Bovine Ocular Squamous Cell Carcinoma Research Articles

The role of oxidative and nitrosative stress in bovine ocular squamous cell carcinomas

Oxidative stress is strongly linked to carcinogenesis, especially head, neck and oral SCCs in humans. In this study, we aimed to evaluate the role of oxidative and nitrosative stress in Bovine ocular squamous cell carcinomas (BOSCCs) using immunohistochemical markers such as Nitrotyrosine (NT), Malondialdehyde (MDA) and 8-hydroxy-2’ -deoxyguanosine (8-OHdG). Tissues were collected from 24 cattle brought to the Pathology Department for routine diagnosis. Tumour samples were fixed in 10% neutral buffered formalin, processed routinely, and embedded in paraffin wax. Tissue sections were cut into 5 μm thickness and stained with Hematoxylin-Eosin to detect histopathological changes. The sections were examined under a light microscope and photographed. The avidin-biotin-peroxidase method was used as immunohistochemical staining. It was observed that the cauliflower-like tumoral masses were mostly located on the upper and lower eyelids, the third eyelid, cornea, limbus, or covering the entire eyeball. NT, MDA and 8-OHdG expressions were statistically increased in poorly-differentiated (PD) cases compared to welldifferentiated (WD) and moderately-differentiated (MD) cases. On the basis of the results of the increase in these markers, we concluded that nitrosative and oxidative stress may have an important role in the carcinogenesis of BOSCCs.

Bovine ocular squamous cell carcinoma of cattle: A case report

Bovine ocular squamous cell carcinoma of cattle: A case report

Current Status of Bovine Ocular Squamous Cell Carcinoma

Bovine ocular squamous cell carcinoma (BOSCC) is one of the important malignant neoplasms of cattle arising from squamous epithelium. The tumour was recorded at the age of three to eight years, and it affects most countries of the world. The possible etiologies include ultraviolet radiation, exposure to sunlight, nutrition, lack of pigmentation, bovine papilloma virus, increased altitude, decreased latitude, and hereditary and outdoor farming systems. Breeds of Hereford, Holstein, Jersey, Holstein cross and Jersey cross cattle are mostly affected, Common sites of the lower eyelid, third eyelid, limbus corneoscleral junction and cornea conjunctival junction were reported. Grossly the tumour is having plague, keratoma, papilloma and carcinoma formation. Cytological and histopathological appearance of BOSCC categorized as poorly differentiated squamous cell carcinoma, moderately squamous cell carcinoma and well differentiated squamous cell carcinoma. Immunohistochemistry using cytokeratin, MNF116, involucrin, P53, ki67 are the useful markers of BOSCC. Surgical extirpation is the choice of management of BOSCC and continued exposure to sunlight to be avoided to control this BOSCC.

Prognostic significance of PCNA, MMP-9 and p53 in bovine ocular squamous cell carcinomas

In this study, we aimed to evaluate PCNA, MMP-9 and p53 expressions according to differentiation degree of BOSCCs by immunohistochemical methods. The material of this study was composed of BOSCC biopsy samples taken from 30 cattle brought to our department. Tissue samples from cattles were fixed in 10% buffered formaldehyde solution, processed routinely, embedded in paraffin and sectioned at 5μm and stained with Hematoxylin & Eosine in order to detect histopathological changes. Sections were examined and photographed under a light microscope. Avidin-Biotin Peroxidase method was used as immunohistochemical method. We observed that the masses were nodular and cauliflower-like appearance. We found that the surfaces of the masses were highly hemorrhagic and ulcerative, sometimes covered with a purulent discharge. We defined cases with excessive and large numbers of keratin pearls, large tumoral islands, and evident squamous differentiation were defined as well-differentiated. In moderately-differentiated cases, we found that the number and size of keratin pearls decreased compared to well-differentiated cases. In addition, we observed that tumoral islets were smaller in these cases, similar to keratin pearls, and the number of poorly differentiated tumor cells increased. In poorly-differentiated cases, we determined that keratinization was either absent or formed in individual cells. As a result of statistical analysis, there was no statistically significant difference between good, moderate and poorly differentiated cases in terms of PCNA and MMP-9 expressions, but we found that the increase in p53 expression correlated with the degree of differentiation of the tumor. In conclusion, we think that p53 is a useful marker in determining the prognosis of BOSCCs.

