BACKGROUND
spontaneous mammary tumours are commonly observed in female dogs, particularly in many european countries where animals are not routinely spayed at a young age.
the majority of malignant tumours arise from epithelial tissue and metastasise to the lungs or other organs  <cit> .
several histopathological variables, such as tumour size, lymph node status, lymphatic or vascular invasion and the tumour grade of differentiation form, have been widely used parameters in prognostic evaluations  <cit> .
in human breast cancer and other malignancies, serum tumour markers play an important role in patient management  <cit> .
tumour cells display protein molecules on their surfaces.
these proteins, called tumour-associated antigens, may be present in higher than usual concentrations in other tissues, serum, urine or body fluids of patients with cancer.
thus, the elevation of these markers may be helpful in early diagnosis, determining prognosis, following a course of treatment, predicting the response or resistance to specific therapies and surveillance after primary surgery  <cit> .
carbohydrate antigen  15– <dig> , the product of the mucin  <dig> gene, is a large transmembrane glycosylated molecule aberrantly overexpressed in many adenocarcinomas in an underglycosylated form and then shed into the circulation  <cit> .
the antigen bears epitopes that are recognised by two murine monoclonal antibodies: the high molecular weight mucin-like glycoprotein df <dig> , raised against a membrane fraction of liver metastasis from breast cancer, and 115d <dig>  raised against a milk fat globule membrane  <cit> .
during a malignant transformation, the membrane expression of the muc <dig> cell surface-associated oncoprotein often changes from apical to circumferential simultaneously with a loss of polarity of the epithelial cells, acting as anti-adhesive molecules and facilitating the detachment of malignant cells, and increasing the metastatic and invasive potential of tumour cells  <cit> .
currently, ca 15– <dig>  the circulating form of df <dig> antigen, is one of the most widely used serum biochemical markers in breast cancer.
high serum levels in pre-operative patients correlate with large-size tumours, the stage of the disease and the presence of lymph node metastases and are associated with an adverse patient outcome  <cit> .
in spite of the advanced diagnostic tools available in human oncology, the methodologies available in veterinary medicine may still be considered to be in progress, and there is a need to develop reliable and easily performed diagnostic tests that may predict an increasing tumour burden or recurrence after a mastectomy in the individual patient and prove more useful in terms of classification and prognosis.
currently, there is a lack of scientific documentation regarding the application of serum tumour markers in the canine mammary oncology field, and only one preliminary report describing the possible use of serum ca 15– <dig> in dogs has been published  <cit> .
the authors showed that the application of direct chemiluminescence using a human commercial kit appears to be a promising method for the determination of ca 15– <dig> levels in canine mammary oncology.
the present study was therefore designed to verify the antigenic correlation of human and canine ca 15– <dig>  to assess the expression profile of this tumour-associated antigen and to establish whether its expression could be associated with several tumour characteristics to evaluate its potential use as a serum tumour marker in the canine mammary oncology field.
methods
cell lines
cell cultures
the seven canine cell lines used for this study have previously been recounted  <cit> .
two of the cell lines used in this study originated from mammary adenocarcinomas , and two of the cell lines originated from their metastases to the lungs ; these cell lines were kindly donated by prof. dr. maciej ugorski and dr. joanna polańska of the wrocław university of environmental and life sciences in poland.
one cell line originated from canine mammary anaplastic cancer  and was kindly provided by dr. gerard rutteman of utrecht university in the netherlands.
two canine cancer cell lines were kindly provided by dr. eva hellmen of the swedish university of agricultural sciences : mammary carcinoma  and spindle-cell mammary tumour .
the cell lines were routinely cultured in rpmi- <dig> medium  enriched with 10% foetal bovine serum  , penicillin-streptomycin  and fungizone   under conditions of 5% co2/95% humidified air at 37 °c.
human breast adenocarcinoma cell line  was kindly provided by dr. luisa lanfrancone of campus ifom-ieo milano .
this cell line was grown in dulbecco’s modified eagle’s medium   supplemented with 10% fbs , 100 iu/ml penicillin and 100 μg/ml streptomycin  at 37 °c in a humidified atmosphere with 5% co <dig>
immunocytochemistry
cells were cultured on lab tek  4-chamber culture slides and were fixed with 70% ethanol after  <dig> hours.
after washing with tris buffered saline  , the samples were incubated in peroxidase blocking reagent  for 10 min at room temperature.
after a 30 min incubation in tbs with 5% bovine serum albumin  , the anti df <dig>  and anti 115d <dig>  monoclonal antibodies, diluted 1: <dig> in 1% bsa, were used, and then the slides were incubated at +4 °c overnight.
for staining, the envision kit  was used.
to develop the coloured product, the  <dig> `-diaminobenzidine  substrate  was used.
finally, haematoxylin was used for nuclei counterstaining.
for immunocytochemical experiments, the negative control samples stained without the use of primary antibodies were set aside.
four independent experiments were conducted.
ten pictures of each slide were taken using olympus microscopy bx <dig> .
the colorimetric intensity of the icc-stained antigen spots  was measured by a computer-assisted image analyser .
the antigen spot colour intensity is expressed as the mean pixel optical density on a 1– <dig> scale.
invasion assay and 3d culture
the invasion assay and 3d culture of the canine mammary cancer cell lines cmt-u <dig>  cmt-u <dig>  p <dig>  cmt-w <dig> and cmt-w <dig> has been previously reported  <cit> .
bd biocoat matrigel™ invasion chambers  pre-coated with bd matrigel matrix were used according to the manufacturer’s protocol.
the assay insert plates were prepared by rehydrating the bd matrigel matrix coating with phosphate buffered saline for two hours at 37 °c.
the rehydration solution was carefully removed, and  <dig> x <dig> cancer cells were added to each apical chamber and  <dig>  ml of rpmi- <dig> containing chemo attractant  was added to the basal chamber.
uncoated insert plates, included as invasion controls, were used without rehydration.
assay plates were incubated for  <dig> hours at standard culturing conditions.
then,  <dig>  μg/ml calcein am was added to 20 μl dmso, and 10 μl of this solution was transferred to 12 ml hanks buffered saline dispense.
next,  <dig>  ml calcein solution was transferred into each well of a 24-well plate.
the medium from the insert was removed, and multiwell inserts were transferred to the plate containing  <dig>  ml/well calcein.
plates were incubated for an hour at standard culture conditions.
the fluorescence of the invaded cells was measured with an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a tecan infinite  <dig> reader .
to characterise the growth of the cancer cell lines, they were treated with trypsin and resuspended in the culture medium.
