for Bcl-2 positivity. RESULTS: The cohort comprises 32% (57/180) luminal FMCs defined by ER and/or PR positivity, and 68% (123/180) triple-negative FMCs (negative for ER, PR, and HER2). Bcl-2 expression was considered as positive when at least 65% of tumor cells were immunohistochemically stained. Thirty-one out of 180 FMCs (17%) were Bcl-2-positive. There was no significant association between Bcl-2 expression, and the tumor size, nodal stage, histological grade, or ER, PR, Ki-67, HER2, and CK5/6 expression. By multivariate survival analysis (Cox proportional-hazards regression), Bcl-2 positivity in FMCs was associated with longer disease-free interval (p = 0.005, HR = 0.38), overall survival (p = 0.028, HR = 0.61), and cancer-specific survival (p = 0.019, HR = 0.54) independently of other powerful prognostic factors such as pathologic tumor size, pathologic nodal stage, and distant metastasis. The positive prognostic value of Bcl-2 was confirmed in both luminal FMCs, of which 9/57 (16%) were Bcl-2-positive, and in basal-like triple-negative (ER-, PR-, HER2-, CK5/6+) FMCs, of which 14/76 (18%) were Bcl-2-positive. CONCLUSIONS: Compared to human breast cancer, Bcl-2 positivity in feline invasive mammary carcinomas is also associated with better outcome, but is less common, and not associated with ER, PR, and HER2 expression. Cats with spontaneous Bcl-2-positive FMCs could be useful in preclinical trials evaluating anti-Bcl-2 strategies for chemoresistant luminal or triple-negative breast cancers.

Androgen receptor and FOXA1 coexpression define a "luminal-AR" subtype of feline mammary carcinomas, spontaneous models of breast cancer.

BACKGROUND: Invasive mammary carcinomas that spontaneously develop in female cats are associated with high mortality, and resemble the most aggressive human breast cancers, especially triple-negative breast cancer (TNBC). Transcriptome studies showed that TNBCs are a heterogeneous group that includes a potentially hormone-dependent subtype named luminal-AR. Some authors proposed an immunohistochemical definition of the luminal-AR subtype, which is not only positive for Androgen Receptor (AR), but also either positive for the transcription factor Forkhead box A1 (FOXA1), or negative for basal markers. The objectives of this study were to describe AR and FOXA1 expressions in feline mammary carcinomas (FMCs), their prognostic value, and if their coexpression could define a "luminal-AR" subtype of triple-negative mammary carcinomas in cats. METHODS: In a previously described retrospective cohort of 180 female cats with FMCs, with a 2-year follow-up post-mastectomy, we assessed AR, FOXA1, ER, PR, Ki-67, HER2, and CK14 expressions by automated immunohistochemistry. RESULTS: Of the 180 FMCs, 57 (32%) were luminal; i.e., ER and/or PR positive, and 123 (68%) were triple-negative (ER-, PR- and HER2-) FMCs. AR overexpression (found in 33 cases/180, 18%) and FOXA1 index ≥1% (64/180, 36%) were associated with a longer disease-free interval, overall survival, and cancer-specific survival in cats with FMC. Analysis of AR, FOXA1 and CK14 coexpression in triple-negative FMCs showed that AR+ triple-negative FMCs were heterogeneous: there existed an AR+ FOXA1+ CK14- subgroup (n = 7) associated with a better cancer-specific survival by multivariate survival analysis (HR = 0.26, 95% CI: 0.07-0.89, p = 0.03) compared to AR+ FOXA1-CK14+ triple-negative FMCs (n = 46) (HR = 1.00), independently of the pathologic tumor size and pathologic nodal stage. The non-basal-like subtype of triple-negative FMCs that coexpresses AR and FOXA1 (the AR+ FOXA1+ CK14- subgroup) could represent the equivalent of the luminal-AR subgroup of human triple-negative breast cancer. CONCLUSIONS: We identified an AR+ FOXA1+ CK14- subgroup of triple-negative FMCs that might correspond to the luminal-AR subgroup of human triple-negative breast cancers. Cats with FMC may be interesting spontaneous animal models to investigate new strategies targeting the androgen receptor, especially in the aggressive subtype of AR+ basal-like triple-negative mammary carcinomas with loss of FOXA1 expression (the AR+ FOXA1-CK14+ subgroup).

Canine invasive mammary carcinomas as models of human breast cancer. Part 2: immunophenotypes and prognostic significance.