Efficacy of Different Treatment Modalities for Eye Cancer in Bovines

Background: Bovine ocular squamous cell carcinoma also called cancer eye, represents the most economically important neoplasm in large animals. Hereditary factors, environmental factors (e.g: latitude, altitude, exposure to sunlight), lack of eyelid pigmentation, age and dietary habits have all been reported to play a role in the etiopathogenesis of bovine ocular squamous cell carcinoma. In addition, in cattle the etiology has been linked to a number of viral agents, especially bovine papilloma virus and bovine herpes virus type 1 and 5. Nevertheless, ultraviolet light, viruses and circumocular pigmentation are the major epidemiologic risk factors for the development of the tumor. The efficacy of different treatment modalities for eye cancer in bovines was studied.Methods: All the animals were divided in to four groups of six animals each. Surgical excision, intra lesion BCG vaccine, surgery with auto vaccine and surgery with mitomycin was the treatment protocols followed. Immunohistochemical studies were conducted to know the rate of proliferation of bovine ye cancer. Immunopositive reaction was observed against Vascular Endothelial Growth Factor and Epidermal Growth Factor Receptor in all the cases.Result: The benefits of different treatment modalities depended on nature, type, location and extensiveness of tumor. Early detection and aggressive treatment were essential in the successful management of these tumors. A multimodal treatment approach was recommended with surgery, immunotherapy and chemotherapy in providing 100% disease free interval.

Efficacy of BCG vaccine and Mitomycin C for the treatment of ocular squamous cell carcinoma in bovines

Bovine ocular squamous cell carcinoma (BOSCC) also called cancer eye, represents the most economically important neoplasm in large animals. Hereditary factors, environmental factors, lack of eyelid pigmentation, age and dietary habits have all been reported to play a role in the etiopathogenesis of bovine ocular squamous cell carcinoma. In group I, six animals with small, localized eye cancer where vision was not affected were included and subjected to intralesional injection of Bacillus Calmette- Guerin (BCG) vaccine at 0, 14, 35, and 56 days interval. In group II (six animals), surgical excision and Mitomycin C 0.04% topically on alternate weeks for two months as adjunctive therapy. All the animals recovered completely with no recurrence for a follow up period of one year.

Immunohistochemical Studies of Cytokeratins and Differentiation Markers in Bovine Ocular Squamous Cell Carcinoma.

Bovine Ocular Squamous Cell Carcinoma is considered the most common bovine tumour, causing significant economic losses, mainly by abattoir condemnations. To obtain a better insight into the genesis and neoplastic transformation, 19 samples collected at slaughter from Holstein Friesian cattle and diagnosed as Ocular Squamous Cell Carcinoma were studied. Tumours were histologically classified into three categories: poorly (26.3%), moderately (26.3%), and well differentiated (47.4%). Expression of keratins (MNF116 and LP34) and of cornified envelope precursors (involucrin and profilaggrin) was studied. Expression of MNF116 was observed in all carcinomas. LP34 immunostaining was seen in all but three carcinomas, one from each degree. Involucrin immunoreaction was observed in all but one poorly differentiated carcinoma. Profilaggrin was present in only two moderately differentiated carcinomas, in all but one well differentiated, and in all but one poorly differentiated. MNF116 is a useful marker to confirm the epithelial origin of the tumour and stain most neoplastic cells in these tumours. The expression of involucrin and LP34 demonstrates that, in all tumours, cells have reached the final program of differentiation, regardless of the grade. The expression of profilaggrin could indicate molecular changes during malignant transformation but their expression does not seem to be of diagnostic value.

Significance of Angiogenic Growth Factors in Bovine Ocular Squamous Cell Carcinoma

Bovine ocular squamous cell carcinoma (BOSCC) is the most common and economically significant neoplasm of the eye in cattle. This study investigated the role of angiogenic growth factors in the pathogenesis of BOSCC. Eighteen cases of BOSCC were classified histopathologically according to the degree of differentiation. Normal upper and lower eyelids and third eyelids collected from the right and left eyes of six healthy cattle aged 1-3 years, that had been presented for slaughter to abattoirs, served as controls. Transcription of genes encoding the angiogenic growth factors vascular endothelial growth factor-C (VEGF-C), basic fibroblast growth factor (bFGF), platelet-derived growth factor-C (PDGF-C) and platelet-derived growth factor receptor-α (PDGFR-α) was determined by quantitative real-time polymerase chain reaction (RT-PCR) in tissue obtained from paraffin wax blocks. Immunohistochemistry (IHC) was utilized to detect intensity of expression and tissue distribution of these growth factors. IHC results revealed that bFGF and PDGF-C were elevated significantly (P>0.05) and VEGF-C expression was decreased in BOSCC compared with healthy control tissue. PDGR-α expression was elevated; however, the difference, compared with control tissues, was not significant. RT-PCR results showed an inverse relationship to the results of IHC; where protein levels were elevated their corresponding mRNA levels were decreased or vice-versa. Angiogenic regulators therefore appear to play a role in the pathogenesis of BOSCC.