thirty-five-mm culture plates  were coated with 100 μl of growth factor reduced matrigel  and left to solidify for 30 min at 37 °c.
the cells were then plated at a concentration of  <dig> cells/ml.
the growth of cells on matrigel was observed each day under a phase-contrast microscope.
sds-page and western blot
the cells were pelleted by centrifugation at  <dig> x g at 4 °c for 5 min.
protein extracts from cultured cells were isolated by lysis of the collected pellets with ripa buffer  supplemented with a protease inhibitor cocktail  and a phosphatase inhibitor cocktail  for 30 min at 4 °c.
lysates were cleared for 20 min at  <dig>  x g, and supernatants were collected.
the protein concentration in the lysates was determined by bio-rad protein assay dye .
cellular proteins extracted from canine and human mammary cancer cell lines were analysed by sds-page and subjected to immunoblotting analysis.
samples were mixed with 4x nupage sample buffer  containing 100 mm ddt and heated to 95 °c for 5 min.
lysates were subjected to electrophoresis using 3-8% nupage tris-acetate precasting gels in tris-acetate sds running buffer and transferred to a pvdf membrane according to the manufacturer’s protocol.
membranes were blocked for  <dig> hours at room temperature in phosphate buffered saline , ph  <dig> , containing  <dig> % tween  <dig>  and 3% skimmed milk.
membranes were then washed with pbst and incubated at 4 °c overnight with the anti df <dig>  and 115d <dig>  monoclonal antibodies, diluted 1: <dig> in 1% milk/pbst and anti-ß-actin antibodies  diluted 1: <dig>  in 1% milk/pbst .
detection was performed by the supersignal west pico chemiluminescence substrate using an anti-mouse antibody conjugated with horseradish peroxidase .
animals, serum biochemistry, histology and immunohistochemistry
fifty tissue samples were obtained from female dogs with malignant mammary tumours during surgery in private clinics;  <dig> were solitary nodules, and  <dig> were multiple nodules presenting simultaneously with  <dig> or more lumps.
in one case, data were not available.
to select only primary tumours without lymph node involvement and lung metastasis, the female dogs were initially submitted to a systematic clinical examination together with a macroscopic evaluation of the tumour and the lymph nodes to accomplish a staging categorisation.
several clinical factors, such as tumour size, the presence/absence of tumour metastasis to regional lymph nodes and the presence/absence of distant metastases, were used as a staging system , according to philbert  <cit> .
as part of the diagnostic workup, the altered regional lymph nodes were evaluated by means of a tough fine needle aspirate  and cytology.
thoracic radiological examinations searching for lung metastases were also performed.
a routine clinicopathological examination  to exclude specific benign conditions that could influence the serum levels of ca 15– <dig>  was also performed in all animals.
to determine factors that possibly influence serum ca 15– <dig> activity in cancer patients, the impact of clinical and pathological parameters were investigated and cases were assessed for tumour size, skin ulceration, histological type, tumour grade, inflammation and necrosis.
unfortunately, the data for the length of survival time were unavailable for most patients.
peripheral venous blood samples were obtained from the patients during the pre-operative stage to measure ca 15– <dig>  the anticoagulated whole blood from patients subjected to a mastectomy was collected for routine diagnostic purposes.
the remaining volume of the blood samples was collected for our analyses .
sera were separated and stored at −20 °c until use.
ca 15– <dig> levels were determined by a direct chemiluminescent technology in the automated immunoanalyser system advia centaur® using a kit for human diagnostic oncology  according to the manufacturer's protocol.
values were expressed as units/ml .
as a control group, we used data obtained in our previous study  <cit> .
for histological examinations, representative portions of tissue samples were fixed in 10% buffered formalin and then dehydrated and embedded in paraffin.
three to 4-micron-thick consecutive sections were cut and stained with haematoxylin and eosin  and subjected to the immunohistochemical procedure using the anti muc <dig> antibodies previously described.
to better investigate the pattern of expression on canine mammary tissues, the immunohistochemical procedure was also applied on  <dig> normal lesions and on  <dig> benign breast lesions  obtained from the archive of the laboratory of histopathology of the istituto zooprofilattico sperimentale dell’umbria e delle marche.
in he sections, tumours were classified according to the who’s criteria for canine mammary neoplasms  <cit>  and coded according to the who’s international classification of disease for oncology system .
in cases of multiple nodules in a chain, the entire chain was resected, and all nodules were subjected to histology.
only the tumour with the higher grade of malignancy was used as the basis for a comparison with ca 15– <dig>
histological grading was evaluated in accordance with elston and ellis  <cit> , the most current method for the histological grading of human breast cancers, recently applied to canine mammary tumours and more practical for the comparison of data between researchers  <cit> .
according to this method, histological findings were recorded and used to classify mammary carcinomas as grade i, grade ii or grade iii tumours.
for immunohistochemistry , tissue sections were hydrated utilising xylene and a graded alcohol series.
successively, the quenching of endogenous peroxidase activity was achieved by incubating the sections for 30 min in 3% hydrogen peroxide in methanol at room temperature.
antigen retrieval was performed using microwave exposure for 5 min at 650 w  and 500 w  in a citrate buffer solution .
primary antibodies  were applied and incubated overnight at 4 °c in a humidified chamber.
a streptavidin-biotin revelation commercial kit  was used, and the reaction was developed by  <dig> ’-diaminobenzidine .
the slides were washed in tap water and counterstained with mayer’s haematoxylin.
a positive control of human malignant breast cancer tissue was included in each staining.
negative control slides were incubated with a specific non immune antibody in parallel with each staining batch to confirm the antibody specificity.
to evaluate muc <dig> expression, a semi-quantitative method was performed; protein expression was assessed estimating the percentage of labelled cells and scored as follows: no staining, weak staining , moderate  and strong  immunostaining.
the images were digitalised using a video camera connected to a microscope , and the semi-quantitative evaluation was estimated by counting  <dig>  positive cells in randomly selected fields , avoiding necrosis.
statistical analysis
the statistical analysis was conducted using prism version  <dig>  software .
the anova and post-hoc dunett`s multiple comparison test and tukey’s honestly significance difference  were applied.
to assess the correlation between the tumour characteristics and the ca 15– <dig> serum level, the pearson correlation coefficient was used.
a p <  <dig>  was regarded as significant, whereas p <  <dig>  was regarded as highly significant.
