Vitamin D receptor (VDR) expression in different molecular subtypes of canine mammary carcinoma.

BACKGROUND: The molecular-based classification of canine mammary carcinomas (CMCs) has been the focus of much current research. Both in canines and humans, the triple-negative (TN) molecular subtype of mammary cancer is defined by a lack of expression of progesterone receptor (PR), oestrogen receptor (ER) and HER2. It has a poor prognosis; no effective targeted therapy is available. Vitamin D displays anticarcinogenic properties, and the expression of its receptor (VDR) has been found in different molecular subtypes, being about 30-40 % of TN breast cancer (TNBC) positive to it. We assessed the VDR expression in the different molecular subtypes of 58 CMCs from 45 female dogs using an immunohistochemical panel for the molecular classification of included: PR, ER, HER2, cytokeratin (CK) 5, CK14, and Ki67. In addition, we studied the relationship among the molecular subtypes of CMCs and clinicopathologic parameters. RESULTS: Investigation showed VDR positivity in 45.0 % of the triple-negative CMCs (TNCMCs), 27.3 % of luminal B and 19.0 % of luminal A. Luminal A was the most molecular subtype represented of the total tumours (36.2 %), followed of TNCMCs (34.5 %), luminal B (20.7 %) and HER2-overexpression (10.3 %). Both HER2-overexpression and TNCMC subtypes were positively related to lymphatic invasion (P = 0.028), simple histologic subtype (P = 0.007), a higher histological grade (P = 0.045) and a trend to higher proliferation index (P = 0.09). CONCLUSIONS: The highest VDR expression was observed in TNCMC, being almost half of them (45 %) positive to this receptor. VDR expression was absent in HER2-overexpression tumours and low in luminal A and B molecular subtypes.

Vitamin D receptor expression in canine mammary gland and relationship with clinicopathological parameters and progesterone/oestrogen receptors.

The vitamin D receptor (VDR) belongs to the nuclear class II receptor family. VDR is a ligand transcription factor and mediates the actions of calcitriol, the active product of vitamin D synthesis. Nowadays, it is known that the biological actions of calcitriol include the capacity to modulate cancer features, such as proliferation and differentiation, apoptosis, angiogenesis, invasion and metastasis. VDR expression has been demonstrated in human breast cancer and vitamin D has emerged as a promising targeted therapy. We analyse the VDR expression in normal and neoplastic canine mammary tissue samples and its relationship with clinicopathological parameters and progesterone/oestrogens receptors (PR/ER). Expression of VDR, Ki67 (to evaluate the proliferation index, PI), PR and ER was assessed in 50 mammary gland tissue samples from 41 female dogs by immunohistochemistry. VDR-positive staining was found in the nuclei of both myoepithelial and luminal epithelial cell layers. VDR expression was higher in normal mammary tissue (37/37 cases, 100%) then followed by benign tumours (6/15 cases, 40%) and malignant tumours (9/34 cases, 26.5%) (P = .001). Female dogs aged ≥10 years had lower VDR expression compared with dogs younger (P = .017). Relationship between VDR and breed, number of tumours, tumour size, histologic subtype, histologic grade of malignancy, PI and PR and ER expression was not observed. Studies with more samples are necessary to further evaluate the possible role of VDR in the biological behaviour of canine mammary tumours, and to corroborate the possibility to use the dog as model for human breast cancer.

Prognostic impact of neoadjuvant aglepristone treatment in clinicopathological parameters of progesterone receptor-positive canine mammary carcinomas.

Neoadjuvant treatment of canine mammary carcinomas with the progesterone receptor (PR) antagonist aglepristone has a PR expression-related inhibiting effect on proliferation index (PI). The aim of this study was to evaluate the effect of the treatment in the disease-free period (DFP) and overall survival (OS) of canine mammary carcinomas. Fifty female dogs with mammary carcinomas were treated with aglepristone (n = 34) or oil vehicle (n = 16) before surgery (day 15). PR expression and PI were analysed by immunohistochemistry in samples taken at days 1 and 15. Epidemiological and clinicopathological data were assessed. DFP and OS data were retrieved every 4-6 months for at least 24 months after surgery. Aglepristone treatment increased DFP of animals bearing PR+ tumours with size smaller than 3 cm, complex and mixed tumours, with histologic grades I and II, and with PI ≤ 10%. Although further studies are necessary, current evidence points to treatment with aglepristone as useful for the management of canine mammary tumours.

