Protein Expression of PI3K/AKT/mTOR Pathway Targets Validated by Gene Expression and its Correlation with Prognosis in Canine Mammary Cancer.

Mammary cancer is the main type of neoplasia in female dogs and is considered an adequate model for the biological and therapeutic study of cancer in women. The PIK3CA/AKT/mTOR pathway plays a central role in cellular homeostasis and is often dysregulated in cancer. The increased expression of PI3K protein in the literature is associated with a poor prognosis, and alterations in the PIK3CA gene can lead to changes in downstream pathways. Thus, the objective of this study was to validate the protein expression to confirm the gene expression of proteins belonging to the main pathway PI3K and PTEN, and their downstream pathways through ZEB1, ZEB2, HIF1A, VHL, CASP3 and PARP1 relating to prognosis in canine mammary cancer. For protein studies, the samples came from 58 female dogs with mammary neoplasia, immunohistochemistry was performed and its analysis by the histoscore method. For the genetic evaluation, the samples came from 13 patients, the DNA was extracted and the analysis for quantitative expression. Through immunohistochemistry, PI3K positivity was significantly associated with affected regional lymph node, distant metastasis, patients with HER2+, Triple Negative and Luminal B phenotypes, and the lowest survival rates. Through gene expression, we observed higher gene expression of ZEB2 and PARP1 both among patients who were alive and who died, which was not true for the expressions of PIK3CA and HIF1A. In conclusion, the data observed in this work are promising in the study of new molecular prognostic markers such as PI3K, ZEB2 and PARP1 for canine mammary cancer.

Presence of human breast cancer xenograft changes the diurnal profile of amino acids in mice.

Human xenografts are extremely useful models to study the biology of human cancers and the effects of novel potential therapies. Deregulation of metabolism, including changes in amino acids (AAs), is a common characteristic of many human neoplasms. Plasma AAs undergo daily variations, driven by circadian endogenous and exogenous factors. We compared AAs concentration in triple negative breast cancer MDA-MB-231 cells and MCF10A non-tumorigenic immortalized breast epithelial cells. We also measured plasma AAs in mice bearing xenograft MDA-MB-231 and compared their levels with non-tumor-bearing control animals over 24 h. In vitro studies revealed that most of AAs were significantly different in MDA-MB-231 cells when compared with MCF10A. Plasma concentrations of 15 AAs were higher in cancer cells, two were lower and four were observed to shift across 24 h. In the in vivo setting, analysis showed that 12 out of 20 AAs varied significantly between tumor-bearing and non-tumor bearing mice. Noticeably, these metabolites peaked in the dark phase in non-tumor bearing mice, which corresponds to the active time of these animals. Conversely, in tumor-bearing mice, the peak time occurred during the light phase. In the early period of the light phase, these AAs were significantly higher in tumor-bearing animals, yet significantly lower in the middle of the light phase when compared with controls. This pilot study highlights the importance of well controlled experiments in studies involving plasma AAs in human breast cancer xenografts, in addition to emphasizing the need for more precise examination of exometabolomic changes using multiple time points.

miR-210 and miR-152 as Biomarkers by Liquid Biopsy in Invasive Ductal Carcinoma.

