Polymorphisms in canine immunoglobulin heavy chain gene cluster: a double-edged sword for diabetes mellitus in the dog.

Insulin deficiency diabetes (IDD) in dogs is an endocrine disease similar to human type 1 diabetes. There are breeds more commonly affected, such as Yorkshire Terrier and Samoyed, suggesting an underlying genetic component. However, the genetic basis for canine diabetes mellitus (DM) is not fully established. We conducted both whole-genome scans for selection signatures and GWASs to compare the genomes of 136 dogs belonging to 29 breeds previously described at low or high risk for developing DM. Candidate variants were tested in dogs with a diagnosis of IDD and controls attending the Complutense Veterinary Teaching Hospital. The only genomic region under selection (CFA8:72 700 000-74 600 000; CanFam3.1) retrieved by our analyses is included in the immunoglobulin heavy chain gene cluster, which has already been related to human human type 1 diabetes susceptibility. This region contains two non-synonymous variants, rs852072969 and rs851728071, showing significant associations with high or low risk for IDD, respectively. The first variant, rs852072969, alters a protein poorly characterised in the dog. In contrast, rs851728071 was predicted to block the synthesis of an immunoglobulin variable (V) domain in breeds at low risk for DM. Although a large and diverse V gene repertoire is thought to offer a fitness advantage, we suggest that rs851728071 prevents the formation of an auto-reactive immunoglobulin V domain probably involved in the pathophysiology of IDD and, thus, decreases the risk for the disease. These results should be interpreted with caution until the functional roles of the proposed variants have been proved in larger studies.

Evaluation of palliative therapy, alone or in combination with toceranib phosphate, in dogs diagnosed with metastatic or recurrent beta-cell neoplasia.

AIMS: To compare survival in dogs with recurrent or metastatic insulinomas that were treated with palliative therapy, alone or in combination with toceranib phosphate and to assess tolerability of the combined therapy in dogs. MATERIALS AND METHODS: Dogs diagnosed with insulinoma were retrospectively identified in the records of the Veterinary Teaching Hospital Complutense (Madrid, Spain). Diagnosis of insulinoma was based on clinical signs of hypoglycaemia, concentrations in serum of glucose <3.3 mmol/L and insulin >10 µIU/mL and presence of a pancreatic mass on diagnostic imaging. Dogs were treated surgically or medically, according to clinical stage established by imaging techniques, and monitored with blood and urine analyses monthly and abdominal ultrasonography every 3 months until death. Dogs that presented with metastatic disease at diagnosis or with recurrent hypoglycaemia after surgery were treated, according to the owner's decision, with one of two treatment protocols: palliative therapy alone (control group, n=7: diet, prednisone, famotidine or omeprazole, ±octreotide) or palliative therapy in combination with toceranib (treatment group, n=5; median dose of toceranib 2.52 mg/kg). Overall survival time (OST) and adverse events were compared between the two treatment groups. RESULTS: The OST was longer in the treatment group (median 399, min 125, max 476 days) compared to the control group (median 67, min 23, max 387 days; p=0.042). Dogs in the treatment group had a higher incidence of grade 1-2 gastrointestinal toxicity (diarrhoea) than dogs in the control group (p=0.010). In all cases, gastrointestinal toxicity was solved by temporarily discontinuing toceranib. CONCLUSIONS AND CLINICAL RELEVANCE: The use of toceranib combined with palliative treatment in dogs with suspect metastatic or recurrent insulinomas increased survival time and was adequate tolerated.

Profile of Steroid Receptors and Increased Aromatase Immunoexpression in Canine Inflammatory Mammary Cancer as a Potential Therapeutic Target.

Canine inflammatory mammary cancer (IMC) has been proposed as a model for the study of human inflammatory breast cancer (IBC). The aims of this study were to compare the immunohistochemical expression of aromatase (Arom) and several hormone receptors [estrogen receptor α (ERα), estrogen receptor ß (ERß), progesterone receptor (PR) and androgen receptor (AR)], in 21 IMC cases vs 19 non-IMC; and to study the possible effect of letrozole on canine IMC and human inflammatory breast cancer (IBC) in vitro using IPC-366 and SUM-149 cell lines. Significant elevations of the means of Arom Total Score (TS), ERß TS and PR TS were found in the IMC group (p = 0.025, p = 0.038 and p = 0.037, respectively). Secondary IMC tumours expressed higher levels of Arom than primary IMC (p = 0.029). Non-IMC PR- tumours contained higher levels of Arom than non-IMC PR+ tumours (p = 0.007). After the addition of letrozole, the number of IMC and IBC cells dropped drastically. The overexpression of Arom found and the results obtained in vitro further support canine IMC as a model for the study of IBC and future approaches to the treatment of dogs with mammary cancer, and especially IMC, using Arom inhibitors.