Ocular squamous cell carcinoma in Holstein cows from the South of Brazil.

Aim:The aim of this study was to investigate 10 cases of bovine ocular squamous cell carcinoma (OSCC) diagnosed in Holstein or Holstein-crosses cows.Materials and Methods:The investigation was performed exclusively in OSCC cases diagnosed in the State of Paraná and Santa Catarina. A combination of two previously existing histopathological classifications systems was used. The tissue samples were tested for immunoexpression of p53 and p16 and polymerase chain reaction (PCR) for bovine herpesvirus and papillomavirus.Results:A positive correlation between number of mitotic figures and tissue invasion was found. Anaplasia parameters did not correlate well with tumor invasion of deeper tissues and mitotic counts. Six of 10 OSCC cases were in animals with heavily pigmented eyes. Immunoexpression of p53 and p16 was observed in 3 cases each. Bovine herpesvirus and papillomavirus were not detected by PCR.Conclusions:Our results indicate that OSCC occurrence is most likely multifactorial with genetic, phenotypic, and environmental influences contributing to the pathogenesis of the disease.

Identification of QTL for UV-Protective Eye Area Pigmentation in Cattle by Progeny Phenotyping and Genome-Wide Association Analysis

Pigmentation patterns allow for the differentiation of cattle breeds. A dominantly inherited white head is characteristic for animals of the Fleckvieh (FV) breed. However, a minority of the FV animals exhibits peculiar pigmentation surrounding the eyes (ambilateral circumocular pigmentation, ACOP). In areas where animals are exposed to increased solar ultraviolet radiation, ACOP is associated with a reduced susceptibility to bovine ocular squamous cell carcinoma (BOSCC, eye cancer). Eye cancer is the most prevalent malignant tumour affecting cattle. Selection for animals with ACOP rapidly reduces the incidence of BOSCC. To identify quantitative trait loci (QTL) underlying ACOP, we performed a genome-wide association study using 658,385 single nucleotide polymorphisms (SNPs). The study population consisted of 3579 bulls of the FV breed with a total of 320,186 progeny with phenotypes for ACOP. The proportion of progeny with ACOP was used as a quantitative trait with high heritability (h2 = 0.79). A variance component based approach to account for population stratification uncovered twelve QTL regions on seven chromosomes. The identified QTL point to MCM6, PAX3, ERBB3, KITLG, LEF1, DKK2, KIT, CRIM1, ATRN, GSDMC, MITF and NBEAL2 as underlying genes for eye area pigmentation in cattle. The twelve QTL regions explain 44.96% of the phenotypic variance of the proportion of daughters with ACOP. The chromosomes harbouring significantly associated SNPs account for 54.13% of the phenotypic variance, while another 19.51% of the phenotypic variance is attributable to chromosomes without identified QTL. Thus, the missing heritability amounts to 7% only. Our results support a polygenic inheritance pattern of ACOP in cattle and provide the basis for efficient genomic selection of animals that are less susceptible to serious eye diseases.

P53 AND KI67 PROTEIN EXPRESSION IN OCULAR SQUAMOUS CELL CARCINOMAS OF DAIRY CATTLE

Role of the protein overexpression is very important in the pathogenesis, diagnosis and prognosis of neoplasia. Cases of overexpression of p53 and Ki67 proteins were investigated in 19 ocular squamous cell carcinomas (OSCC) in dairy cows. Microscopically, in six cases the tumors were carcinoma in situ (noninvasive carcinoma, G0), and in the remaining 13 cases the tumors were invasive carcinoma. The distribution of invasive carcinoma was as follows: G1, 1 (7.6%); G2, 1 (7.6%); G3, 3 (23.0%); G4, 8 (61.8%). Of the 19 tumors, 18 were immunoreactive for p53 (++), and 14 showed Ki67 expression (++). A significant positive correlation was found between the percentage of p53 stained nuclei and the degree of differentiation. The Ki67 index showed a significant negative correlation with the histological pattern, where the greatest proliferation was found in the most poorly differentiated OSCC. As in human squamous cell carcinoma(SCC), p53 overexpression is frequent in bovine OSCC, providing support for a possible role of the protein in the pathogenesis of this neoplasm.