Detecting circulating microRNAs (miRNAs; miRs) by means of liquid biopsy is an important tool for the early diagnosis and prognosis of breast cancer (BC). We aimed to identify and validate miR-210 and miR-152 as non-invasive circulating biomarkers, for the diagnosis and staging of BC patients, confirming their involvement in tumor angiogenesis. METHODS: RT-qPCR was performed and MiRNA expression analysis was obtained from plasma and fragments of BC and benign breast condition (BBC) women patients, plus healthy subjects. Additionally, the immunohistochemistry technique was carried out to analyze the expression of target proteins. RESULTS: Tumor fragments showed increased expression of oncomiR-210 and decreased expression of miR-152 tumoral suppressor. Both miRNAs were increased in plasma samples from BC patients. The receiver operating characteristic (ROC) curve analysis revealed that only the expression of oncomiR-210 in tissue samples and only the expression of the miR-152 suppressor in plasma have the appropriate sensitivity and specificity for use as differential biomarkers between early/intermediate and advanced stages of BC patients. In addition, there was an increase in the expression of hypoxia-inducible factor 1-alpha (HIF-1α), insulin-like growth factor 1 receptor (IGF-1R), and vascular endothelial growth factor (VEGF) in BC patients. On the contrary, a decrease in Von Hippel-Lindau (VHL) protein expression was observed. CONCLUSIONS: This study showed that increased levels of miR-210 and decreased levels of miR152, in addition to the expressions of their target proteins, could indicate, respectively, the oncogenic and tumor suppressive role of these miRNAs in fragments. Both miRNAs are potential diagnostic biomarkers for BC by liquid biopsy. In addition, miR-152 proved to be a promising biomarker for disease staging.

Melatonin Treatment Combined with TGF-ß Silencing Inhibits Epithelial- Mesenchymal Transition in CF41 Canine Mammary Cancer Cell Line.

BACKGROUND: Mammary cancer is the most prevalent type of cancer in female dogs. The main cause of mortality is the occurrence of metastasis. The metastatic process is complex and involves the Epithelial- Mesenchymal Transition (EMT), which can be activated by Transforming Growth Factor beta (TGF-ß) and involves changes in cellular phenotype, as well as, in the expression of proteins such as E-cadherin, N-cadherin, vimentin and claudin-7. Melatonin is a hormone with oncostatic and anti-metastatic properties and appears to participate in the TGF-ß pathway. Thus, the present work aimed to evaluate the expression of EMT markers, E-cadherin, N-cadherin, vimentin and claudin-7, as well as, the cell migration of the canine mammary cancer cell line, CF41, after treatment with melatonin and TGF-ß silencing. METHODS: Canine mammary cancer cell line, CF41, was cultured and characterized in relation to markers ER, PR and HER2. Cell line CF41 with reducing expression level of TGF-ßwas performed according to Leonel et al. (2017). Expression of the protein E-caderin, N-cadherin, vimentin and claudin-7 was evaluated by immunocytochemistry and quantified by optical densitometry. The analysis of cell migration was performed in transwell chambers with 8µM pore size membrane. RESULTS: CF41 cells present a triple negative phenotype, which is an aggressive phenotype. Immunocytochemistry staining showed increased expression of E-caderin and claudin-7 (P˂0.05) and decreased expression of N-cadherin and vimentin (P˂0.05) in CF41 cells after treatment with 1mM melatonin and TGF-ß silencing. Moreover, treatment with melatonin and TGF-ß silencing was able to reduce migration in cell line CF41 (P˂0.05). CONCLUSION: Our data suggests that therapies combining TGF- ß1 silencing and melatonin may be effective in suppressing the process of EMT, corroborating the hypothesis that melatonin acts on the TGF-ß1 pathway and can reduce the metastatic potential of CF41 cells. This is so far the first study that reports melatonin treatment in CF41 cells with TGF-ß1 silencing and its effect on EMT. Thus, further studies are needed to confirm this hypothesis.

The role of melatonin on miRNAs modulation in triple-negative breast cancer cells.