Progesterone receptor isoform analysis by quantitative real-time polymerase chain reaction in formalin-fixed, paraffin-embedded canine mammary dysplasias and tumors.

Cloning and sequencing of the progesterone receptor gene in dogs have revealed 2 isoforms, A and B, transcribed from a single gene. Distribution of isoforms A and B in canine mammary lesions has hitherto been investigated only by Western blot analysis. This study analyzed progesterone receptor and its isoforms in formalin-fixed, paraffin-embedded tissue samples from canine mammary lesions (4 dysplasias, 10 benign tumors, and 46 carcinomas) using 1-step SYBR Green quantitative real-time polymerase chain reaction (RT-qPCR). Progesterone receptor was expressed in 75% of dysplasias, all benign tumors, and 59% of carcinomas. Carcinomas, and particularly simple epithelial-type carcinomas, displayed the lowest levels of expression. A high rate of agreement was recorded between RT-qPCR and immunohistochemical labeling. Isoforms A and B were successfully amplified, with correlation coefficients of 0.99 and amplification efficiencies close to 2, and were expressed in all lesion types analyzed. Predominance of A over B expression was observed in carcinomas and complex adenomas. Low-grade tumors exhibited higher progesterone receptor messenger RNA (mRNA) levels, but no difference was observed in the expression of isoform A versus B. Analysis of progesterone receptor mRNA isoforms by RT-qPCR was successful in routinely formalin-fixed, paraffin-embedded tissue samples and enabled the distribution of isoforms A and B to be identified for the first time in dysplasias, benign tumors, and malignant tumors of the canine mammary gland. These findings will facilitate future research into the role of progesterone receptor isoforms in the progression of canine mammary tumors.

Canine mammary tumors: a review and consensus of standard guidelines on epithelial and myoepithelial phenotype markers, HER2, and hormone receptor assessment using immunohistochemistry.

Although there have been several studies on the use of immunohistochemical biomarkers of canine mammary tumors (CMTs), the results are difficult to compare. This article provides guidelines on the most useful immunohistochemical markers to standardize their use and understand how outcomes are measured, thus ensuring reproducibility of results. We have reviewed the biomarkers of canine mammary epithelial and myoepithelial cells and identified those biomarkers that are most useful and those biomarkers for invasion and lymph node micrometastatic disease. A 10% threshold for positive reaction for most of these markers is recommended. Guidelines on immunolabeling for HER2, estrogen receptors (ERs), and progesterone receptors (PRs) are provided along with the specific recommendations for interpretation of the results for each of these biomarkers in CMTs. Only 3+ HER2-positive tumors should be considered positive, as found in human breast cancer. The lack of any known response to adjuvant endocrine therapy of ER- and PR-positive CMTs prevents the use of the biological positive/negative threshold used in human breast cancer. Immunohistochemistry results of ER and PR in CMTs should be reported as the sum of the percentage of positive cells and the intensity of immunolabeling (Allred score). Incorporation of these recommendations in future studies, either prospective or retrospective, will provide a mechanism for the direct comparison of studies and will help to determine whether these biomarkers have prognostic significance. Finally, these biomarkers may ascertain the most appropriate treatment(s) for canine malignant mammary neoplasms.

Isolation, purification, culture and characterisation of myoepithelial cells from normal and neoplastic canine mammary glands using a magnetic-activated cell sorting separation system.

Mammary gland tumours, the most common malignant neoplasm in bitches, often display myoepithelial (ME) cell proliferation. The aim of this study was to isolate, purify, culture and characterise ME cells from normal and neoplastic canine mammary glands. Monodispersed cells from three normal canine mammary glands and five canine mammary tumours were incubated with an anti-Thy1 antibody and isolated by magnetic-activated cell sorting (MACS). Cells isolated from two normal glands (cell lines CmME-N1 and CmME-N2) and four tumours (cell lines CmME-K1 from a complex carcinoma, CmME-K2 from a simple tubulopapillary carcinoma, and CmME-K3 and CmME-K4 from two carcinomas within benign tumours) were cultured in supplemented DMEM/F12 media for 40days. Cell purity was >90%. Tumour-derived ME cell lines exhibited heterogeneous morphology, growth patterns and immunocytochemical expression of cytokeratins, whereas cell lines from normal glands retained their morphology and levels of cytokeratin expression during culture. Cell lines from normal glands and carcinomas within benign tumours grew more slowly than those from simple and complex carcinomas. This methodology has the potential to be used for in vitro analysis of the role of ME cells in the growth and progression of canine mammary tumours.

