Proposal of a 2-tier histologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior.

Currently, prognostic and therapeutic determinations for canine cutaneous mast cell tumors (MCTs) are primarily based on histologic grade. However, the use of different grading systems by veterinary pathologists and institutional modifications make the prognostic value of histologic grading highly questionable. To evaluate the consistency of microscopic grading among veterinary pathologists and the prognostic significance of the Patnaik grading system, 95 cutaneous MCTs from 95 dogs were graded in a blinded study by 28 veterinary pathologists from 16 institutions. Concordance among veterinary pathologists was 75% for the diagnosis of grade 3 MCTs and less than 64% for the diagnosis of grade 1 and 2 MCTs. To improve concordance among pathologists and to provide better prognostic significance, a 2-tier histologic grading system was devised. The diagnosis of high-grade MCTs is based on the presence of any one of the following criteria: at least 7 mitotic figures in 10 high-power fields (hpf); at least 3 multinucleated (3 or more nuclei) cells in 10 hpf; at least 3 bizarre nuclei in 10 hpf; karyomegaly (ie, nuclear diameters of at least 10% of neoplastic cells vary by at least two-fold). Fields with the highest mitotic activity or with the highest degree of anisokaryosis were selected to assess the different parameters. According to the novel grading system, high-grade MCTs were significantly associated with shorter time to metastasis or new tumor development, and with shorter survival time. The median survival time was less than 4 months for high-grade MCTs but more than 2 years for low-grade MCTs.

Some prognostic and epidemiologic factors in canine osteosarcoma.

Naturally occurring skeletal osteosarcomas in a series of 144 untreated dogs were found especially to involve the ends of the long bones of the forelimbs and affected predominantly older male dogs of giant and large breeds. Most tumors were large and partially necrotic and had extended into soft tissues. Of 12 host and tumor characteristics tested in the first part of the study, tumor diameter and volume were significantly associated with the presence of pulmonary metastases at autopsy. The second part of the study revealed that extension of the tumor into the soft tissues and localization of the tumor in the hind legs were associated with a poor prognosis, whereas the fibrosarcomatous type of tumor was associated, as in man, with a favorable prognosis. An association between the 12 characteristics tested was found in 11 of 78 combinations at the 5% level and in 5 combinations at the 1% level. Affected giant dogs were generally younger than affected small and medium-sized dogs. Especially in giant dogs, the osteosarcomas involved the long bones and were of relatively large diameters. The sarcomas in female dogs were larger in volume than those in males. Pure osteoblastic osteosarcomas were generally smaller than combined (chondroblastic and fibroblastic) osteosarcomas. Peritumorous lymphocytes and plasma cells were present in 50% of the dogs, especially in small and young dogs. When compared with a reference population, great Danes, rottweilers, German shepherds, and boxers were found to be overrepresented in the osteosarcoma group.

Prognostic factors in canine mammary cancer.

From a follow-up study of dogs surgically treated for mammary cancer, ten characteristics were analyzed statistically with special reference to their association with prognosis (expressed as survival for 2 years). The interrelations among five of the characteristics were also tested. The histologic type (descending range in malignancy: sarcomas greater than simple carcinomas greater than complex carcinomas), mode of growth (highly infiltrating greater than moderately infiltrating greater than expansive), clinical stage of complex carcinomas (large tumors and/or tumors involving the skin or underlying tissue greater than small, well-defined tumors), and size (greater than 15 cm greater than 11-15 cm greater than 5-10 cm greater than 0-5 cm) were of definite prognostic importance. The histologic grade was of possible prognostic importance. Localization, type of surgical therapy (mastectomy, block-dissection), growth in lymph vessels, involvement of regional lymph nodes, and duration of symptoms before treatment were not important to prognosis. A comparison between the factors associated with the prognosis of canine and human mammary cancer showed many similarities. However, the involvement of regional lymph nodes, important in women, was not so in bitches.

Isolation of autonomously growing dog mammary tumor cell lines cultured in medium supplemented with serum treated to inactivate growth factors.