Melatonin, a hormone secreted by pineal gland, exerts antimetastatic effects by reducing tumor cell proliferation, migration and invasion. MicroRNAs (miRNAs) are small, non-coding RNAs that play a crucial role in regulation of gene expression and biological processes of the cells. Herein, we search for a link between the tumor/metastatic-suppressive actions of melatonin and miRNA expression in triple-negative breast cancer cells. We demonstrated that melatonin exerts its anti-tumor actions by reducing proliferation, migration and c-Myc expression of triple negative breast cancer cells. By using Taqman-based assays, we analyzed the expression levels of a set of miRNAs following melatonin treatment of triple negative breast cancer cells and we identified 17 differentially expressed miRNAs, 6 down-regulated and 11 up-regulated. We focused on the anti-metastatic miR-148b and the oncogenic miR-210 both up-regulated by melatonin treatment and studied the effect of their modulation on melatonin-mediated impairment of tumor progression. Surprisingly, when miR-148b or miR-210 were depleted in triple-negative breast cancer cells, using a specific miR-148b sponge or anti-miR-210, melatonin effects on migration inhibition and c-myc downregulation were still visible suggesting that the increase of miR-148b and miR-210 expression observed following melatonin treatment was not required for the efficacy of melatonin action. Nevertheless, ours results suggest that melatonin exhibit a compound for metastatic trait inhibition, especially in MDA-MB-231 breast cancer cells even if a direct link between modulation of expression of certain proteins or miRNAs and melatonin effects has still to be established.

Immunohistochemical Expression of Melatonin Receptor MT1 and Glucose Transporter GLUT1 in Human Breast Cancer.

BACKGROUND: Breast cancer is a heterogeneous disease and is the leading cause of cancer-related deaths among women. Even after diagnosis, the prognosis cannot be concluded since patients can develop resistance to therapy, which favors tumor growth, invasion and metastasis. In recent years, research has focused on identifying significant markers that can be used to determine the prognosis. Melatonin can act through G protein- coupled MT1 receptor, which controls selected protein kinases, influences the levels of transcription factor phosphorylation, specific genes expression, proliferation, angiogenesis, cell differentiation, migration, and indirectly controls the transport of glucose in cancer cells. It is known that glucose enters the cells by glucose transporters, such as GLUT1 which shows wide tissue distribution and appears to be altered in human breast carcinoma. High GLUT1 expression is associated with increased malignant potential, invasiveness and poor prognosis in some cancers including breast cancer. OBJECTIVE: The aim of this study was to evaluate the expression of MT1 receptor and GLUT1 in breast tumors and correlate with molecular subtypes and prognostic characteristics. METHOD: Protein expression was performed by an immunohistochemical procedure with specific antibodies and positive and negative controls. RESULTS: We found that MT1 high expression was associated with good prognosis subtype (Luminal A), while GLUT1 high expression was related to poor prognosis subtype (triple-negative). In addition, we found high expression of MT1 in ER+ and the inverse in GLUT1 expression. GLUT1 is also highly expressed in tumor ≥20mm. CONCLUSION: These results indicate MT1 and GLUT1 as potential targets for breast cancer subtypes and prognosis.

ZEB1 and ZEB2 transcription factors are potential therapeutic targets of canine mammary cancer cells.

Mammary tumours are the most frequent in female dogs as in women and half are malignant. Tumorigenicity and invasiveness are important acquired characteristics for the development and progression of cancers and could be regulated by transcription factors associated with epithelial-mesenchymal transition (EMT) as ZEB1, ZEB2, SNAI1, SLUG and STAT3. Thus, here, we evaluate the expression of EMT-associated transcription factors in canine mammary cancer (CMC) cell lines characterized for invasiveness and tumorigenicity to determine if these could be considered good targets for future development of therapies. Five CMC cell lines were characterized regarding their morphology, doubling time and expression of intermediate and actin filaments. In addition, gene expression of SLUG, STAT3, ZEB1, ZEB2 and CDH1, tumorigenicity and invasiveness were assessed. Two of these cells presented an epithelial-like morphology (E20 and E37) and three a mesenchymal-like morphology (M5, M25 and CF41.Mg). M25 and CF41.Mg presented higher invasiveness. Furthermore, only mesenchymal-like cells formed tumorspheres and CF41.Mg made more and larger tumorspheres. The mesenchymal-like cells are more malignant than the epithelial-like cells being the CF41.Mg the most malignant. This cell presented higher ZEB1 and ZEB2 and lower CDH1 gene expression. Finally, our results revealed that there is a positive correlation between ZEBs and the tumorsphere number and size. In conclusion, these findings support ZEB1 and ZEB2 as potential therapeutic targets for CMC cells, demonstrating a great potential of canine models for comparative and translational studies.