Local therapy of cancer with free IL-2.

This is a position paper about the therapeutic effects of locally applied free IL-2 in the treatment of cancer. Local therapy: IL-2 therapy of cancer was originally introduced as a systemic therapy. This therapy led to about 20% objective responses. Systemic therapy however was very toxic due to the vascular leakage syndrome. Nevertheless, this treatment was a break-through in cancer immunotherapy and stimulated some interesting questions: Supposing that the mechanism of IL-2 treatment is both proliferation and tumoricidal activity of the tumor infiltrating cells, then locally applied IL-2 should result in a much higher local IL-2 concentration than systemic IL-2 application. Consequently a greater beneficial effect could be expected after local IL-2 application (peritumoral = juxtatumoral, intratumoral, intra-arterial, intracavitary, or intratracheal = inhalation). Free IL-2: Many groups have tried to prepare a more effective IL-2 formulation than free IL-2. Examples are slow release systems, insertion of the IL-2 gene into a tumor cell causing prolonged IL-2 release. However, logistically free IL-2 is much easier to apply; hence we concentrated in this review and in most of our experiments on the use of free IL-2. Local therapy with free IL-2 may be effective against transplanted tumors in experimental animals, and against various spontaneous carcinomas, sarcomas, and melanoma in veterinary and human cancer patients. It may induce rejection of very large, metastasized tumor loads, for instance advanced clinical tumors. The effects of even a single IL-2 application may be impressive. Not each tumor or tumor type is sensitive to local IL-2 application. For instance transplanted EL4 lymphoma or TLX9 lymphoma were not sensitive in our hands. Also the extent of sensitivity differs: In Bovine Ocular Squamous Cell Carcinoma (BOSCC) often a complete regression is obtained, whereas with the Bovine Vulval Papilloma and Carcinoma Complex (BVPCC) mainly stable disease is attained. Analysis of the results of local IL-2 therapy in 288 cases of cancer in human patients shows that there were 27% Complete Regressions (CR), 23% Partial Regressions (PR), 18% Stable Disease (SD), and 32% Progressive Disease (PD). In all tumors analyzed, local IL-2 therapy was more effective than systemic IL-2 treatment. Intratumoral IL-2 applications are more effective than peritumoral application or application at a distant site. Tumor regression induced by intratumoral IL-2 application may be a fast process (requiring about a week) in the case of a highly vascular tumor since IL-2 induces vascular leakage/edema and consequently massive tumor necrosis. The latter then stimulates an immune response. In less vascular tumors or less vascular tumor sites, regression may require 9–20 months; this regression is mainly caused by a cytotoxic leukocyte reaction. Hence the disadvantageous vascular leakage syndrome complicating systemic treatment is however advantageous in local treatment, since local edema may initiate tumor necrosis. Thus the therapeutic effect of local IL-2 treatment is not primarily based on tumor immunity, but tumor immunity seems to be useful as a secondary component of the IL-2 induced local processes. If local IL-2 is combined with surgery, radiotherapy or local chemotherapy the therapeutic effect is usually greater than with either therapy alone. Hence local free IL-2 application can be recommended as an addition to standard treatment protocols. Local treatment with free IL-2 is straightforward and can readily be applied even during surgical interventions. Local IL-2 treatment is usually without serious side effects and besides minor complaints it is generally well supported. Only small quantities of IL-2 are required. Hence the therapy is relatively cheap. A single IL-2 application of 4.5 million U IL-2 costs about 70 Euros. Thus combined local treatment may offer an alternative in those circumstances when more expensive forms of treatment are not available, for instance in resource poor countries.

Prognosis Indicators and Economic Implications of Advanced Bovine Ocular Squamous Cell Carcinoma After Radical Surgery

Prognosis Indicators and Economic Implications of Advanced Bovine Ocular Squamous Cell Carcinoma After Radical Surgery

Treatment of ocular squamous cell carcinomas in cattle with interleukin-2

In total, 174 bovine ocular squamous cell carcinomas of varying sizes (20 to 2800 mm2 in area) were treated daily with peritumoural injections of solvent, or solvent containing 5000 U,...