Conventional methods for isolation of cell lines from carcinomas suffer inherently from a lack of advantage for proliferation of transformed cells as opposed to contaminating fibroblasts and normal epithelial cells. To isolate cell lines from metastases of estrogen receptor-negative mammary carcinomas in dogs, we applied a novel method using medium supplemented with serum treated to inactivate growth factors. Under these conditions, autonomously growing tumor cells are selectively allowed to proliferate. In this way, four autonomously growing tumor cell lines were obtained from metastases of two dogs. Tumors formed from cells implanted in C3H nude mice closely resembled the original dog tumors, indicating that the main result of this selective procedure was suppression of normal cell proliferation. Serum treated to inactivate growth factors seems to be an important medium supplement for isolation of autonomously growing tumor cell lines, which may be valuable tools for future studies on regulation of cell proliferation in advanced hormone-independent mammary tumors.

Immunotherapy by intralesional injection of BCG cell walls or live BCG in bovine ocular squamous cell carcinoma: a preliminary report.

Cows of the Dutch Frisian and Maas-Rijn-IJssel breed with histologically confirmed ocular squamous cell carcinoma showed complete regression of the primary tumor in 70 or 60% of the cases after intralesional injection of a BCG cell wall or live BCG vaccine, respectively. Recurrence of the tumor was observed in 57% of the animals treated with BCG cell walls and in 25% of the animals treated with live BCG vaccine. Spontaneous regression was seen in 20% of the untreated cows. In a second control group, radical surgery, the most successful treatment for primary stage I tumors in humans, resulted in a 90% cure. Influence of immunotherapy on metastases could not yet be fully evaluated. White blood cell counts were not changed after therapy. It was not possible to link a favorable response to BCG therapy with the intensity of the delayed type hypersensitivity (DTH) reaction to purified protein derivative of mycobacteriae (PPD) or the formation of antibodies to BCG as determined by a micro-enzyme-linked immunosorbent assay. However, in animals that showed tumor regression, the DTH reaction to PPD had a tendency to persist for a longer period of time. It was concluded that 1) block resection was the best method of treatment for this tumor, 2) a single intralesional injection of a BCG cell wall vaccine was as effective as live BCG vaccine in the induction of complete regression of the primary tumor, 3) in this preliminary study BCG cell wall vaccine was less effective than live BCG vaccine in the prevention of recurrence, and 4) this naturally occurring tumor model is well suited for the study of the influence of BCG immunotherapy in a primary stage I tumor.

Flow cytometric analysis of DNA ploidy in canine mammary tumors.

DNA ploidy has been determined using flow cytometry in 23 nonmalignant and 34 malignant (primary and metastatic) mammary tumors from 46 dogs. This parameter was compared with clinical stage, histology, and estrogen and progesterone receptor analysis. Twenty-one of 34 cancers (61.8%) from 32 dogs were DNA aneuploid. Aneuploidy was also found in 4 of 23 nonmalignant tumors (17.4%) from 20 dogs. Regional lymph nodes were involved in 6 of 10 diploid and 3 of 9 aneuploid cancers of dogs with operable disease. The aneuploidy incidence was higher in dogs that had distant metastasis at initial diagnosis (8 of 11) than in those presented with local or locoregional disease (9 of 19), although this difference was not statistically significant. DNA aneuploidy incidence was not found to be related to histological tumor type, histological malignancy grade, nuclear grade, or steroid receptor presence. Heterogeneity in DNA content was found in 4 of 32 cancers (30 dogs) in samples from primary or locally recurrent lesions. In 3 of 16 cancers that were analyzed both at the primary and at secondary sites of growth, a significant variation in DNA content was observed. The degree of aneuploidy in the dog cancers was much lower than seen for human breast carcinomas with a relatively high frequency of hypoploid stemlines (7 of 34 cancers, 20.6%). The frequency distribution of DNA indices in dog mammary cancers indicates that aneuploidy evolution probably differs from that of human breast cancer.

Flow cytometric DNA ploidy analysis of feline mammary tumors.