Melatonin restrains angiogenic factors in triple-negative breast cancer by targeting miR-152-3p: In vivo and in vitro studies.

AIMS: Breast cancer represents the second most prevalent tumor-related cause of death among women. Although studies have already been published regarding the association between breast tumors and miRNAs, this field remains unclear. MicroRNAs (miRNAs) are defined as non-coding RNA molecules, and are known to be involved in cell pathways through the regulation of gene expression. Melatonin can regulate miRNAs and genes related with angiogenesis. This hormone is produced naturally by the pineal gland and presents several antitumor effects. The aim of this study was to understand the action of melatonin in the regulation of miRNA-152-3p in vivo and in vitro. MAIN METHODS: In order to standardize the melatonin treatment in the MDA-MB-468 cells, we carried out the cell viability assay at different concentrations. PCR Array plates were used to identify the differentiated expression of miRNAs after the treatment with melatonin. The relative quantification of the target gene expression (IGF-IR, HIF-1α and VEGF) was performed by real-time PCR. For the tumor development, MDA-MB-468 cells were implanted in female BALB/c mice, and treated or not treated with melatonin. Moreover, the quantification of the target genes protein expression was performed by immunocytochemistry and immunohistochemistry. KEY FINDINGS: Relative quantification shows that the melatonin treatment increases the gene expression of miR-152-3p and the target genes, and decreased protein levels of the genes both in vitro and in vivo. SIGNIFICANCE: Our results confirm the action of melatonin on the miR-152-3p regulation known to be involved in the progression of breast cancer.

Melatonin Differentially Modulates NF-кB Expression in Breast and Liver Cancer Cells.

BACKGROUND: NF-kB (nuclear factor kappa B) is a transcription factor composed of two subunits, p50 and p65, which plays a key role in the inflammatory process. Melatonin has oncostatic, antiangiogenic and antimetastatic properties, and some recent studies have indicated an inhibitory effect of melatonin on NF-kB in some types of cancer. This work aims to investigate the effects of melatonin treatment on the expression of NFkB in breast and liver cancer models. METHOD: The breast cancer xenographic model was performed using female Balb/c nude athymic mice injected with MDA-MB-231 cells. The animals were treated with 40 mg/Kg of melatonin for 21 days. Volume of the tumors was measured with a digital caliper. Hepatocarcinoma model was developed by using the HepG2 cells in vitro, treated with 1 mM melatonin for 24 h. The expression of NF-kB protein was verified by immunohistochemistry and immunocytochemistry and quantified by optical densitometry, in vivo study and in vitro study, respectively. NF-kB gene expression was performed by quantitative RT-PCR. RESULTS: The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size (P=0.0022). There was a decrease in NF-kB protein staining (P=0.0027) and gene expression (P=0.0185) in mice treated with melatonin. The opposite results were observed for the hepatocarcinoma model. HepG2 cells treated with melatonin showed an increase in the NF-kB immunostaining when compared to control cells (P=0.0042). CONCLUSION: Our results indicated that the treatment with melatonin was able to decrease both gene and protein expressions of NF-kB in breast cancer cells and, conversely, increase the transcription factor protein expression in hepatocarcinoma cells. These data highlighted a double role in the expression of NF-kB, depending on the cell type. Further studies are needed to better elucidate the action of melatonin in NF-kB, since this transcription factor acts on different signaling pathways that are fundamental for carcinogenesis.