RESULTS
cell lines
immunocytochemistry
each tumour cell line used in this study showed cytoplasmic expression of muc <dig>  and the positivity was mainly observed in a granular pattern.
frequently, cytoplasmic staining was observed together with a membrane reaction in a mixed pattern.
cmt-w <dig>  cmt-w1m, cmt-w <dig> and cmt-w2m cell lines showed a high expression of muc <dig>  whereas cmt-u <dig>  cmt-u <dig> and p <dig> cell lines showed weak 115d <dig> expression.
the same results were obtained using antibodies against df <dig> .
the mean optical density  of brown coloured antigen reflecting 115d <dig> expression was counted.
as a control, the mcf <dig> cell line was used, which expresses the antigen at the level of  <dig>  .
no significant differences were found between the expression of the df <dig> antigen in the control cell line and in the following cell lines: cmt-w <dig> , cmt-w1m , cmt-w <dig> , and cmt-w2m .
a significant difference  was found between the 115d <dig> expression in the mcf <dig> cell line and the cmt-u <dig> cell line , whereas highly significant differences  were found in the 115d <dig> expression in the cmt-u <dig> and p <dig> cell lines  in comparison with mcf <dig> .
invasion of canine mammary cancer cell lines
the invasion assay results and growth characteristics on the matrigel matrix of the canine mammary cancer cell lines cmt-u <dig>  cmt-u <dig>  p <dig>  cmt-w <dig> and cmt-w <dig> have previously been reported  <cit> ; however, these cell lines have not been compared with cmt-w1m and cmt-w2m cell lines.
the assay showed that only cmt-w <dig>  cmt-w1m, cmt-w <dig> and cmt-w2m cells migrated through the matrigel .
the fluorescence intensity related to the migration of the cmt-w <dig> cells was  <dig>  , and the migration of the cmt-w1m cells was  <dig>  , whereas migration of the cmt-w <dig> and cmt-w2m cells was  <dig>   and  <dig>  , respectively.
to confirm the ability of these cell lines to invade the matrix, we have assessed their growth characteristics on the matrigel matrix .
after  <dig> hours of culturing  on matrigel, the cmt-u <dig>  cmt-u <dig> and p <dig> cell lines formed colonies, whereas the cmt-w <dig>  cmt-w1m, cmt-w <dig> and cmt-w2m cell lines formed branching structures , which indicated their invasive phenotype.
sds-page and western blot
immunoblot analysis using anti 115d <dig> antibodies revealed the presence of two reactive protein bands in the human mcf <dig> cell line, used as a positive control .
the band with a lower molecular weight  is approximately 250 kda, while the mw of the major protein band appears to be more than 300 kda.
a single protein band, with the same high mw as the positive control, was detected for the cmt-w <dig> and cmt-w <dig> cell lines , as well as for the cmt-w1m and cmt-w2m cell lines .
no bands were observed for the cmt-u <dig>  cmt-u <dig> and p <dig> cell lines .
immunoblotting performed with the anti df <dig> antibody gave overlapping results .
the western blot analysis with anti ß-actin antibodies showed that variability in protein loading could not account for the observed differences in 115d <dig> expression.
animals, serum biochemistry, histology and immunohistochemistry
most dogs in our series were mixed breeds , followed by german shepherd dogs .
the mean age was  <dig>  ± <dig>  years , with an increased prevalence among intact females .
the mean diameter of nodules was  <dig>  ±  <dig>  cm , and skin ulceration was present in 22% of cases.
none of these patients had apparent nodal involvement.
with respect to the histopathological analyses based on the who’s classification, complex carcinoma was the most common type of tumour  followed by simple carcinoma  and carcinosarcoma ; necrosis and inflammation were observed in 62% and 42% of cancer cases, respectively.
according to elston and ellis’s method, 66% of tumours were classified as grade i, 28% were classified as grade ii and 6% were classified as grade iii.
ca 15– <dig> was detected in all of the analysed sera.
furthermore, dogs with malignant mammary tumours had a significantly higher median activity of serum ca 15– <dig>  than the control group  .
when we compared tumour carbohydrate antigen 15– <dig> serum levels and the pathological variables considered in this study, no statistically significant differences between ca 15– <dig> and age, history of ovariohysterectomy, numbers and sizes of tumour and histological types were observed .
furthermore, because the inflammatory and necrotic processes were often associated with cancer, they did not appear to have influenced the serum levels of ca 15– <dig>  interestingly, a highly significant correlation was found between ca 15– <dig> serum levels and the tumour grade .
in grade i tumours, the average concentration of this marker was approximately half  compared with that of grades ii  and iii .
given the fact that grade iii neoplasms were represented by a small number  and that their corresponding mean serum level was not significantly different from grade ii tumours, they were included in the same group.
relation between tumour size, skin ulceration, histopathological diagnosis, inflammation, necrosis and histological grade of canine mammary cancer tissues and serum ca 15– <dig> levels.
the pearson correlation coefficient was applied using prism version  <dig>  software .
with immunohistochemistry, df <dig> antigen was detected in all normal gland tissues, and, as expected, the immunoreactivity was almost restricted to the apical membrane of epithelial cells, which displayed a weak to moderate staining.
the positivity was more evident in ductal epithelial cells and in luminal secretions .
in benign mammary lesions , the staining was observed in 7/ <dig>  of the analysed cases, and the pattern of reaction was weak-to-moderate, usually restricted to the apical membrane of the epithelial cells.
approximately 20% of normal ducts also showed a slight apical staining.
in complex adenomas, simple adenoma and benign mixed tumours, a cytoplasmic stain was also detected , while in the dysplastic lesions , no evidence of reaction was found.
in malignant mammary tumours, the apical staining was almost always observed together with a cytoplasmic expression in a mixed pattern with variable intensity and mostly in diffuse or granular pattern.
it is important to note that the reaction affected all of the cells in  <dig>  carcinoma samples, including  <dig>  with strong and  <dig>  with moderate staining.
in complex carcinomas, epithelial cells showed a diffuse positivity in  <dig>  cases, with a greater intensity at the periphery of the neoplastic foci .
in the marginal areas of invasive tumours, markedly positive neoplastic cells invading the surrounding adipose tissue were frequently observed .
in simple carcinomas, a diffuse expression was detected in  <dig>  cases and most evident in the apical membrane of neoplastic cells that formed tubular structures .
this tumour had a strong tendency to infiltrate the surrounding vessels, and lymphatic emboli, when present, exhibited a strong cytoplasmic positivity .
in carcinosarcomas, df <dig> expression was very weak in 100% of cases because the sarcomatous cells were preponderant and only a few stained epithelial cells were observed.
in hyperplastic or normal lobules that sometimes circumscribed the tumour proliferated areas, a weak positivity was also detected.
in all of the tissues examined, myoepithelial and stromal cells, lymphocytes, macrophages and blood vessels did not express immunoreactivity.