Effect of different extenders on in vitro characteristics of feline epididymal sperm during cryopreservation.

To evaluate and compare the efficacy of various extenders for the cryopreservation of epididymal cat spermatozoa, two experiments were planned. Bovine and equine commercial extenders in the experiment 1 and TRIS-egg yolk-based extenders in experiment 2 were separately studied since the number of sperm collected per cat is reduced. Epididymal sperm samples were packaged into 0.25-ml straws and frozen. Vigour, motility, morphology, acrosome status, sperm viability and functional membrane integrity were assessed at collection, after cooling and after thawing, while DNA integrity was evaluated at 0- and 6-h post-thaw. Experiment 1 compared the effect of three non-feline commercial extenders - based on TRIS-egg yolk (Triladyl), egg-yolk-free medium (AndroMed) and skimmed milk-egg yolk (Gent) - on the quality of frozen-thawed epididymal cat sperm. Values for sperm motility and functional membrane integrity in cooled sperm diluted in Triladyl were higher (p < 0.001) than those recorded for Andromed and Gent. Except sperm morphology, the other assessed characteristics showed significant higher values in frozen-thawed sperm diluted in Triladyl than in Andromed and Gent extenders. Experiment 2 analysed the effects of three TRIS-egg yolk-based extenders, one non-feline commercial (Triladyl) and the other two prepared using different monosaccharides (glucose and fructose), on freezing-thawed sperm. Results showed that specifically prepared extenders for cryopreservation of feline spermatozoa performed better than the commercial extender Triladyl, although sperm quality during the freezing-thawing process did not significantly differ associated with the type of monosaccharide (glucose vs fructose) added to the mentioned extenders. Although TRIS-egg yolk-based extenders prepared in experiment 2 improved sperm cryoprotection, Triladyl remains a good option for practitioners who, for ease of use and availability, prefer to work with commercial extenders.

Use of CD10 as a marker of canine mammary myoepithelial cells.

CD10 is an important cell marker in the diagnosis of acute lymphoblastic leukaemia and of breast myoepithelial (ME) cells in humans. The objective of this study was to assess the value of CD10 as a marker of canine ME cells using immunohistochemistry on routinely processed normal, dysplastic and neoplastic mammary tissue. Five different CD10 positive cell types were identified on the basis of cell morphology, pattern of immunoreactivity, and on the co-expression of additional cell lineage-specific markers. Type 1 cells were typical fusiform cells with a ME cell phenotype (calponin- and cytokeratin [CK] 14-positive, CK8/18-negative). Type 2 cells were typical or atypical polyhedral cells with a luminal epithelial (LE) cell phenotype (calponin- and CK14-negative, CK8/18-positive). Type 3 cells had a type 1 phenotype with variable morphology, and type 4 were atypical neoplastic cells with a mixed ME/LE phenotype. Type 5 cells were typical fusiform cells with a stromal phenotype. Type 1 cells were considered normal ME cells and were found in all sample types; type 2 cells were considered normal or neoplastic LE cells and were also found in all sample types; types 3 and 4 cells were restricted to tumour samples and to malignant tumours, respectively, and type 5 cells were found in all sample types, although predominantly in neoplastic tissue. The findings indicate that the CD10 antigen is a sensitive (although not specific) marker of canine ME cells in normal, dysplastic and neoplastic mammary tissue. Differences in the distribution and staining intensity of CD10-positive cells suggest a number of potential roles for this protein in the pathogenesis of canine mammary neoplasia.

Activation of AKT in feline mammary carcinoma: a new prognostic factor for feline mammary tumours.