Consensus for the Diagnosis, Prognosis and Treatment of Canine Mammary Tumors

The purpose of this paper is to establish criteria that could guide the diagnosis, prognosis and treatment of canine mammary neoplasias. It was elaborated during the Mammary Pathology Meeting: Diagnosis, Prognosis and Treatment of the Canine Mammary Neoplasm, held on November 6th and 7th, 2010 in Belo Horizonte ­ MG, Brazil, sponsored by the Laboratory of Comparative Pathology ­ UFMG, with the support of the Brazilian Association of Veterinary Pathology (ABPV) and Brazilian Association of Veterinary Oncology (ABROVET). Academics from several regions of Brazil were present and contributed to this work.(AU)

The maspin expression in canine mammary tumors: an immunohistochemical and molecular study / A expressão do maspin nos tumores mamários caninos: um estudo imuno-histoquímico e molecular

The serpin maspin, a tumor suppressor in breast cancer was described as an inhibitor of cell migration and inducer of cell adhesion between the basement membrane and extracellular matrix resulting in inhibition of tumor metastasis. In contrast, overexpression of maspin is correlated with poor prognosis in other types of cancer. Little is known about expression, regulation and function of maspin in canine mammary tumors. It was demonstrated in this study, a loss of maspin expression in malignant canine mammary cells compared with a pool of normal canine mammary tissue, analyzed by quantitative real-time PCR; weak maspin expression in malignant canine mammary tumors were observed by immunohistochemistry. It was also demonstrated that a correlation with nuclear maspin expression and a good prognosis. It is suggested that maspin could be used as a prognostic marker in canine mammary neoplasia.(AU)

O serpin maspin, um supressor tumoral no câncer de mama foi descrito como inibidor de migração celular e indutor de adesão celular entre a membrana basal e a matriz extracelular resultando na inibição da metástase tumoral. Por outro lado, a alta expressão do maspin está relacionada com um mau prognóstico em outros tipos de câncer. Pouco se sabe sobre a expressão, regulação e função do maspin nos tumores mamários caninos. Neste estudo, foi demonstrada uma perda da expressão de maspin nas células mamárias malignas de cães quando comparadas com um pool de tecido mamário normal de cães, analisado por PCR quantitativa em tempo real. Houve uma expressão fraca maspin em preparações de tumores mamários malignos observadas por imuno-histoquímica. Também foi verificado que a expressão nuclear do maspin em tumores mamários caninos está relacionada a um bom prognóstico. Assim, o maspin pode ser utilizado como um marcador prognóstico nas neoplasias mamárias em cães.(AU)

The maspin expression in canine mammary tumors: an immunohistochemical and molecular study / A expressão do maspin nos tumores mamários caninos: um estudo imuno-histoquímico e molecular

The serpin maspin, a tumor suppressor in breast cancer was described as an inhibitor of cell migration and inducer of cell adhesion between the basement membrane and extracellular matrix resulting in inhibition of tumor metastasis. In contrast, overexpression of maspin is correlated with poor prognosis in other types of cancer. Little is known about expression, regulation and function of maspin in canine mammary tumors. It was demonstrated in this study, a loss of maspin expression in malignant canine mammary cells compared with a pool of normal canine mammary tissue, analyzed by quantitative real-time PCR; weak maspin expression in malignant canine mammary tumors were observed by immunohistochemistry. It was also demonstrated that a correlation with nuclear maspin expression and a good prognosis. It is suggested that maspin could be used as a prognostic marker in canine mammary neoplasia.

O serpin maspin, um supressor tumoral no câncer de mama foi descrito como inibidor de migração celular e indutor de adesão celular entre a membrana basal e a matriz extracelular resultando na inibição da metástase tumoral. Por outro lado, a alta expressão do maspin está relacionada com um mau prognóstico em outros tipos de câncer. Pouco se sabe sobre a expressão, regulação e função do maspin nos tumores mamários caninos. Neste estudo, foi demonstrada uma perda da expressão de maspin nas células mamárias malignas de cães quando comparadas com um pool de tecido mamário normal de cães, analisado por PCR quantitativa em tempo real. Houve uma expressão fraca maspin em preparações de tumores mamários malignos observadas por imuno-histoquímica. Também foi verificado que a expressão nuclear do maspin em tumores mamários caninos está relacionada a um bom prognóstico. Assim, o maspin pode ser utilizado como um marcador prognóstico nas neoplasias mamárias em cães.