no significant correlations were found between df <dig> overexpression and serum ca 15– <dig> levels.
discussion
in veterinary medicine, a variety of assays and techniques have been used in an attempt to identify the prognostic markers for neoplastic diseases, including clinical parameters, histologic features, immunohistochemistry, quantitative pcr, evaluation for genomic mutations and specific serum protein levels or enzyme activity analyses  <cit> .
to investigate the potential functional relevance of ca 15– <dig> as a serum tumour marker in veterinary oncology, we performed a multidisciplinary study on canine neoplastic mammary tissues and cell lines.
the main finding in western blot analysis regarded the positive interaction between the human antibodies and the canine muc <dig> protein.
the protein sequence of epitope df <dig> in humans is trpapgs  <cit> , whereas the canine df <dig> epitope sequence is srpapss .
we used the blosum <dig> amino acid substitution matrix  <cit>  to score alignments between evolutionarily divergent protein sequences.
it revealed a high similarity between these two epitopes in humans and in dogs .
thus, theoretically, the similarity of the protein sequence of the df <dig> epitope is very high in these two species.
nevertheless, the results of wb showed that anti 115d <dig> and anti df <dig> antibodies display cross-reactivity with canine samples.
the icc analysis of the muc <dig> expression  in canine mammary cancer cell lines showed staining patterns similar to those observed in humans.
in wb, a double protein band with a high molecular weight was observed in mcf- <dig> cells  <cit> .
in the canine tumour cell lines cmt-w <dig>  cmt-w <dig>  cmt-w1m and cmt-w2m, we found a single protein band of muc <dig> with a molecular weight similar to the higher human form.
however, when we extended the time of the chemical exposure, some films revealed an apparent reactive point with a molecular weight of approximately 250 kda.
these data are still under investigation because it is difficult to determine whether it is an actual protein band or an artefact of the analysis, considering the low sensitivity of the test.
our immunocytochemistry results differ from those obtained using western blot.
the immunocytochemical examination revealed muc <dig> expression in all of the examined cell lines.
however, only the cmt-w <dig>  cmt-w1m, cmt-w <dig> and cmt-w2m cell lines expressed muc <dig> at high levels, similar to mcf <dig> .
the remaining cell lines exhibited a weaker expression of muc <dig>  which differed significantly from the mcf <dig> cell line.
according to the subject data  <cit> , a higher expression of muc <dig> is observed in more invasive metastatic cancer.
our findings support this hypothesis, as cmt-w <dig>  cmt-w1m, cmt-w <dig> and cmt-w2m cell lines with invasive phenotypes  <cit>   showed a higher muc <dig> expression .
thus, a lack of muc <dig> expression in non-invasive p <dig>  cmt-u <dig> and cmt-u <dig> cell lines in western blot analysis may be due to low levels of this antigen expression and/or the low sensitivity of the western blot analysis.
although both methods detect a specific protein expression, they significantly differ from each other on what may explain the differences in the obtained results.
similar variations between the results obtained using western blot and immunocytochemistry have previously been reported  <cit> , showing that immunocytochemistry is a more sensitive method than the western blot in cases of weak antigen expression.
statistical analysis allowed us to demonstrate that ca 15– <dig> serum levels correlate positively with the degree of tumour proliferation and differentiation, as a significantly higher concentration of ca 15– <dig> was found in grades ii-iii than in grade i carcinomas .
tumour size, skin ulceration, necrosis, inflammation and the histological type of mammary cancer were not significantly related to the serum levels of ca 15– <dig>
the existence of a relationship between ca 15– <dig> serum levels and tumour grading appears to be logical because we assumed that tumour markers reflect the number and the activity of neoplastic cells as well as their products that access the circulation where the marker is measured.
therefore, this parameter could be taken into consideration as potentially enabling a better evaluation of a tumour’s malignancy, particularly for establishing the preoperative stage of the neoplasm and providing useful guidelines when determining the surgical approach.
another finding of our study, as expected, is the expression of muc <dig> in normal canine mammary tissues and in mammary gland benign lesions.
the reaction was primarily restricted to the apical membrane of epithelial cells.
however, in benign tumours, the pattern of reaction was extended to the cytoplasm of the luminal epithelial cells.
these results are in absolute parallel to those observed in human mammary tissues.
<cit> .
among the carcinomas, we observed muc <dig> activity in 68% of the analysed cases that displayed a strong immunostaining in a mixed pattern , which comprises the whole cell population in several carcinoma samples.
this finding is in accordance with the observation of lacunza  <cit> , which demonstrated a correlation between muc <dig> protein over-expression and gene amplifications in benign and malignant breast tumours.
many studies have revealed that this event could be linked to the presence of membrane-associated muc <dig> molecular portions that are shed from the surface of tumour cells to the cytosol by modulation of its glycosylation state  <cit> .
in the present study, there was no significant correlation between the over-expression of muc <dig> and ca 15– <dig>  the circulating form of the df <dig> antigen.
however, a significant correlation has been found between the histopathological grade of the tumour malignancy and muc <dig> expression and between the histopathological grade of the tumour malignancy and the circulating ca 15– <dig> level.
we suppose that a lack of a correlation between muc <dig> over-expression in tissues and the serum level of ca 15– <dig> may be related to the different antibodies used in the ihc and ca 15– <dig> tests.
CONCLUSIONS
to the best of our knowledge, this is the first report demonstrating a significant correlation between serum ca 15– <dig> expression and tumour malignancy in dogs with mammary cancer.
our results may provide the basis for developing a novel approach in veterinary research, although additional studies using a large number of healthy and pathological clinical samples to validate these preliminary results and to establish normal and pathological serum ranges of ca 15– <dig> antigen in dogs.
clearly, this pilot study is only the beginning of understanding the potential application of this serum tumour marker in canine mammary cancer management, and we hope that these findings will encourage future research work in this field.
competing interest
the authors declare that they have no competing interests.
authors’ contributions
em and mk conceived the study and drafted the manuscript.
em performed the histopathology and the scoring process and supervised the immunohistochemical procedure.
mk, kp and cc performed cell culture, immunocytochemistry and contributed to western blot analysis.
mk performed the 3d culture.
km performed the invasion assay.
adg and kf performed the western blot.
ff and mk performed the statistical analyses.
mcm and ep performed the chemiluminescent assay.
all authors read and approved the final manuscript.
acknowledgments
we thank prof. dr. maciej ugorski, dr. joanna polańska, dr. gerard rutteman, dr. eva hellmen and dr. luisa lanfrancone for providing the cell lines used in this study.
this work was partially supported by grant no.
n n <dig> from the ministry of sciences and higher education .