Flow cytometric DNA analysis was performed on biopsies from 9 nonmalignant and 111 malignant (primary and metastatic) feline mammary lesions. In our series, 46.3% of the primary mammary carcinomas appeared to be aneuploid, whereas all but one benign breast lesion were diploid. The degree of aneuploidy in carcinomas was low, with a relatively high number of primary tumors (12 of 82) displaying hypodiploidy. Aneuploidy was not found to be correlated with any specific histological tumor type, vascular invasion, tumor size, or histological malignancy grade or with the separate components thereof. Comparison of the ploidy in primary and metastatic tumors from the same cases revealed a remarkable stability, both in time and location of appearance of the metastases. It is concluded that with respect to DNA ploidy feline mammary carcinoma has more in common with canine mammary carcinoma than with human mammary carcinoma. Further prospective studies are necessary to clarify the implications of aneuploidy in feline mammary carcinoma for tumor behavior and prognosis.

Isolation of two distinct epithelial cell lines from a single feline mammary carcinoma with different tumorigenic potential in nude mice and expressing different levels of epidermal growth factor receptors.

From a single spontaneous feline mammary carcinoma, two subpopulations of epithelial tumor cells have been isolated. The variant cells were established as cell lines designated K248C and K248P. DNA ploidy analysis showed that the two cell lines represented cell populations already present in the original tumor. Chromosome analysis confirmed the feline origin of K248C and K248P and demonstrated that in addition to unique marker chromosomes characteristic for each cell line, both cell lines had several marker chromosomes in common. These data suggest that the two cell populations arose from a hypothetical single ancestor which diverged during tumor progression. The K248C and K248P cell lines differed from one another with respect to their tumorigenicity in athymic mice and epidermal growth factor (EGF) receptor content. The K248C cells were highly tumorigenic as indicated by a short latency period and high take rate. The K248P cells were poorly tumorigenic. Southern blot analysis revealed that the K248C cells contained an amplified EGF receptor gene that was accompanied by elevated levels of EGF receptor RNA and protein. The K248C cells were growth inhibited in vitro at EGF concentrations that stimulated growth of K248P cells. The amplification of the EGF receptor gene could be detected only in DNA derived from K248C cells at high passage numbers and not in DNA derived from the original tumor and K248C cells at low passage numbers. These data suggest that amplification of the EGF receptor gene occurred during establishment of the K248C cell line.

Expression of the gene encoding growth hormone in the human mammary gland.

Progestins cause a syndrome of growth hormone (GH) excess and enhanced mammary tumorigenesis in the dog. This has been regarded as being specific for the dog. Recently we reported that progestin-induced GH excess originates from foci of hyperplastic ductular epithelium of the mammary gland in the dog. In the present report we demonstrate by reverse-transcriptase PCR and immunohistochemistry that a main factor involved in tissue growth, i.e. GH, is also expressed in normal and neoplastic human mammary glands. The gene expressed in the human mammary gland proved to be identical to the gene encoding GH in the pituitary gland. The role of progesterone in the GH expression of the human mammary gland needs, however, to be proven. It is hypothesized that this locally produced hGH may play a pathogenetic role in breast cancer.

Polyethylene glycol-L-asparaginase versus native L-asparaginase in canine non-Hodgkin's lymphoma.

42 dogs with non-Hodgkin's lymphoma (NHL) were randomized for treatment with either PEG-L-asparaginase 10 IU/kg intramuscularly (n = 22) or L-asparaginase 400 IU/kg intraperitoneally (n = 20). Another 20 dogs were treated with either PEG-L-asparaginase 30 IU/kg (n = 10) or L-asparaginase 400 IU/kg (n = 10). Each treatment protocol consisted of two asparaginase treatments followed by a 10-week period of induction chemotherapy and then maintenance on asparaginase until progression occurred. No significant differences were found between treatments in the response rates after 2 weeks of asparaginase therapy or in the time to relapse, the time to treatment failure or the remission period. The reaction to asparaginase after the initial 2 weeks was a prognostic factor for the total duration of remission under asparaginase maintenance therapy. No side-effects were noted in the dogs treated with PEG-L-asparaginase, whereas 14 (48%) of the L-asparaginase treated dogs had side-effects related to this drug, including anaphylactic shock (9), anorexia or vomiting (4), hypersensitivity-related oedema (3), seizures (1) and acute pancreatitis (1). No abnormalities in clotting times, fibrinogen levels or antithrombin-III levels were found in any of the 62 dogs. PEG-L-asparaginase has the same anti-tumour activity as native L-asparaginase in dogs with NHL, but lacks side-effects.