Prognostic value of histological grading in noninflammatory canine mammary carcinomas in a prospective study with two-year follow-up: relationship with clinical and histological characteristics.

In this prospective study, a canine-adapted histological grading method was compared with histopathological and clinical characteristics and was evaluated as a prognostic indicator in canine mammary carcinomas (CMCs). Recruited dogs with at least 1 malignant mammary tumor (n = 65) were clinically evaluated, surgically treated, and followed up (minimum follow-up 28 months, maximum 38 months). Histopathological diagnoses were performed according to Goldschmidt et al (2011). Tumors were graded as grade I (29/65), grade II (19/65), and grade III (17/65). The tumor size, clinical stage, histological diagnosis, presence/absence of myoepithelial proliferation, and regional lymph node metastases at diagnosis were significantly associated with histological grade. The histological grade, age, clinical stage, tumor subtype group, and lymph node metastases at time of diagnosis were significantly associated with the development of recurrences and/or metastases, cancer-associated death, and survival times (disease-free survival and overall survival) in univariate analyses. A subdivision of clinical stage I (T1N0M0) into stages IA and IB was proposed in terms of prognosis. The clinical stage, histological grade, and spay status were selected as independent prognostic variables (multivariate analyses) with disease-free survival as the dependent variable. When overall survival was evaluated as a dependent variable, clinical stage and histological grade were selected as the independent covariates. This grading system is a useful prognostic tool, facilitates histological interpretation, and offers uniform criteria for veterinary pathologists. Comparative studies on CMCs performed in different countries should take into account possible changes in the prognoses due to different proportions of spayed females among the selected dog population.

Cell adhesion molecules E-cadherin and CADM1 are differently expressed in canine inflammatory mammary cancer.

Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (IMC) are the most aggressive and lethal types of mammary tumors with specific characteristics such as exacerbated angiogenesis, lymphangiogenesis and lymphangiotropism. E-cadherin expression is another specific feature of IBC not previously studied in canine IMC. In this study, the expression of E-cadherin and CADM1 (Cell Adhesion molecule 1) and their possible role as key molecules involved in the pathogenesis of IMC were immunohistochemically analyzed in 19 canine IMC and 15 grade III non-IMC cases. E-cadherin and CADM1 expression was higher in IMC cases (p = 0.002, p = 0.008, respectively). In the IMC group, E-cadherin cytoplasmic immunolabeling was more frequent (p = 0.035) and it was associated to the expression of the angiogenic and lymphangiogenic factors COX-2 (p = 0.009), VEGF-A (p = 0.031) and VEGF-D (p = 0.008). The differential mRNA expression between IMC and non-IMC was studied by microarray analysis in 6 cases. E-cadherin gene (CDH1) was not up-regulated in IMC cases at a transcriptional level; interestingly CADM1 was 7-fold upregulated. The differential expression of E-cadherin protein in IMC suggests a possible role of E-cadherin in the characteristic exacerbated angiogenesis and lymphangiogenesis and further support IMC as a natural model for the study of human IBC. Future studies in IBC and IMC including a broad panel of adhesion molecules are necessary to elucidate their role in the metastatic process and angiogenesis.

Histological, immunohistological, and ultrastructural description of vasculogenic mimicry in canine mammary cancer.