 spontaneous mammary tumours are commonly observed in female dogs, particularly in many european countries where animals are not routinely spayed at a young age. the majority of malignant tumours arise from epithelial tissue and metastasise to the lungs or other organs. several histopathological variables, such as tumour size, lymph node status, lymphatic or vascular invasion and the tumour grade of differentiation form, have been widely used parameters in prognostic evaluations. in human breast cancer and other malignancies, serum tumour markers play an important role in patient management. tumour cells display protein molecules on their surfaces. these proteins, called tumour-associated antigens, may be present in higher than usual concentrations in other tissues, serum, urine or body fluids of patients with cancer. thus, the elevation of these markers may be helpful in early diagnosis, determining prognosis, following a course of treatment, predicting the response or resistance to specific therapies and surveillance after primary surgery. carbohydrate antigen 15–, the product of the mucin gene, is a large transmembrane glycosylated molecule aberrantly overexpressed in many adenocarcinomas in an underglycosylated form and then shed into the circulation. the antigen bears epitopes that are recognised by two murine monoclonal antibodies: the high molecular weight mucin-like glycoprotein df, raised against a membrane fraction of liver metastasis from breast cancer, and 115d raised against a milk fat globule membrane. during a malignant transformation, the membrane expression of the muc cell surface-associated oncoprotein often changes from apical to circumferential simultaneously with a loss of polarity of the epithelial cells, acting as anti-adhesive molecules and facilitating the detachment of malignant cells, and increasing the metastatic and invasive potential of tumour cells. currently, ca 15– the circulating form of df antigen, is one of the most widely used serum biochemical markers in breast cancer. high serum levels in pre-operative patients correlate with large-size tumours, the stage of the disease and the presence of lymph node metastases and are associated with an adverse patient outcome. in spite of the advanced diagnostic tools available in human oncology, the methodologies available in veterinary medicine may still be considered to be in progress, and there is a need to develop reliable and easily performed diagnostic tests that may predict an increasing tumour burden or recurrence after a mastectomy in the individual patient and prove more useful in terms of classification and prognosis. currently, there is a lack of scientific documentation regarding the application of serum tumour markers in the canine mammary oncology field, and only one preliminary report describing the possible use of serum ca 15– in dogs has been published. the authors showed that the application of direct chemiluminescence using a human commercial kit appears to be a promising method for the determination of ca 15– levels in canine mammary oncology. the present study was therefore designed to verify the antigenic correlation of human and canine ca 15– to assess the expression profile of this tumour-associated antigen and to establish whether its expression could be associated with several tumour characteristics to evaluate its potential use as a serum tumour marker in the canine mammary oncology field. methods cell lines cell cultures the seven canine cell lines used for this study have previously been recounted. two of the cell lines used in this study originated from mammary adenocarcinomas, and two of the cell lines originated from their metastases to the lungs; these cell lines were kindly donated by prof. dr. maciej ugorski and dr. joanna polańska of the wrocław university of environmental and life sciences in poland. one cell line originated from canine mammary anaplastic cancer and was kindly provided by dr. gerard rutteman of utrecht university in the netherlands. two canine cancer cell lines were kindly provided by dr. eva hellmen of the swedish university of agricultural sciences: mammary carcinoma and spindle-cell mammary tumour. the cell lines were routinely cultured in rpmi- medium enriched with 10% foetal bovine serum, penicillin-streptomycin and fungizone under conditions of 5% co2/95% humidified air at 37 °c. human breast adenocarcinoma cell line was kindly provided by dr. luisa lanfrancone of campus ifom-ieo milano. this cell line was grown in dulbecco’s modified eagle’s medium supplemented with 10% fbs, 100 iu/ml penicillin and 100 μg/ml streptomycin at 37 °c in a humidified atmosphere with 5% co immunocytochemistry cells were cultured on lab tek 4-chamber culture slides and were fixed with 70% ethanol after hours. after washing with tris buffered saline, the samples were incubated in peroxidase blocking reagent for 10 min at room temperature. after a 30 min incubation in tbs with 5% bovine serum albumin, the anti df and anti 115d monoclonal antibodies, diluted 1: in 1% bsa, were used, and then the slides were incubated at +4 °c overnight. for staining, the envision kit was used. to develop the coloured product, the `-diaminobenzidine substrate was used. finally, haematoxylin was used for nuclei counterstaining. for immunocytochemical experiments, the negative control samples stained without the use of primary antibodies were set aside. four independent experiments were conducted. ten pictures of each slide were taken using olympus microscopy bx. the colorimetric intensity of the icc-stained antigen spots was measured by a computer-assisted image analyser. the antigen spot colour intensity is expressed as the mean pixel optical density on a 1– scale. invasion assay and 3d culture the invasion assay and 3d culture of the canine mammary cancer cell lines cmt-u cmt-u p cmt-w and cmt-w has been previously reported. bd biocoat matrigel™ invasion chambers pre-coated with bd matrigel matrix were used according to the manufacturer’s protocol. the assay insert plates were prepared by rehydrating the bd matrigel matrix coating with phosphate buffered saline for two hours at 37 °c. the rehydration solution was carefully removed, and x cancer cells were added to each apical chamber and  ml of rpmi- containing chemo attractant was added to the basal chamber. uncoated insert plates, included as invasion controls, were used without rehydration. assay plates were incubated for hours at standard culturing conditions. then,  μg/ml calcein am was added to 20 μl dmso, and 10 μl of this solution was transferred to 12 ml hanks buffered saline dispense. next,  ml calcein solution was transferred into each well of a 24-well plate. the medium from the insert was removed, and multiwell inserts were transferred to the plate containing  ml/well calcein. plates were incubated for an hour at standard culture conditions. the fluorescence of the invaded cells was measured with an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a tecan infinite reader. to characterise the growth of the cancer cell lines, they were treated with trypsin and resuspended in the culture medium. thirty-five-mm culture plates were coated with 100 μl of growth factor reduced matrigel and left to solidify for 30 min at 37 °c. the cells were then plated at a concentration of cells/ml. the growth of cells on matrigel was observed each day under a phase-contrast microscope. sds-page and western blot the cells were pelleted by centrifugation at x g at 4 °c for 5 min. protein extracts from cultured cells were isolated by lysis of the collected pellets with ripa buffer supplemented with a protease inhibitor cocktail and a phosphatase inhibitor cocktail for 30 min at 4 °c. lysates were cleared for 20 min at x g, and supernatants were collected. the protein concentration in the lysates was determined by bio-rad protein assay dye. cellular proteins extracted from canine and human mammary cancer cell lines were analysed by sds-page and subjected to immunoblotting analysis. samples were mixed with 4x nupage sample