The PI3K/AKT/PTEN pathway is involved in the pathogenesis of several human cancers. This study investigated the biological and prognostic value of PI3K/AKT/PTEN pathway dysregulation in feline mammary tumours. Expression of p-AKT, HER2, PTEN and steroid receptors was assessed by immunohistochemistry (IHC) in 27 malignant and 12 benign mammary tumours from 39 female cats followed up over a 24-month period. Feline mammary carcinoma (FMC) cell lines were analyzed by Western blot and the feline AKT gene sequence was characterized. p-AKT expression statistically correlated with tumour malignancy, histological dedifferentiation and clinical recurrence. The animals with tumours expressing p-AKT had a shorter disease-free period than those with p-AKT-negative tumours. AKT activation was associated with HER2 expression and PTEN down-regulation, as occurs in human breast cancer, and feline AKT sequencing showed high homology with the human AKT gene. No AKT activation was observed in relation to either oestrogen receptor α (ERα) or progesterone receptor expression. Taken together, these data offer an explanation for AKT signalling and its role in FMC pathogenesis and prognosis, shedding new light on similarities between feline mammary tumours and hormone-independent breast cancer.

Aglepristone decreases proliferation in progesterone receptor-positive canine mammary carcinomas.

BACKGROUND: Progesterone receptor (PR) antagonist aglepristone (RU534) has been used successfully for pregnancy termination and therapy of pyometra, vaginal tumors, and mammary hyperplasia in bitches and queens. All of these conditions share with canine mammary carcinomas the expression of PR. OBJECTIVES: To study the effect of RU534 on proliferation and apoptosis in canine mammary carcinomas in relation to PR expression. ANIMALS: Twenty-seven nonspayed bitches with mammary carcinomas were treated with either 2 doses of 20 mg/kg RU534 (n = 22, RU534-treated group) or oil placebo (n = 5, control group) on days 1 and 8. METHODS: Tumor samples were collected before (day 1) and after (day 15) treatment for immunohistochemistry. PR expression, proliferation index (PI), and apoptotic index (AI) were determined using antibodies against PR, Ki67, and cleaved lamin A/C antigens, respectively. The effect of treatment on these parameters was analyzed. RESULTS: Differential expression of PR between day 1 (59.1% PR-positive tumors) and day 15 (36.4% PR-positive tumors) was observed in RU534-treated tumors exclusively. After RU534 treatment, mean PI was significantly decreased in PR-positive but unchanged in PR-negative RU534-treated tumors. A reduction of ≥20% in PI was found in 61.5% of RU534-treated tumors with PR expression. Conversely, no effect on AI was observed after RU534 treatment. CONCLUSIONS AND CLINICAL IMPORTANCE: Neoadjuvant RU534 treatment had PR expression-related inhibiting effects on proliferation of canine mammary carcinoma cells.

Influence of anaesthetic drugs on the epididymal sperm quality in domestic cats.

The present study investigated the effect of different anaesthetic agents commonly used in cats on the fresh and frozen-thawed epididymal sperm. Seventeen male domestic cats were castrated using pentobarbital, ketamine HCl or isoflurane. Sperm samples were recovered from epididymides and evaluated before and after freezing, determining the vigor, motility, morphology, acrosome status, sperm viability and functional membrane integrity. Fresh epididymal sperm was influenced by the drugs used, noting that motility features, i.e. vigor (p≤0.05) and progressive motility (p≤0.05), were higher for the inhalation anaesthetic while the others did not showed statistical differences. In frozen-thawed sperm samples, cats treated with barbiturics showed lower values for acrosome status (p≤0.05) and integrity and functionality of membrane (p≤0.05 and p≤0.01, respectively) than in the others groups. Results suggested that drugs used for castration in cats could affect the sperm quality and this should be considered when implementing sperm cryopreservation in the feline.

Myoepithelial cell layer integrity in canine mammary carcinoma.

The aim of this study was to determine whether the myoepithelial (ME) cell marker calponin could be used to analyze the integrity of the ME cell layer as a means of identifying canine mammary carcinoma in situ. Tissue from 74 canine mammary lesions was evaluated (two dysplasia, eight benign tumours and 64 carcinomas including one carcinoma in situ). The 63 carcinomas included examples of histological grade 1 (n=32), grade 2 (n=23) and grade 3 (n=8). Expression of calponin was determined by immunohistochemistry. The percentage of proliferating cells surrounded by a single layer of calponin-positive cells formed the basis of classification as type I (≥ 90%), type II (70-90%) and type III (≤ 70%). Expression of Ki67 was used to determine the proliferation index (PI). The malignant tumours comprised of an approximately equal mixture of type I, II and III lesions. The two examples of dysplasia, the carcinoma in situ and two thirds of the benign tumours were classified as type I lesions. Some overlap in the level of calponin expression was observed between benign and malignant tumours. Positive correlations between the degree of calponin expression and the type of lesion (i.e. benign versus malignant; R=+0.3, P=0.08) and the histological grade of malignancy (R=+0.54, P=0.000001) were found. A negative correlation between the degree of calponin expression and PI (R=+0.027, P=0.016) was found. The ME cell marker calponin may be used as an aid in the identification of canine carcinoma in situ, but the study of the ME cell layer integrity is not definitive for the diagnosis of malignancy in canine mammary tumours.