P53 gene mutations in osteosarcomas in the dog.

Osteosarcomas in 18 dogs were examined for the presence of p53 mutations in exons 4-8 by single strand conformation polymorphism (SSCP) analysis, followed by sequence analysis in tumors demonstrating abnormal bands in the SSCP analysis. P53 mutations were found in four of the primary tumors in 17 dogs. Metastases studied in two of these dogs in which the primary tumor contained only wild type p53 did not contain mutations, nor those of one dog in which the primary tumor was not studied. The alterations that were found included three missense mutations and one 3 bp insertion.

Tyrosinase gene expression in clear cell sarcoma indicates a melanocytic origin: insight from the first reported canine case.

The aim of this study was to characterize a metastasizing soft tissue tumor in a dog, which clinically, grossly and histologically showed a close resemblance to human clear cell sarcoma, a soft tissue variant of malignant melanoma. Ultrastructurally, melanosomes were found, indicating a melanocytic origin of the tumor. Using reverse-transcription polymerase chain reaction, expression of the gene encoding tyrosinase was determined in tumor cells. With this first case of canine clear cell sarcoma, as well as the earlier report from our laboratory on amelanotic melanomas in the cat, we demonstrate that expression of the tyrosinase gene may occur in a broader range of less differentiated melanocytic tumors in different species, including man.

Feline mammary tumors: a case-control study of hormonal factors.

Feline mammary carcinomas are, like human breast cancers, spontaneous, locally infiltrative and metastasizing tumors. Therefore, this tumor disease in the cat can serve as a pathogenetic and experimental-therapeutic model for the human counterpart. In the cat, as in the woman, little is so far known with certainty about the hormonal background of mammary tumors. In order to elucidate the role of endogenous and exogenous hormonal factors, a case-control study was conducted. Data on age, history of castration, parity and progestogen administration were compared in cats with malignant or benign mammary tumors on one hand, and in a control group on the other. The statistical relative risks and their significance were assessed using conditional logistic regression analysis. In our study there was a tendency for mammary carcinomas to be found in cats that were older than those bearing benign mammary tumors. Ovariectomy was found to protect against mammary carcinomas but not against benign mammary tumors. No association between parity and mammary tumor risk was found. Regular administration of progestogens was associated with an increased risk of both mammary carcinoma and benign mammary tumors. However, this was not true of irregular progestogen administration and, in general, the administration of progestogens was not associated with an earlier appearance of mammary tumors.

The occurrence of estrogen and progestin receptors and anti-estrogen binding sites (AEBS) in canine non-Hodgkin's lymphomas.

In the present study the occurrence of estrogen and progestin receptors in the lymphoid tissue of 20 canine non-Hodgkin's Lymphoma patients was investigated. Lesions in all dogs were histologically classified according to criteria used in human patients. No progestin receptors could be demonstrated. Estrogen receptors were found only in the cytosol of two canine lymphomas. Antiestrogen binding sites were present in all 18 cases examined (range 37-356, median 110 fmol/mg protein). No correlation with age, sex or histological classification was found. A pilot study was carried out in five of these dogs that were negative for estrogen receptors and positive for antiestrogen binding sites. These dogs were treated with the antiestrogen tamoxifen (20-30 mg/day) for 2-3 weeks, but the treatment had no apparent effect. It was concluded that if antiestrogens do play a role in the treatment of non-Hodgkin's lymphoma in the dog, it is most likely not by means of antiestrogen binding sites.

Anterior pituitary function in female dogs with spontaneous mammary tumors: I. Growth hormone.