buffer containing 100 mm ddt and heated to 95 °c for 5 min. lysates were subjected to electrophoresis using 3-8% nupage tris-acetate precasting gels in tris-acetate sds running buffer and transferred to a pvdf membrane according to the manufacturer’s protocol. membranes were blocked for hours at room temperature in phosphate buffered saline, ph, containing % tween and 3% skimmed milk. membranes were then washed with pbst and incubated at 4 °c overnight with the anti df and 115d monoclonal antibodies, diluted 1: in 1% milk/pbst and anti-ß-actin antibodies diluted 1: in 1% milk/pbst. detection was performed by the supersignal west pico chemiluminescence substrate using an anti-mouse antibody conjugated with horseradish peroxidase. animals, serum biochemistry, histology and immunohistochemistry fifty tissue samples were obtained from female dogs with malignant mammary tumours during surgery in private clinics; were solitary nodules, and were multiple nodules presenting simultaneously with or more lumps. in one case, data were not available. to select only primary tumours without lymph node involvement and lung metastasis, the female dogs were initially submitted to a systematic clinical examination together with a macroscopic evaluation of the tumour and the lymph nodes to accomplish a staging categorisation. several clinical factors, such as tumour size, the presence/absence of tumour metastasis to regional lymph nodes and the presence/absence of distant metastases, were used as a staging system, according to philbert. as part of the diagnostic workup, the altered regional lymph nodes were evaluated by means of a tough fine needle aspirate and cytology. thoracic radiological examinations searching for lung metastases were also performed. a routine clinicopathological examination to exclude specific benign conditions that could influence the serum levels of ca 15– was also performed in all animals. to determine factors that possibly influence serum ca 15– activity in cancer patients, the impact of clinical and pathological parameters were investigated and cases were assessed for tumour size, skin ulceration, histological type, tumour grade, inflammation and necrosis. unfortunately, the data for the length of survival time were unavailable for most patients. peripheral venous blood samples were obtained from the patients during the pre-operative stage to measure ca 15– the anticoagulated whole blood from patients subjected to a mastectomy was collected for routine diagnostic purposes. the remaining volume of the blood samples was collected for our analyses. sera were separated and stored at −20 °c until use. ca 15– levels were determined by a direct chemiluminescent technology in the automated immunoanalyser system advia centaur® using a kit for human diagnostic oncology according to the manufacturer's protocol. values were expressed as units/ml. as a control group, we used data obtained in our previous study. for histological examinations, representative portions of tissue samples were fixed in 10% buffered formalin and then dehydrated and embedded in paraffin. three to 4-micron-thick consecutive sections were cut and stained with haematoxylin and eosin and subjected to the immunohistochemical procedure using the anti muc antibodies previously described. to better investigate the pattern of expression on canine mammary tissues, the immunohistochemical procedure was also applied on normal lesions and on benign breast lesions obtained from the archive of the laboratory of histopathology of the istituto zooprofilattico sperimentale dell’umbria e delle marche. in he sections, tumours were classified according to the who’s criteria for canine mammary neoplasms and coded according to the who’s international classification of disease for oncology system. in cases of multiple nodules in a chain, the entire chain was resected, and all nodules were subjected to histology. only the tumour with the higher grade of malignancy was used as the basis for a comparison with ca 15– histological grading was evaluated in accordance with elston and ellis, the most current method for the histological grading of human breast cancers, recently applied to canine mammary tumours and more practical for the comparison of data between researchers. according to this method, histological findings were recorded and used to classify mammary carcinomas as grade i, grade ii or grade iii tumours. for immunohistochemistry, tissue sections were hydrated utilising xylene and a graded alcohol series. successively, the quenching of endogenous peroxidase activity was achieved by incubating the sections for 30 min in 3% hydrogen peroxide in methanol at room temperature. antigen retrieval was performed using microwave exposure for 5 min at 650 w and 500 w in a citrate buffer solution. primary antibodies were applied and incubated overnight at 4 °c in a humidified chamber. a streptavidin-biotin revelation commercial kit was used, and the reaction was developed by ’-diaminobenzidine. the slides were washed in tap water and counterstained with mayer’s haematoxylin. a positive control of human malignant breast cancer tissue was included in each staining. negative control slides were incubated with a specific non immune antibody in parallel with each staining batch to confirm the antibody specificity. to evaluate muc expression, a semi-quantitative method was performed; protein expression was assessed estimating the percentage of labelled cells and scored as follows: no staining, weak staining, moderate and strong immunostaining. the images were digitalised using a video camera connected to a microscope, and the semi-quantitative evaluation was estimated by counting positive cells in randomly selected fields, avoiding necrosis. statistical analysis the statistical analysis was conducted using prism version software. the anova and post-hoc dunett`s multiple comparison test and tukey’s honestly significance difference were applied. to assess the correlation between the tumour characteristics and the ca 15– serum level, the pearson correlation coefficient was used. a p <  was regarded as significant, whereas p <  was regarded as highly significant.  cell lines immunocytochemistry each tumour cell line used in this study showed cytoplasmic expression of muc and the positivity was mainly observed in a granular pattern. frequently, cytoplasmic staining was observed together with a membrane reaction in a mixed pattern. cmt-w cmt-w1m, cmt-w and cmt-w2m cell lines showed a high expression of muc whereas cmt-u cmt-u and p cell lines showed weak 115d expression. the same results were obtained using antibodies against df. the mean optical density of brown coloured antigen reflecting 115d expression was counted. as a control, the mcf cell line was used, which expresses the antigen at the level of. no significant differences were found between the expression of the df antigen in the control cell line and in the following cell lines: cmt-w, cmt-w1m, cmt-w, and cmt-w2m. a significant difference was found between the 115d expression in the mcf cell line and the cmt-u cell line, whereas highly significant differences were found in the 115d expression in the cmt-u and p cell lines in comparison with mcf. invasion of canine mammary cancer cell lines the invasion assay results and growth characteristics on the matrigel matrix of the canine mammary cancer cell lines cmt-u cmt-u p cmt-w and cmt-w have previously been reported; however, these cell lines have not been compared with cmt-w1m and cmt-w2m cell lines. the assay showed that only cmt-w cmt-w1m, cmt-w and cmt-w2m cells migrated through the matrigel. the fluorescence intensity related to the migration of the cmt-w cells was, and the migration of the cmt-w1m cells was, whereas migration of the cmt-w and cmt-w2m cells was and, respectively. to confirm the ability of these cell lines to invade the matrix, we have assessed their growth characteristics on the matrigel matrix. after hours of culturing on matrigel, the cmt-u cmt-u and p cell lines formed colonies, whereas the