Canine inflammatory mammary cancer (IMC) and human inflammatory breast cancer (IBC) are the most aggressive and lethal type of mammary cancer in female dogs and in women. The generation of microvascular channels by malignant tumor cells (endothelial-like cells [ELCs]) without endothelial cell participation (vasculogenic mimicry) has been reported in human breast cancer, including IBC, and is considered a new type of tumor angiogenesis. The aim of this study was to investigate the presence of ELCs in highly malignant canine mammary tumors (IMC and non-IMC) by histology, inmunohistochemistry (pancytokeratin, cytokeratin 14, vimentin, actin, desmin, vWF, CD31, and CD34), and electron microscopy. This retrospective study included 21 female dogs with diagnoses of IMC and 20 animals with metastatic grade III noninflammatory malignant mammary tumors (MMT). IMC tumors (33.33%) and MMT (5%) showed ELCs forming structures similar to small capillaries. The histological, immunohistochemical (positive to AE1/AE3 and cytokeratin 14, mostly negative to endothelial markers), and ultrastructural characteristics of these cells indicated vasculogenic mimicry. The higher frequency of this phenomenon in inflammatory versus noninflammatory canine mammary cancer is in agreement with previous studies in experimental and spontaneous human IBC, and it could be in relation with the extremely high lymphangiogenic capacity and metastatic lymphangiotropism characteristics of inflammatory breast cancer.

Survival time of dogs with inflammatory mammary cancer treated with palliative therapy alone or palliative therapy plus chemotherapy.

Seven of 30 female dogs diagnosed with inflammatory mammary cancer were given chemotherapy and palliative treatment, and the other 23 received only palliative treatment. The median survival time of the seven dogs given chemotherapy was 57 days, compared with 35 days for the 23 given only palliative treatment.

Comparison of non-selective adrenocorticolysis with mitotane or trilostane for the treatment of dogs with pituitary-dependent hyperadrenocorticism.

Forty-six dogs with pituitary-dependent hyperadrenocorticism were treated with mitotane by the non-selective adrenocorticolysis protocol and 40 were treated twice a day with trilostane. The treatment groups were compared by chi-squared tests, and survival data were analysed using Kaplan-Meier survival plots and a Cox proportional hazard method. The non-selective adrenocorticolysis protocol was very effective (89 per cent), its toxicity was moderate (24 per cent) and there were fewer recurrences (29 per cent) than reported with the classical selective adrenocorticolysis protocol (58 per cent). In a multivariate model, age and bodyweight at diagnosis were significantly negatively correlated with survival time. The median survival time of the dogs treated with trilostane twice a day (900 days) was longer (P=0.05) than that of the dogs treated with mitotane (720 days).

Quantification of epidermal growth factor receptor (EGFR) in canine mammary tumours by ELISA assay: clinical and prognostic implications.

The involvement of epidermal growth factor receptor (EGFR) is well established in human breast cancer, however, in canine mammary tumours (CMT), including inflammatory mammary carcinomas (IMC), still needs to be clarified. Enzyme immune assay techniques were used for EGFR determinations in tumour tissue from 45 bitches with CMT and in normal mammary glands from eight control dogs. Higher tissue EGFR levels were found in CMT compared with controls (P < 0.05). In malignant CMT, tissue EGFR elevated concentrations were statistically significantly associated with tumour relapse and/or distant metastasis during follow-up and with reduced disease-free and overall survival times. The IMC cases had the highest tissue EGFR levels compared with other malignant non-IMC tumours (P < 0.001). The results support the hypothesis that EGFR levels influence prognosis in malignant CMT, suggesting that EGFR may represent a therapeutic target in cases of high histological aggressiveness and especially in cases of metastatic phenotype and poor prognosis.

Adjuvant therapy for highly malignant canine mammary tumours: Cox-2 inhibitor versus chemotherapy: a case-control prospective study.

Cyclooxygenase-2 (Cox-2) enzyme participates in different steps of the carcinogenetic process and in canine mammary tumours (CMTs), a high expression of Cox-2 is associated with malignancy and tumour angiogenesis. The objectives of the study were to evaluate the disease-free survival (DFS) and overall survival (OS) of a Cox-2 inhibitor as adjuvant therapy in dogs with highly malignant (HM)-CMTs and compare it with that of dogs treated with chemotherapy and with control dogs. Twenty-eight dogs were prospectively included. After surgery, dogs were alternatively allocated into two treatment groups (chemotherapy with mitoxantrone n=8; Cox-2 inhibitor, firocoxib n=7). Control group (n=13) included dogs whose owners rejected adjuvant therapy. All dogs were followed up for two years or until death. The DFS was significantly higher in dogs that received adjuvant treatment (mitoxantrone or firocoxib) (P=0.030) than in control dogs. Dogs on firocoxib treatment had significantly higher DFS (P=0.015) and OS (P=0.048) than control dogs. The DFS and OS of dogs on mitoxantrone treatment were not statistically different from controls. In conclusion, this study supports the use of firocoxib for the treatment of HM-CMTs. Further studies are needed to compare the efficacy of chemotherapy drugs versus Cox-2 inhibitors as adjuvant treatment in these cases.