Local interleukin-2 and interleukin-12 therapy of bovine ocular squamous cell carcinomas

Interleukin-2 and interleukin-12 have been used independently to successfully treat the induced and the spontaneous tumours in animals. This trial was done to determine if a combination of IL-2 and IL-12 in the treatment of spontaneous bovine ocular squamous cell carcinomas (BOSCC) would be more successful than IL-2 or IL-12 therapy by themselves. For this trial, we selected 25 BOSCC tumours seen on Holstein Fresian cows in Beatrice, Zimbabwe. The cows were randomly assigned to a treatment group of 5 days of IL-2 (200,000 U/day), 5 days of IL-12 (0.5 μg/day) or 5 days of IL-2 (200,000 U/day) and IL-12 (0.5 μg/day). At 20 months after treatment, the IL-2 therapy group had 63% complete regressions; the combination group had 38% complete regressions, which were significantly higher than the IL-12 group, which had 0% complete regressions at 20 months, despite having 29% complete regressions at 6 months. These results show that IL-2 therapy by itself and in combination with IL-12 is more successful than IL-12 by itself. However, combination therapy does not improve the outcome in comparison to IL-2 as a single therapy. It also proves that IL-2 is consistently successful in the therapy of BOSCC with over 60% complete regression, which corresponds to a number of other studies we have done on IL-2 therapy of BOSCC [Rutten, V.P.M.G., Klein, W.R., De Jong, W.A., Misdorp, W., Den Otter, W., Steerenberg, P.A., De Jong, W.H., Ruitenberg, E.J., 1989. Local interleukin-2 therapy in bovine ocular squamous cell carcinoma. A pilot study. Cancer Immunol. Immunother. 30, 165–169; Stewart, R.J.E., Hill, F.W.G., Masztalerz, A., Jacobs, J.J.L., Koten, J.W., Den Otter, W., 2003. Local low dose interleukin-2 therapy of bovine ocular squamous cell carcinomas in cattle in Zimbabwe, submitted for publication; Den Otter, W., Hill, F.W.G., Klein, W.R., Koten, J.W., Steerenberg, P.A., De Mulder, P.H.M., Rutten, V.P.M.G., Ruitenberg, E.J., 1993. Low doses of interleukin-2 can cure large bovine ocular squamous cell carcinoma. Anticancer Res. 13, 2453–2455; Den Otter, W., Hill, F.W.G., Klein, W.R., Koten, J.W., Steerenberg, P.A., De Mulder, P.H., Rhode, C., Stewart, R., Faber, J.A., Ruitenberg, E.J., 1995. Therapy of bovine ocular squamous cell carcinoma with local doses of interleukin-2: 67% complete regressions after 20 months of follow-up. Cancer Immunol. Immunother. 41, 10–14].

Immunohistochemical Studies of Epithelial Cell Proliferation and p53 Mutation in Bovine Ocular Squamous Cell Carcinoma

Bovine ocular squamous cell carcinoma (OSCC) is the second most common cause of rejection due to neoplasia in slaughterhouses on Sao Miguel Island, Azores, and accounts for significant economic losses. To obtain a better insight into the genesis and neoplastic transformation process of bovine OSCC, abnormal protein expression and proliferation index were assessed by the immunohistochemical evaluation of p53 and Ki67, respectively. OSCC samples were collected from 15 bovines and were classified histologically according to the degree of differentiation into three categories: poorly, moderately, and well differentiated. Immunohistochemistry using polyclonal anti-human p53 antibody and polyclonal anti-human Ki67 antibody was performed. Ten of 15 tumors tested were immunoreactive for p53. Twelve tumors demonstrated Ki67 expression. As in human squamous cell carcinoma, p53 overexpression is frequent in bovine OSCC, providing support for a possible role of the protein in the pathogenesis of this neoplasia. No correlation between the percentage of p53 stained nuclei and the degree of differentiation was observed, although different patterns of staining were seen according to the degree of keratinization of the tumor cells. With the exception of the moderately differentiated OSCC group, Ki67 index showed significant correlation with the histologic pattern, increased proliferation being found in poorly differentiated OSCC (P = 0.013).