PURPOSE: Relevant animal models of human breast cancer are currently needed, especially for the aggressive triple-negative breast cancer subtype. Recent studies and our results (Part 1) indicate that spontaneous canine invasive mammary carcinomas (CMCs) resemble human breast cancer by clinics and pathology as well as behavior and prognostic indicators. We hypothesized that the current molecular classifications of human breast cancer, used for therapeutic decision, could be relevant to dogs. METHODS: Three hundred and fifty female dogs with spontaneous CMC and a 2-year follow-up were retrospectively included. By immunohistochemistry, CMCs were classified according to Nielsen (Clin Cancer Res 10:5367-5374, 2004) and Blows (PlosOne doi: 10.1371/journal.pmed.1000279, 2010) into the subtypes of human breast cancer. RESULTS: Four immunophenotypes were defined either according to Nielsen classification (luminal A 14.3%, luminal B 9.4%, triple-negative basal-like 58.6%, and triple-negative nonbasal-like 17.7% CMCs); or to Blows classification (luminal 1-: 11.4%, luminal 1+: 12.3%, Core basal phenotype: 58.6%, and five-negative phenotype: 17.7%). No HER2-overexpressing CMC as defined by a 3 + immunohistochemical score was observed in our cohort. By univariate and multivariate analyses, both immunophenotypical classifications applied to CMCs showed strong prognostic significance: luminal A or luminal 1+ CMCs showed a significantly longer disease-free interval (HR = 0.46), Overall (HR = 0.47), and Specific Survival (HR = 0.56) compared to triple-negative carcinomas, after adjustment for stage. CONCLUSIONS: In our cohort, triple-negative CMCs largely predominated (76%), were much more prevalent than in human beings, and showed an aggressive natural behavior after mastectomy. Dogs are thus potent valuable spontaneous models to test new therapeutic strategies for this particular subtype of breast cancer.

Canine invasive mammary carcinomas as models of human breast cancer. Part 1: natural history and prognostic factors.

PURPOSE: Dogs have been proposed as spontaneous animal models of human breast cancer, based on clinicopathologic similarities between canine and human mammary carcinomas. We hypothesized that a better knowledge of the natural history and prognostic factors of canine invasive mammary carcinomas would favor the design of preclinical trials using dogs as models of breast cancer. METHODS: The 2-year outcome of 350 female dogs with spontaneous invasive mammary carcinoma was studied. The investigated prognostic factors included age at diagnosis, pathologic tumor size, pathologic nodal stage, lymphovascular invasion, histological grade, and expression of Estrogen Receptor alpha (ERα), Progesterone Receptor, Ki-67, Human Epidermal Growth Factor Receptor 2, basal cytokeratins 5/6, and Epidermal Growth Factor Receptor. Multivariate survival analyses were performed using the Cox proportional hazards model. RESULTS: The overall survival after mastectomy was 11 months. Within 1 year post mastectomy, 41.5% of dogs (145/350) died from their mammary carcinoma. By multivariate analysis, the significant prognostic factors for overall survival included a pathologic tumor size larger than 20 mm [HR 1.47 (95% confidence interval 1.15-1.89)], a positive nodal stage [pN+, HR 1.89 (1.43-2.48)], a histological grade III [HR 1.32 (1.02-1.69)], ERα negativity [HR 1.39 (1.01-1.89)], a high Ki-67 proliferation index [HR 1.32 (1.04-1.67)], and EGFR absence [HR 1.33 (1.04-1.69)]. CONCLUSION: The short natural history of spontaneous canine invasive mammary carcinomas and high rate of cancer-related death allow for rapid termination of preclinical investigations. The prognostic factors of invasive mammary carcinomas are remarkably similar in dogs and humans, highlighting the similarities in cancer biology between both species.

The dog as a naturally-occurring model for insulin-like growth factor type 1 receptor-overexpressing breast cancer: an observational cohort study.

BACKGROUND: Dogs spontaneously develop invasive mammary carcinoma with a high prevalence of the triple-negative (TN) subtype (lack of ER-Estrogen Receptor and PR-Progesterone Receptor expression, lack of HER2-Human Epidermal Growth Factor Receptor 2 overexpression), making this animal model relevant for investigating new therapeutic pathways. Insulin-like growth factor Type-1 receptor (IGF1R) is frequently overexpressed in primary human breast cancers, with a growing role in the TN phenotype. The purpose of this study was to investigate the Dog as a candidate model for IGF1R-overexpressing mammary carcinoma. METHODS: 150 bitches with canine mammary carcinoma (CMC) and a known 2-year follow-up were retrospectively included. IGF1R expression was assessed by immunohistochemistry (IHC) using a similar scoring system as for HER2 in breast cancer. The prognostic value of the IGF1R expression was assessed in terms of overall and specific survival as well as disease-free interval (DFI). RESULTS: 47 CMC (31 %) were classified as luminal and 103 (69 %) as triple-negative (TN-CMC). 41 % of CMC overexpressed IGF1R (IHC score 3+) of which 76 % were TN-CMC and 62 % grade III. IGF1R overexpression was associated with aggressive features including lymphovascular invasion, histological grade III, low ER expression and the TN phenotype. Univariate and multivariate analyses revealed that IGF1R overexpression was associated with shorter overall and specific survivals and shorter DFI in TN-CMC. CONCLUSIONS: IGF1R overexpression is common and related to a poor outcome in canine invasive mammary carcinoma, particularly in the triple negative subtype, as in human breast cancer. Preclinical studies using the Dog as a spontaneous animal model could be considered to investigate new therapies targeting IGF1R in triple-negative breast cancer.