Role of steroid hormones and prolactin in canine mammary cancer.

In several animal studies, prolactin has been found to be essential for mammary epithelial development, and its administration has been consistently shown to increase the rate of mammary tumours. High levels of steroid hormones have also been suggested to enhance mammary cancer development. The present study investigates the levels of the following hormones in serum and in tissue homogenates in dogs bearing canine mammary tumours: prolactin (PRL), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 17beta-estradiol (17beta-E2) and estrone sulfate (S04E1). Eighty mammary tumours (40 dysplasias and benign and 40 malignant tumours) from 32 female dogs, and 10 normal mammary glands from eight female dogs without history of mammary tumours, were analysed. Prolactin and steroid hormones in serum and tissue homogenates, were analysed by enzyme immunoassays (EIA) techniques, previously validated for this animal species. Levels of prolactin in tissue homogenates were significantly different between malignant and benign mammary tumours (p<0.01). Serum prolactin concentrations were lower in the control group as compared with the group of dogs with benign tumours and in dogs with malignant tumours (p=0.01). Serum prolactin levels in dogs with benign lesions were not significantly different than those obtained from dogs with malignant tumours. Levels of steroid hormones were significantly higher in malignant tumours compared with the benign tumours and normal mammary glands (p<0.01) both in serum and homogenate determinations. Our results suggest that the canine neoplastic mammary gland could be a source of prolactin. Our hypothesis is that both prolactin and steroid hormones are involved in the growth of canine mammary cancer, and that they might have an autocrine/paracrine role in the maintenance of this disease.

H-ras immunohistochemical expression and molecular analysis of urinary bladder lesions in grazing adult cattle exposed to bracken fern.

Chronic ingestion of bracken fern (Pteridium spp.) by cattle produces upper alimentary tract and urinary bladder tumours causing a syndrome called bovine enzootic haematuria (BEH). Previous studies demonstrated ptaquiloside-DNA adducts and mutations in the h-ras gene in ileal epithelial cells of bracken fern-fed calves. Systematic inspection of the bladder mucosa of grazing cattle (n=126) from bracken-fern areas was carried out in a slaughterhouse. Of the 126 slaughterhouse cattle, 46 showed macroscopical lesions of the bladder. These bladders, together with six others known to have BEH, were examined histopathologically and by H-ras immunohistochemistry. Thirteen affected bladders were also examined by H-ras molecular analysis to detect mutations. Macroscopical and histological study of urinary bladder lesions found at the slaughterhouse revealed chronic cystitis (34.1%) and tumours (2.4%). There was significantly increased immunohistochemical expression of H-ras (P<0.05) in chronic cystitis (H-ras=53.24%) and bladder tumours (H-ras=63.60%) as compared with normal urinary bladders (H-ras=4.32%). A silent mutation (D38D) was detected in one animal with a mixed bladder tumour. The prevalence of urinary bladder lesions (chronic cystitis and tumours) obtained at the slaughterhouse was higher than expected. This study demonstrates that close inspection of urinary bladders of adult grazing cows is necessary to prevent possible human exposure to bracken-fern carcinogens. The absence of mutations in the codons of h-ras studied did not exclude the presence of polymorphisms in other regions of the gene (promoter or regulation sequences) or in other genes (belonging or not to the ras family) that significantly affect the H-ras protein.

Steroid hormone profile of canine inflammatory mammary carcinoma: a preliminary study.