cmt-w cmt-w1m, cmt-w and cmt-w2m cell lines formed branching structures, which indicated their invasive phenotype. sds-page and western blot immunoblot analysis using anti 115d antibodies revealed the presence of two reactive protein bands in the human mcf cell line, used as a positive control. the band with a lower molecular weight is approximately 250 kda, while the mw of the major protein band appears to be more than 300 kda. a single protein band, with the same high mw as the positive control, was detected for the cmt-w and cmt-w cell lines, as well as for the cmt-w1m and cmt-w2m cell lines. no bands were observed for the cmt-u cmt-u and p cell lines. immunoblotting performed with the anti df antibody gave overlapping results. the western blot analysis with anti ß-actin antibodies showed that variability in protein loading could not account for the observed differences in 115d expression. animals, serum biochemistry, histology and immunohistochemistry most dogs in our series were mixed breeds, followed by german shepherd dogs. the mean age was ±  years, with an increased prevalence among intact females. the mean diameter of nodules was  ±   cm, and skin ulceration was present in 22% of cases. none of these patients had apparent nodal involvement. with respect to the histopathological analyses based on the who’s classification, complex carcinoma was the most common type of tumour followed by simple carcinoma and carcinosarcoma; necrosis and inflammation were observed in 62% and 42% of cancer cases, respectively. according to elston and ellis’s method, 66% of tumours were classified as grade i, 28% were classified as grade ii and 6% were classified as grade iii. ca 15– was detected in all of the analysed sera. furthermore, dogs with malignant mammary tumours had a significantly higher median activity of serum ca 15– than the control group. when we compared tumour carbohydrate antigen 15– serum levels and the pathological variables considered in this study, no statistically significant differences between ca 15– and age, history of ovariohysterectomy, numbers and sizes of tumour and histological types were observed. furthermore, because the inflammatory and necrotic processes were often associated with cancer, they did not appear to have influenced the serum levels of ca 15– interestingly, a highly significant correlation was found between ca 15– serum levels and the tumour grade. in grade i tumours, the average concentration of this marker was approximately half compared with that of grades ii and iii. given the fact that grade iii neoplasms were represented by a small number and that their corresponding mean serum level was not significantly different from grade ii tumours, they were included in the same group. relation between tumour size, skin ulceration, histopathological diagnosis, inflammation, necrosis and histological grade of canine mammary cancer tissues and serum ca 15– levels. the pearson correlation coefficient was applied using prism version software. with immunohistochemistry, df antigen was detected in all normal gland tissues, and, as expected, the immunoreactivity was almost restricted to the apical membrane of epithelial cells, which displayed a weak to moderate staining. the positivity was more evident in ductal epithelial cells and in luminal secretions. in benign mammary lesions, the staining was observed in 7/ of the analysed cases, and the pattern of reaction was weak-to-moderate, usually restricted to the apical membrane of the epithelial cells. approximately 20% of normal ducts also showed a slight apical staining. in complex adenomas, simple adenoma and benign mixed tumours, a cytoplasmic stain was also detected, while in the dysplastic lesions, no evidence of reaction was found. in malignant mammary tumours, the apical staining was almost always observed together with a cytoplasmic expression in a mixed pattern with variable intensity and mostly in diffuse or granular pattern. it is important to note that the reaction affected all of the cells in carcinoma samples, including with strong and with moderate staining. in complex carcinomas, epithelial cells showed a diffuse positivity in cases, with a greater intensity at the periphery of the neoplastic foci. in the marginal areas of invasive tumours, markedly positive neoplastic cells invading the surrounding adipose tissue were frequently observed. in simple carcinomas, a diffuse expression was detected in cases and most evident in the apical membrane of neoplastic cells that formed tubular structures. this tumour had a strong tendency to infiltrate the surrounding vessels, and lymphatic emboli, when present, exhibited a strong cytoplasmic positivity. in carcinosarcomas, df expression was very weak in 100% of cases because the sarcomatous cells were preponderant and only a few stained epithelial cells were observed. in hyperplastic or normal lobules that sometimes circumscribed the tumour proliferated areas, a weak positivity was also detected. in all of the tissues examined, myoepithelial and stromal cells, lymphocytes, macrophages and blood vessels did not express immunoreactivity. no significant correlations were found between df overexpression and serum ca 15– levels. discussion in veterinary medicine, a variety of assays and techniques have been used in an attempt to identify the prognostic markers for neoplastic diseases, including clinical parameters, histologic features, immunohistochemistry, quantitative pcr, evaluation for genomic mutations and specific serum protein levels or enzyme activity analyses. to investigate the potential functional relevance of ca 15– as a serum tumour marker in veterinary oncology, we performed a multidisciplinary study on canine neoplastic mammary tissues and cell lines. the main finding in western blot analysis regarded the positive interaction between the human antibodies and the canine muc protein. the protein sequence of epitope df in humans is trpapgs, whereas the canine df epitope sequence is srpapss. we used the blosum amino acid substitution matrix to score alignments between evolutionarily divergent protein sequences. it revealed a high similarity between these two epitopes in humans and in dogs. thus, theoretically, the similarity of the protein sequence of the df epitope is very high in these two species. nevertheless, the results of wb showed that anti 115d and anti df antibodies display cross-reactivity with canine samples. the icc analysis of the muc expression in canine mammary cancer cell lines showed staining patterns similar to those observed in humans. in wb, a double protein band with a high molecular weight was observed in mcf- cells. in the canine tumour cell lines cmt-w cmt-w cmt-w1m and cmt-w2m, we found a single protein band of muc with a molecular weight similar to the higher human form. however, when we extended the time of the chemical exposure, some films revealed an apparent reactive point with a molecular weight of approximately 250 kda. these data are still under investigation because it is difficult to determine whether it is an actual protein band or an artefact of the analysis, considering the low sensitivity of the test. our immunocytochemistry results differ from those obtained using western blot. the immunocytochemical examination revealed muc expression in all of the examined cell lines. however, only the cmt-w cmt-w1m, cmt-w and cmt-w2m cell lines expressed muc at high levels, similar to mcf. the remaining cell lines exhibited a weaker expression of muc which differed significantly from the mcf cell line. according to the subject data, a higher expression of muc is observed in more invasive metastatic cancer. our findings support this hypothesis, as cmt-w cmt-w1m, cmt-w and cmt-w2m cell lines with invasive phenotypes showed a higher muc expression. thus, a lack of muc expression in non-invasive p cmt-u and cmt-u cell lines in western blot analysis may be due to low levels of this antigen expression and/or the low sensitivity of the western blot analysis. although both methods detect a specific protein