Immunohistochemical evaluation of e-cadherin, Ki-67 and PCNA in canine mammary neoplasias: correlation of prognostic factors and clinical outcome / Avaliação imuno-histoquímica da e-caderina, Ki-67 e PCNA nas neoplasias mamárias caninas: correlação dos fatores prognósticos com a evolução clínica

E-cadherin is a cell-cell adhesion molecule and low e-cadherin expression is related to invasiveness and may indicate a bad prognosis in mammary neoplasms. The expression of cell proliferation markers PCNA and especially Ki-67, has also proved to have a strong prognostic value in this tumor class. The expression of these markers was related to the clinical-pathological characteristics of 73 surgically removed mammary tumors in female dogs by immunohistochemistry. There was no statistical correlation between these markers and death by neoplasm, survival time and disease-free interval. However, the loss of e-cadherin expression and marked Ki-67 expression (p=0.016) were considered statistically significant for the diagnosis (p=0.032). When evaluated as independent factors, there was evidence of the relationship between the loss of e-cadherin expression and high PCNA expression with changes in the body status (divided into obese, normal and cachectic) of female dogs (p=0.030); there was also evidence of the relationship between pseudopregnancy and e-cadherin alone (p=0.021) and for ulceration and PCNA alone (p=0.035). The significant correlation between the markers expression and these well known prognostic factors used individually or in combination suggests their prognostic value in canine mammary tumors.(AU)

A e-caderina é uma molécula de adesão celular e a perda de sua expressão esta relacionada à invasão tumoral podendo indicar um prognóstico ruim nas neoplasias mamárias. A expressão dos marcadores de proliferação celular PCNA e especialmente o Ki-67, também têm mostrado forte valor prognóstico nesta classe tumoral. A expressão imuno-histoquímica destes marcadores foi relacionada com as características clinico-patológicas de 73 tumores removidos cirurgicamente de fêmeas caninas. Não houve correlação estatística entre estes marcadores e a morte por neoplasia, tempo de sobrevida e intervalo livre de doença. Entretanto, a perda da expressão da ecaderina e a forte expressão do Ki-67 (p=0,016) foram considerados estatisticamente significativos quando relacionados com o diagnóstico (p=0,032). Quando avaliados os fatores independentes, houve evidência de associação entre a perda de expressão da e-caderina e a alta expressão do PCNA com as mudanças no estado nutricional das cadelas (divididas em obesas, normais e caquéticas) (p=0,030); houve também evidência de associação entre a pseudociese e a expressão da e-caderina (p=0,021) e com a ulceração e a expressão do PCNA (p=0,035). A correlação significativa entre a expressão dos marcadores e estes, bem conhecidos fatores prognósticos usados individualmente ou em associação, sugere importante valor prognóstico destes marcadores nos tumores mamários caninos.(AU)

Immunohistochemical evaluation of e-cadherin, Ki-67 and PCNA in canine mammary neoplasias: correlation of prognostic factors and clinical outcome / Avaliação imuno-histoquímica da e-caderina, Ki-67 e PCNA nas neoplasias mamárias caninas: correlação dos fatores prognósticos com a evolução clínica