Oligodendroglial gliomatosis cerebri in a poodle.

Gliomatosis cerebri (GC) is a rare, diffusely infiltrating, glial cell tumour of neuroepithelial origin. This report describes a case of oligodendroglial GC in a 6-year-old male Poodle with central nervous system symptoms. Computed tomography revealed anomalous parenchyma density and ventricular asymmetry. Cerebrospinal fluid showed elevated protein (30 mg dL(-1)) and nucleated cell count (20 µL(-1)). Presumptive diagnosis of necrotizing meningoencephalitis was made. Because of rapid deterioration of the general condition of the animal, the dog was euthanized. Histologically there was an infiltration of round or ovoid neoplastic cells in the white matter of the left cerebral hemisphere and in leptomeninges. Immunohistochemistry showed that 80% of the neoplastic cells expressed Olig2 and some 50% expressed glial fibrilary acidic protein. On the basis of clinical, histological and immunohistochemical features, a diagnosis of oligodendoglial GC was done. This case represents the first report of a case of oligodendroglial GC in the canid.

Hypomyelination in three Weimaraner dogs.

Hypomyelination syndrome of the Weimaraner dog is a disease characterised by a reduction or absence of myelin in the axons of the central nervous system (CNS) exclusively. The objective of this study was to analyse the cause of this deficiency of myelin. Tissue samples of the CNS of three Weimaraner dogs with neurological signs were fixed in 10% formalin and embedded in paraffin wax, and histochemical, immunohistochemical and ultrastructural studies were performed. Histochemical staining with haematoxylin and eosin and Kluver-Barrera techniques showed generalised pallor in the peripheral areas of the ventral and lateral funiculi of the spinal cord. Immunohistochemical analysis showed a weak expression of both proteolipid protein (PLP) and myelin basic protein (MBP) and a marked decrease of Olig2(+) cells in the demyelinated areas. The immunohistochemical findings suggested a myelination or remyelination failure because of the smaller population of oligodendrocytes. However, PLP gene mutations may also be the cause of the decrease of PLP expression as described in other species.

Human follicular fluid from superovulated women inhibits progesterone receptor-dependent gonadotropin-releasing hormone self-priming in an estrous cycle-dependent manner in the rat.

These experiments investigated the involvement of gonadotrope progesterone receptor (PR) in the effects of the putative gonadotropin surge-attenuating factor (GnSAF) on gonadotropin (LH and FSH) secretion. Human follicular fluids (hFF) used in this study were aspirated from follicles in gonadotropin-treated women for in vitro fertilization. Samples were subjected to two-fold charcoal extraction of steroid hormones and two-fold inhibin immunoprecipitation. Gonadotropin secretion parameters were assessed by specific radioimmunoassays. In the first experiment, the effects of hFF on both basal and GnRH-stimulated gonadotropin secretion and GnRH self-priming were studied in incubated hemipituitaries from rats on each day of the 4-day estrous cycle. hFF inhibited only GnRH self-priming in pituitaries from rats in diestrus. In the second experiment, immunohistochemical PR expression and action were evaluated in pituitaries from rats in diestrus. PR-positive (PR10A9 antibody) gonadotropes were detected (4-5/field 40x), and antiprogestins added to the incubation media blocked the ligand-independent (GnRH) activation of PR effects on GnRH selfpriming. Finally, the third experiment evaluated the effects of hFF on P-induced potentiation of GnRH-stimulated LH secretion. GnSAF bioactivity, as evidenced by inhibition of PR-induced potentiation of GnRH-stimulated LH secretion, was found in diestrous pituitaries incubated with hFF. The results indicate that GnSAF attenuated GnRH-dependent LH secretion in diestrus through the inhibition of PR-dependent GnRH self-priming.

Expression of steroid receptors and calponin in a cervical leiomyoma in a young pig.