Inflammatory mammary carcinoma (IMC) is the most aggressive spontaneous type of mammary malignant tumor both in women and dogs. Latest studies in dogs indicate that different endocrine mechanisms seem to be involved in inflammatory carcinomas (IMCs). The aim of the present study was to characterize the steroid hormone profile of inflammatory carcinoma, and to compare it with mammary dysplasias, benign tumors and other malignant tumors. Eighty-six mammary samples (10 normal mammary tissue, 21 dysplasias, 26 benign, 22 malignant, and 7 IMC) from 30 female dogs were used. Hormone levels of progesterone (P4), 17beta-estradiol (E2), androstenedione (A4), dehydroepiandrosterone (DHEA), and estrone sulphate (E1SO4) in tissue homogenates were measured by enzyme immunoassays (EIAs) techniques, previously validated for this species. IMC displayed the following steroid profile: P4: 13.80+/-0.56 microg/g; E2: 675.19+/-33.00 ng/g; A4: 631.73+/-70.73 microg/g; DHEA: 702.22+/-89.93 microg/g, and E1SO4: 2.84+/-0.32 mg/g. All of these hormones were significantly higher (P<0.001) compared with the hormone steroid profile determined for malignant, benign, dysplasias, and normal mammary tissue. The most relevant finding was the increased levels, two or three times, of both DHEA and E1SO4 in IMC respect to other groups (P<0.001). These results, together with the highest immunohistochemical expression of P450scc found in IMC, suggest the hypothesis that an autocrine mechanism could be especially involved in the development of canine inflammatory carcinoma.

BRCA1 expression in canine mammary dysplasias and tumours: relationship with prognostic variables.

BRCA1 is a nuclear phosphoprotein that participates in the regulation of the cell cycle. The role of the BRCA1 gene in canine mammary tissue and mammary tumours has not been studied. The present study examined immunohistochemically the expression and intracellular distribution of BRCA1 protein in two normal, seven dysplastic and 44 neoplastic canine mammary glands and its relationship with clinical and pathological variables and other prognostic parameters. Strong nuclear immunolabelling of BRCA1 protein was observed in the epithelial cells of the normal mammary glands and mammary dysplasias. The majority of benign tumours, and more especially of malignant tumours, showed a significant reduction in the nuclear expression of BRCA1 protein and an increase in cytoplasmic expression. Loss of BRCA1 expression was associated with high proliferation marker Ki-67 and ER-alpha negative tumours. The reduction and aberrant distribution of BRCA1 in canine mammary tumours were significantly associated with malignant characteristics. The results may indicate that BRCA1 has a role in the malignant behaviour of these tumours.

Expression of fibronectin and its integrin receptor alpha 5 beta 1 in canine mammary tumours.

Fibronectin and its integrin receptor alpha 5 beta 1 were studied by immunohistochemical methods in five normal canine mammary glands, four dysplastic glands and 18 mammary tumours. The aim of the study was to evaluate the possible changes in the alpha 5 beta 1 integrin receptor and its ligand fibronectin in relation to the metastatic capacity of canine mammary neoplasms. The immunostaining of alpha 5 beta 1 was very uniform in the hyperplastic glands but uneven in the mammary tumours. The expression of alpha 5 and beta 1 was diminished in metastatic tumours but there were some alpha 5-positive cells with pronounced features of malignancy and immaturity. Stromal fibronectin was increased in most cases and cytoplasmic staining of fibronectin was observed in epithelial and myoepithelial cells in mammary neoplasms but not in normal or dysplastic mammary tissue. There was no relationship between the content of alpha 5 beta 1 and the expression of fibronectin in canine mammary tumours.

DNA flow cytometry of canine mammary tumours: the relationship of DNA ploidy and S-phase fraction to clinical and histological features.

The DNA ploidy status and S-phase fraction (SPF) of benign proliferative lesions (BPL) and malignant tumours (MT) in the mammary glands of dogs were determined by flow cytometric analysis and the results were related to their clinical and histological features. Seven (14.3 per cent) of 49 BPL and 16 (48.5 per cent) of 33 primary MT had aneuploid G0,1 peaks (P < 0.001). Hypodiploid G0,1 peaks were found in one BPL and in five primary MT. The DNA ploidy status of primary MT was not found to be associated with their size, nodal status, grade of histological malignancy or nuclear grade. In several cases there was intra-tumour heterogeneity in ploidy status independent of histological heterogeneity. The SPF was significantly higher in 27 primary MT than in 45 BPL when diploid and aneploid cases were combined for comparison (P < 0.05), but not when only diploid cases were compared. Among the primary MT the SPF was higher in aneuploid than in diploid tumours (P < 0.05) and it was higher in five MT from five dogs with regional disease than in 22 MT from 19 dogs with local disease (P < 0.05). The SPF was positively correlated with the grade of histological malignancy (P < 0.05) but not with nuclear grade.

Inflammatory mammary carcinoma in dogs: 33 cases (1995-1999).