Search for bovine papilloma virus DNA in bovine ocular squamous cell carcinomas (boscc) and boscc-derived cell lines

SUMMARY Although the cause of bovine ocular squamous cell carcinoma (boscc) is attributed to viruses in addition to cofactors (eg, uv light), to our knowledge, the final causative agent has not been described. Bovine papilloma virus (bpv)- like particles were detected in approximately 33% of various putative precursor lesions of boscc. In contrast, it was reported that, using bpv-specific antibodies, it was not possible to detect viral antigens in boscc. Fourteen established boscc and 9 boscc-derived cell lines were examined for bpv dna. Probes of all 6 known bpv types were used in various hybridization assays. Neither Southern blot analysis, under high and low stringency conditions, nor in situ hybridization resulted in detection of bpv dna. Papilloma viruses were not observed in electron microscopic studies. Results exclude direct association between boscc and bpv types 1 to 6, or as yet unknown closely related bpv types. However, bpv may contribute to induction of precursor lesions or events leading to carcinogenic transformation, without being relevant for maintenance of the tumor.

Local interleukin-2 therapy in bovine ocular squamous cell carcinoma. A pilot study.

Five cows bearing bovine ocular squamous cell carcinoma (BOSCC) were treated with low doses of recombinant human interleukin-2 (rhIL-2). A dose of 2500 U rhIL-2 was injected intralesionally and another 2500 U were injected into the subparotid regional lymph node once a day during a period of 5 consecutive days. This cycle of 5 days was repeated after an interval of 2 days. Total regression of the tumor was observed in three out of five animals. One cow showed tumor regression (greater than 80%) accompanied by metastases to the regional lymph node that were observed from the fifth week after the beginning of the treatment. Growth of the tumor of the fifth animal was retarded after treatment. In vitro proliferation of peripheral blood lymphocytes was investigated in two animals and tumor-infiltrating lymphocytes in one animal during incubation in various rhIL-2 concentrations. Cytotoxic activity of both cell populations against P815, Yac-1 and BOSCC-derived cell lines increased during incubation with rhIL-2. Cultured BOSCC-infiltrating lymphocytes showed predominant killing of the BOSCC-derived autologous cell line after 4 weeks of culture. Preliminary phenotype analysis did not give conclusive results with respect to the types of cells responsible for killing.

Depression of cell-mediated immunity by tumour cell products: induction of resistance by immunotherapeutically active extracts of bovine ocular squamous cell carcinoma.

Tumours produce substances that inhibit the expression of cell-mediated immunity, in the form of delayed-type hypersensitivity in mice. Phenol-saline extracts of bovine ocular squamous cell carcinoma (BOSCC) which have immunotherapeutic activity in cattle were able to immunize mice against this depressive effect. Such immunization was effective against products of BOSCC, a spontaneous rat tumour, three of four human tumour cell lines and (in other experiments) mouse tumours. Phenol-saline extracts of mouse tumour cell lines were immunogenic (protective against depression of delayed-type hypersensitivity) in mice. Fractions of BOSCC phenol-saline extracts which were immunotherapeutically active in cattle were generally also protective in mice. The protective activity was lost after treatment with proteinase K, and was present in the supernatant after precipitation with 55% ammonium sulphate. It was not affected by treatment with RNase or DNase or by heating to 50 degrees C for 2 h. It was present in gel filtration fractions with an apparent molecular weight of 10,000-37,000 daltons. The immunogenic factor in mice and the immunotherapeutic factor in cattle may be related to each other.

Clinico-pathological aspects of immunotherapy by intralesional injection of BCG cell walls or live BCG in bovine ocular squamous cell carcinoma.

Bovine ocular squamous cell carcinoma (BOSCC) of clinical stage I, mostly situated in the third eyelid, was chosen as a therapy model for squamous cell carcinoma of the head and neck in humans. Block resection was found to be the best method of treatment. Regression was noticed in 19 out of 30 cows treated intratumourously with a single injection of live BCG or BCG cell wall vaccine, followed by recurrence in 8 cases. In 2 untreated cows, complete lasting regression occurred. Regression was significantly more frequently encountered in intratumourously treated cows than in controls. Regression was associated with a high mitotic index, severe infiltrating growth and small amounts of cellular (lymphoid) infiltration. Metastasis was found in 14 out of 50 cows: 5 in 10 untreated controls, 8 in 30 BCG treated cows and 1 in 10 surgically treated cows. The growth rate of progressively growing untreated and of some treated tumours was not associated with the mitotic index nor with other morphological characteristics tested. The mitotic index was found to be higher in the deep infiltrating layer than in the superficial layer of the primary tumour, suggesting that a single biopsy is not sufficiently representative for cell kinetic studies.