expression, they significantly differ from each other on what may explain the differences in the obtained results. similar variations between the results obtained using western blot and immunocytochemistry have previously been reported, showing that immunocytochemistry is a more sensitive method than the western blot in cases of weak antigen expression. statistical analysis allowed us to demonstrate that ca 15– serum levels correlate positively with the degree of tumour proliferation and differentiation, as a significantly higher concentration of ca 15– was found in grades ii-iii than in grade i carcinomas. tumour size, skin ulceration, necrosis, inflammation and the histological type of mammary cancer were not significantly related to the serum levels of ca 15– the existence of a relationship between ca 15– serum levels and tumour grading appears to be logical because we assumed that tumour markers reflect the number and the activity of neoplastic cells as well as their products that access the circulation where the marker is measured. therefore, this parameter could be taken into consideration as potentially enabling a better evaluation of a tumour’s malignancy, particularly for establishing the preoperative stage of the neoplasm and providing useful guidelines when determining the surgical approach. another finding of our study, as expected, is the expression of muc in normal canine mammary tissues and in mammary gland benign lesions. the reaction was primarily restricted to the apical membrane of epithelial cells. however, in benign tumours, the pattern of reaction was extended to the cytoplasm of the luminal epithelial cells. these results are in absolute parallel to those observed in human mammary tissues.. among the carcinomas, we observed muc activity in 68% of the analysed cases that displayed a strong immunostaining in a mixed pattern, which comprises the whole cell population in several carcinoma samples. this finding is in accordance with the observation of lacunza, which demonstrated a correlation between muc protein over-expression and gene amplifications in benign and malignant breast tumours. many studies have revealed that this event could be linked to the presence of membrane-associated muc molecular portions that are shed from the surface of tumour cells to the cytosol by modulation of its glycosylation state. in the present study, there was no significant correlation between the over-expression of muc and ca 15– the circulating form of the df antigen. however, a significant correlation has been found between the histopathological grade of the tumour malignancy and muc expression and between the histopathological grade of the tumour malignancy and the circulating ca 15– level. we suppose that a lack of a correlation between muc over-expression in tissues and the serum level of ca 15– may be related to the different antibodies used in the ihc and ca 15– tests.  to the best of our knowledge, this is the first report demonstrating a significant correlation between serum ca 15– expression and tumour malignancy in dogs with mammary cancer. our results may provide the basis for developing a novel approach in veterinary research, although additional studies using a large number of healthy and pathological clinical samples to validate these preliminary results and to establish normal and pathological serum ranges of ca 15– antigen in dogs. clearly, this pilot study is only the beginning of understanding the potential application of this serum tumour marker in canine mammary cancer management, and we hope that these findings will encourage future research work in this field. competing interest the authors declare that they have no competing interests. authors’ contributions em and mk conceived the study and drafted the manuscript. em performed the histopathology and the scoring process and supervised the immunohistochemical procedure. mk, kp and cc performed cell culture, immunocytochemistry and contributed to western blot analysis. mk performed the 3d culture. km performed the invasion assay. adg and kf performed the western blot. ff and mk performed the statistical analyses. mcm and ep performed the chemiluminescent assay. all authors read and approved the final manuscript. acknowledgments we thank prof. dr. maciej ugorski, dr. joanna polańska, dr. gerard rutteman, dr. eva hellmen and dr. luisa lanfrancone for providing the cell lines used in this study. this work was partially supported by grant no. n n from the ministry of sciences and higher education.
as a positive control, the human breast carcinoma cell line mcf and tissue were used. the results of this study reveal that ca 15– is expressed in both canine mammary tumour cell lines and tissues and that serum levels significantly correlate with the histological grade of the malignancy. we also investigated serum ca 15– activity to establish whether its expression could be assigned to several tumour characteristics to evaluate its potential use as a serum tumour marker in the canine mammary oncology field. mammary tumours are the most common malignancy diagnosed in female dogs and a significant cause of mortality and morbidity in this species. immunohistochemical expression of ca 15– was evaluated in canine mammary cancer cell lines and malignant mammary tumours. immunocytochemical analysis revealed ca 15– expression in all examined canine mammary cancer cell lines, whereas its expression was confirmed by immunoblot only in the most invasive cells. carbohydrate antigen 15– is a mucinous glycoprotein aberrantly over-expressed in human mammary neoplasms and one of the most widely used serum tumour markers in women with breast cancer. to assess ca 15– staining, a semi-quantitative method was applied. a high statistical correlation between serum ca 15– levels and the degree of tumour proliferation and differentiation was shown, which indicates that the values of this serum marker increase as the tumour stage progresses. in the tissue, an immunohistochemical staining pattern was observed in of the malignant tumours. the aim of this study was to investigate the antigenic analogies of human and canine ca 15– and to assess its expression in canine mammary cancer tissues and cell lines.
 mammary tumours are the most common malignancy diagnosed in female dogs and a significant cause of mortality and morbidity in this species. carbohydrate antigen 15– is a mucinous glycoprotein aberrantly over-expressed in human mammary neoplasms and one of the most widely used serum tumour markers in women with breast cancer. the aim of this study was to investigate the antigenic analogies of human and canine ca 15– and to assess its expression in canine mammary cancer tissues and cell lines. immunohistochemical expression of ca 15– was evaluated in canine mammary cancer cell lines and malignant mammary tumours. as a positive control, the human breast carcinoma cell line mcf and tissue were used. to assess ca 15– staining, a semi-quantitative method was applied. to confirm the specificity and cross-reactivity of an anti-human ca 15– antibody to canine tissues, an immunoblot analysis was performed. we also investigated serum ca 15– activity to establish whether its expression could be assigned to several tumour characteristics to evaluate its potential use as a serum tumour marker in the canine mammary oncology field.  immunocytochemical analysis revealed ca 15– expression in all examined canine mammary cancer cell lines, whereas its expression was confirmed by immunoblot only in the most invasive cells. in the tissue, an immunohistochemical staining pattern was observed in of the malignant tumours. a high statistical correlation between serum ca 15– levels and the degree of tumour proliferation and differentiation was shown, which indicates that the values of this serum marker increase as the tumour stage progresses.  the results of this study reveal that ca 15– is expressed in both canine mammary tumour cell lines and tissues and that serum levels significantly correlate with the histological grade of the malignancy. 