E-cadherin is a cell-cell adhesion molecule and low e-cadherin expression is related to invasiveness and may indicate a bad prognosis in mammary neoplasms. The expression of cell proliferation markers PCNA and especially Ki-67, has also proved to have a strong prognostic value in this tumor class. The expression of these markers was related to the clinical-pathological characteristics of 73 surgically removed mammary tumors in female dogs by immunohistochemistry. There was no statistical correlation between these markers and death by neoplasm, survival time and disease-free interval. However, the loss of e-cadherin expression and marked Ki-67 expression (p=0.016) were considered statistically significant for the diagnosis (p=0.032). When evaluated as independent factors, there was evidence of the relationship between the loss of e-cadherin expression and high PCNA expression with changes in the body status (divided into obese, normal and cachectic) of female dogs (p=0.030); there was also evidence of the relationship between pseudopregnancy and e-cadherin alone (p=0.021) and for ulceration and PCNA alone (p=0.035). The significant correlation between the markers expression and these well known prognostic factors used individually or in combination suggests their prognostic value in canine mammary tumors.

A e-caderina é uma molécula de adesão celular e a perda de sua expressão esta relacionada à invasão tumoral podendo indicar um prognóstico ruim nas neoplasias mamárias. A expressão dos marcadores de proliferação celular PCNA e especialmente o Ki-67, também têm mostrado forte valor prognóstico nesta classe tumoral. A expressão imuno-histoquímica destes marcadores foi relacionada com as características clinico-patológicas de 73 tumores removidos cirurgicamente de fêmeas caninas. Não houve correlação estatística entre estes marcadores e a morte por neoplasia, tempo de sobrevida e intervalo livre de doença. Entretanto, a perda da expressão da ecaderina e a forte expressão do Ki-67 (p=0,016) foram considerados estatisticamente significativos quando relacionados com o diagnóstico (p=0,032). Quando avaliados os fatores independentes, houve evidência de associação entre a perda de expressão da e-caderina e a alta expressão do PCNA com as mudanças no estado nutricional das cadelas (divididas em obesas, normais e caquéticas) (p=0,030); houve também evidência de associação entre a pseudociese e a expressão da e-caderina (p=0,021) e com a ulceração e a expressão do PCNA (p=0,035). A correlação significativa entre a expressão dos marcadores e estes, bem conhecidos fatores prognósticos usados individualmente ou em associação, sugere importante valor prognóstico destes marcadores nos tumores mamários caninos.

Immunocytochemical study of Ki-67 as a prognostic marker in canine mammary neoplasia.

BACKGROUND: Canine mammary tumors are challenging for clinicians and pathologists because of complex histologic classification, low specificity of cytologic diagnosis, and unpredictable biological behavior. In histologic specimens, expression of tumor proliferation marker Ki-67, a nuclear nonhistone protein, has been shown to have prognostic value for canine mammary tumors and to correlate with malignancy and low survival rates. OBJECTIVE: The objective of this study was to measure the proliferation index of canine mammary tumors by immunochemical detection of Ki-67 in cytologic specimens and to determine its relationship to clinical and pathologic variables and patient outcome. METHODS: Spontaneous mammary tumors from 31 female dogs were surgically excised. Imprint specimens for cytologic evaluation were wet-fixed in ethanol; histologic specimens were prepared routinely. Immunostaining was performed with the PH 177 monoclonal antibody against Ki-67; proliferation index was graded from negative to +++. Dogs were followed for 18 months. Multivariate logistic regression analysis was used to determine correlations between immunocytochemical results, tumor and clinical variables, and patient outcome. RESULTS: Ki-67 proliferation indices in cytologic specimens were significantly lower for nonmalignant tumors than for malignant tumors. High index values of Ki-67 were positively correlated with metastasis, death from neoplasia, low disease-free survival rates, and low overall survival rate. With the exception of 4 specimens for which cellularity was insufficient, positive expression of Ki-67 in cytologic specimens correlated with that of histologic specimens. CONCLUSIONS: The prognostic value of the Ki-67 index in canine mammary tumors by using wet-fixed cytology imprint specimens was similar to that observed previously for histologic specimens. Immunocytochemical detection of Ki-67 could improve the accuracy and value of cytology by providing safe and rapid information about malignancy and patient outcome.