OBJECTIVE: To determine epidemiologic, clinical, and pathologic characteristics of inflammatory mammary carcinoma (IC) in dogs. DESIGN: Retrospective study. ANIMALS: 33 dogs with IC and 153 dogs with malignant mammary tumors other than IC. PROCEDURES: Medical records were reviewed, and signalment, history, physical examination findings, and results of thoracic radiography and necropsy were obtained. RESULTS: 33 of 436 (7.6%) dogs examined at a veterinary teaching hospital because of dysplasia or tumors of the mammary glands and 33 of 186 (17.7%) dogs with at least 1 malignant tumor had IC. Thirty-two of the 33 dogs were sexually intact. Dogs with IC were significantly older than were dogs with other malignant mammary tumors, and in dogs with IC, the tumor was initially noticed a mean of 52 days after the beginning of the last observed estrus, whereas in dogs with other mammary tumors, the tumor was initially noticed a mean of 137 days after the beginning of the last observed estrus. Dogs with IC were more likely to be anorectic and to have generalized weakness, weight loss, and thoracic metastases. Dogs with IC survived a mean of 25 days with palliative treatment. Histologically, involvement of dermal lymphatic vessels was identified in 14 of 19 (74%) dogs with IC. Two clinical forms of IC (primary and secondary) were identified. Dogs with primary IC had a worse clinical condition. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that IC is an uncommon but distinct entity in dogs. A histologic finding of dermal lymphatic involvement should be considered a hallmark for the pathologic diagnosis of IC in dogs.

Clinical and pathological prognostic factors in canine mammary tumors.

In the present study, clinical and reproductive variables and pathological features were evaluated as prognostic factors in canine mammary tumor disease. Seventy-four female dogs free of distant metastases at diagnosis were followed-up during a period of 18 months after the surgical excision of mammary nodules. Age at presentation, tumor size, lymph node involvement, histological malignant grade and nuclear grade were proven to act as variables able to predict the overall survival time in dogs with mammary neoplasms. Disease free survival time was associated to the age, the presence of multiple malignant tumors, tumor size and ulceration of the skin.

Use of aminoglutethimide in the treatment of pituitary-dependent hyperadrenocorticism in the dog.

The aim of this study was to evaluate the efficacy and safety of aminoglutethimide in the treatment of dogs with pituitary-dependent hyperadrenocorticism (PDH). Ten dogs were diagnosed with PDH based on clinical and laboratory data, adrenal function tests (adrenocorticotropic hormone [ACTH] stimulation test and urinary cortisol/creatinine ratio [UCCR] combined with a high dose oral dexamethasone suppression test) and ultrasonographic evaluation of the adrenal glands. Aminoglutethimide was administered daily at a dose of 15 mg/kg bodyweight for one month. Median basal cortisol concentration and post-ACTH cortisol concentration one month after treatment were significantly lower than pretreatment values. Complete response was achieved in one dog, and partial response was obtained in three dogs. Severe side effects of anorexia, vomiting and weakness occurred in one dog and medication was withdrawn. Two further dogs developed decompensations of concurrent diseases and medication was stopped in these animals as well. Mild toxicity occurred in four dogs. Moderate to severe elevations in liver enzymes occurred in all dogs. The efficacy of this drug is lower than that observed using mitotane and ketoconazole, and adverse effects limit its use. Aminoglutethimide, using the protocol described, cannot be recommended for long-term management of PDH in the dog.

Factors influencing the incidence and prognosis of canine mammary tumours.

Factors relating to the incidence of canine mammary tumours are reviewed. Increased age, intact status or ovariectomy after 2.5 years of age, as well as progestagen treatment, can all lead to an increased risk of mammary neoplasia in the bitch. In addition, obesity early in life, and a habitual diet based on home-made food (rich in beef and pork, and poor in chicken) as opposed to commercial food, are also associated with the occurrence of mammary tumours. Other aspects related to incidence are also discussed. Increased age at diagnosis, invasive growth (fixed to adjacent tissues), large tumour size, ulceration of skin, and axillary or inguinal node involvement are clinical parameters associated with a low chance of survival after surgical excision of mammary tumours. Histological typing and grading of the tumour allows the establishment of a prognosis, which is poor where there is tumour proliferation as measured by S-phase fraction determination and Ki-67 immunostaining.