Thickening of the uterine cervix and bilateral ovarian cystic change was identified in a 6-month-old pig during routine abattoir inspection. Microscopically, the cervical lesion comprised a non-encapsulated mass of densely packed, large and monomorphic spindle cells within the myometrium. Immunohistochemically, the majority of these neoplastic cells expressed the cytoplasmic terminal smooth muscle differentiation marker calponin, the nuclear oestrogen receptor alpha and the progesterone receptor. The ovarian cysts were classified as follicular cysts. A diagnosis of leiomyoma of the uterine cervix with bilateral ovarian follicular cysts was made. The expression of calponin as a marker of smooth muscle differentiation in tumours of the genital tract of the pig has not previously been reported. The expression of steroid hormone receptors suggests a role for steroid hormones derived from the ovarian follicular cysts in tumourigenesis.

Bilateral retiform Sertoli-Leydig cell tumour in a bitch. Alpha-inhibin and epithelial membrane antigen as useful tools for differential diagnosis.

Sertoli-Leydig cell tumours with a retiform pattern similar to the pattern of the rete testis are a subtype of sex cord-stromal tumours recognized in the human WHO histological classification of ovarian tumours but not in the equivalent classification for domestic animals. The morphology of the tumour may be confused with that of the more common ovarian epithelial tumours. The gross, microscopical and immunohistochemical features of a canine retiform Sertoli-Leydig cell tumour and its comparison with the human counterpart are presented in this report. Both ovaries were enlarged and cystic. Microscopically, the tumour was cystic with tubulopapillary growth characterized by narrow, elongated branching tubules. Immunohistochemically, the tumour cells expressed alpha-inhibin, while epithelial membrane antigen was not detected, indicating a sex cord-stromal origin of the tumour. Additionally, the tumour cells expressed cytokeratin and vimentin in addition to oestrogen receptor alpha and progesterone receptor.

Effects of aglepristone, a progesterone receptor antagonist, in a dog with a vaginal fibroma.

A 12-year-old, entire, nulliparous crossbreed female dog was presented with a history of vulval bleeding, bulging of the perineum and faecal tenesmus. A firm, non-painful perineal mass, measuring 9.11x5.4 cm, with erythema was detected. Abdominal radiography showed compression and elevation of the rectal ampulla. A dose of 10 mg/kg aglepristone was administered subcutaneously on days 1, 2, 8, 15, 28 and 35. An incision biopsy was taken on day 15 and immunohistochemical analysis showed that the majority of neoplastic cells expressed progesterone receptors. Both the cutaneous erythema and the faecal tenesmus had resolved by day 28. A 50 per cent reduction in size was observed by day 60 (surgical excision). This study shows that benign tumours of the vagina of the dog that contain progesterone receptors can be reduced in size in a palliative or neoadjuvant setting using the progesterone receptor antagonist aglepristone.

Steroid receptors in canine and human female genital tract tumours with smooth muscle differentiation.

The expression of oestrogen receptor-alpha (ERalpha) and progesterone receptor (PR) was examined in 32 canine genital tract tumours diagnosed as smooth muscle tumours (benign or malignant, pure or mixed). The immunohistochemical expression of calponin was used to assess the smooth muscle differentiation of the tumours. Nineteen human uterine leiomyomas were also examined. Calponin expression was detected in 89.3% of canine and 100% of human genital tract tumours diagnosed as leiomyomas, as well as in the majority of other tumours examined (canine or human, genital or extragenital, benign or malignant) with the exception of canine negative control tumours (cutaneous fibroma and hepatoid gland adenoma). ERalpha was found in 56.3% of canine and 52.6% of human leiomyomas, while PR was found in 84.4% of canine and 94.7% of human tumours. These results indicate that calponin is a good marker for differentiating neoplasia of the canine genital system of uncertain origin, as in human patients. They also show that canine tumours with smooth muscle differentiation of the genital tract of the bitch express steroid hormone receptors, a finding that opens up the possibility of hormone therapy.

Intestinal glandular inclusions (glandular choristoma) in the mesenteric lymph node of a goat.

A glandular choristoma found in the mesenteric lymph node of a goat would appear to represent the first reported case of non-neoplastic glandular inclusions in domestic animals. The origin of this type of lesion may be difficult to determine, but in the present case cytokeratin expression patterns suggested that the inclusions had an intestinal origin.