Comparative analysis of BRAF, NRAS and c-KIT mutation status between tumor tissues and autologous tumor cell-lines of stage III/IV melanoma.

In the last decade, advances in molecular biology have provided evidence of the genotypic heterogeneity of melanoma. We analysed BRAF, NRAS and c-KIT alterations in tissue samples from 63 stage III/IV melanoma patients and autologous cell-lines, using either allele-specific or quantitative PCR. The expression of BRAF V600E protein was also investigated using an anti-BRAF antibody in the same tissue samples. 81% of FFPE samples and tumor cell-lines harboured a genetic alteration in either BRAF (54%) or NRAS (27%) oncogenes. There was a strong concordance (100%) between tissue samples and tumor cell-lines. The BRAF V600E mutant-specific antibody showed high sensitivity (96%) and specificity (100%) for detecting the presence of a BRAF V600E mutation. The correlation was of 98% between PCR and immunohistochemistry results for BRAF mutation. These results suggest that BRAF and NRAS mutation status of tumor cells is not affected by culture conditions.

A compartmental model of mouse thrombopoiesis and erythropoiesis to predict bone marrow toxicity after internal irradiation.

UNLABELLED: In targeted radionuclide radiotherapy, the relationship between bone marrow (BM) toxicity and absorbed dose seems to be elusive. A compartmental model of mouse thrombopoiesis and erythropoiesis was set up to predict the depletion of hematopoietic cells as a function of the irradiation dose delivered to BM by injected radiopharmaceuticals. All simulated kinetics were compared with experimental toxicity for several stages of differentiation of the 2 hematopoietic lineages. METHODS: C57BL/6 mice were injected either with (18)FNa (37 and 60 MBq), a bone-seeking agent, or with saline. BM mean absorbed doses were calculated according to the MIRD formalism from small-animal PET/CT images. Hematologic toxicity was monitored over time, after (18)FNa injection, by studying BM progenitors and precursors in addition to blood cells. The compartmental model takes into account the pharmacokinetics of the compound, in addition to cellular kinetics and cell radiosensitivities for the 2 studied lineages. RESULTS: Because biodistribution studies showed an uptake of (18)FNa in bones, the skeleton was considered as the principal source organ of BM irradiation. The time-activity curve obtained from validated quantification of PET/CT images allowed for the calculation of mean absorbed doses to the whole BM of 2.1 and 3.4 Gy for (18)FNa injections of 37 and 60 MBq, respectively. Concerning hematologic toxicity, the model was in good agreement for the 2 absorbed doses with experimental measurements of cell depletion for platelets, progenitors, and precursors within the BM in terms of time to nadir, depletion intensity, and time to recovery. The same agreement was obtained for red blood cells and their precursors. Model predictions demonstrated that BM toxicity was in correlation with the mean absorbed dose as higher depletions at nadir and longer delays to recovery were noticed for 3.4 Gy than for 2.1 Gy. CONCLUSION: The developed compartmental model of thrombopoiesis and erythropoiesis in a BM toxicity context, after internal irradiation, allowed for the prediction of cell kinetics of BM progenitors, precursors, and mature blood cells in a dose-dependent manner. This model could therefore be used to predict hematologic toxicity in preclinical internal radiotherapy to study the dose-response relationship.

Microvessel density in muscles of dogs with golden retriever muscular dystrophy.

Due to the abundance of muscle, intravascular administration seems required for efficient gene or cell therapy of muscular dystrophy. Here, we examined the skeletal muscle microvasculature to assess if it is altered with dystrophin deficiency. Image analysis of capillaries was performed in three muscles of one- to ten-month-old golden retriever muscular dystrophy (GRMD) dogs and compared with healthy controls. In the gracilis muscle (and in the biceps brachii muscle) of 4- to 10-month-old GRMD dogs, the microvessel density (445+/-47 microvessels per mm(2)), the capillary to fiber ratio (111+/-26 capillaries per 100 myofibers), and the mean intercapillary distance (49+/-3 microm), were similar in affected and control dogs. The sartorius cranialis muscle in GRMD dogs showed microvessel depletion and increased intercapillary distance, but unaltered capillary to fiber ratio, relative to the controls. The mean diameter of microvessels and the total vascular area were higher in GRMD muscles than in control ones. In severely affected GRMD muscles at 7-10 months of age, fibrosis was associated with decreased microvessel density, increased intercapillary distance and microvessel diameter, but normal capillary to fiber ratio and total vascular area.