It has been hypothesized that growth hormone (GH) plays a major role in the pathogenesis of canine mammary tumor disease. In order to test this hypothesis, plasma GH levels were measured at rest and during dynamic function tests in dogs with benign or malignant proliferative mammary lesions and in control dogs. Both in control dogs and in dogs with benign disease, basal GH levels were found to be elevated during both metestrus and progestin treatment, as compared to anestrus. Dogs with benign or malignant disease did not have higher basal GH levels than control dogs matched for the effect of endogenous or exogenous progestin exposure. The GH response to glucose or thyrotropin releasing hormone (TRH) did not vary significantly with progestin exposure, nor between the 3 groups of animals. The stimulatory effect of clonidine upon GH secretion was reduced in dogs with benign or malignant disease as compared to controls matched for the effect of progestin exposure. These findings indicate that mammary tumor disease in the dog is associated with a disturbance in the regulation of GH release.

Anterior pituitary function in female dogs with mammary tumors: II. Prolactin.

Plasma prolactin (PRL) concentrations at rest and during dynamic tests were measured in dogs with benign or malignant proliferative mammary lesions and in age-matched healthy control dogs. Basal PRL concentrations of dogs with benign or malignant lesions were not found to be elevated in comparisons controlled for the effects of estrous cycle phase or progestin treatment. A statistically non-significant increase of the PRL response to thyrotropin-releasing hormone (TRH) was seen in separate groups of affected dogs as compared to matched controls. A tendency towards a significantly elevated response to TRH appeared in the combined (benign plus malignant disease) group of affected dogs in anestrus (P = 0.05) and in metestrus (P = 0.09). The PRL response to clonidine was very variable and did not differ between the 3 groups of dogs. The bromocriptine-induced suppression of PRL secretion did not vary significantly among the three groups. These results do not indicate an unequivocal association of mammary tumor development and altered PRL secretion. It is only warranted to conclude that in some dogs the occurrence of benign or malignant mammary lesions is associated with hyper-responsiveness of the PRL secretion to stimulation with TRH.

Liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE): a randomized clinical trial in dogs with mammary carcinoma.

In this prospective randomized double-blind clinical study the anti-tumour activity of liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) was evaluated as an adjuvant immunotherapy in dogs with mammary tumours of the simple carcinoma type. Dogs were randomized after surgery to one of two treatment groups, in which they were treated with either L-MTP-PE (2 mg/m2 i.v.; Ciba Geigy Basel, Switzerland) twice weekly for eight weeks, or with empty liposomes according to the same protocol. The minimal follow-up period was one year. Thirteen dogs were entered in the L-MTP-PE group and fourteen dogs in the placebo control group. Only minor toxicities (fever and shivering during 10-24 hours) were seen in six dogs treated with L-MTP-PE, these being mainly of the smaller breeds. At the time of evaluation seven dogs were still disease free. In the other twenty dogs the disease-free period (DFP) was ended by local recurrences in 16 and by distant metastases in 4. The difference in DFP between dogs treated with L-MTP-PE (median 165 days, range 15-905) and dogs in the placebo group (median 133 days, range 27-659) was not significant. The difference in overall survival between the dogs treated with L-MTP-PE (median 222 days, range 36-905) and those receiving the placebo (median 182 days, range 54-659) was also not significant. It was concluded that liposome-encapsulated MTP-PE was not efficacious in the treatment of dogs with mammary carcinoma.

Feline mammary carcinomas as a model for human breast cancer. II. Comparison of in vivo and in vitro adriamycin sensitivity.

Feline mammary carcinomas were tested for their in vitro and in vivo sensitivity to Adriamycin. In vitro, twenty randomly chosen tumor islands were studied to test the effect of concentrations of the drug ranging from 0.25-2.00 micrograms/ml. In vivo, parts of primary tumors left in situ after surgical biopsy were used to assess the response to five i.v. injections of 30 mg/m2 Adriamycin. When comparing the in vitro and in vivo response, the best sensitivity (100%) was obtained using 2.00 micrograms/ml Adriamycin and the best specificity (75%) using 1.00 microgram/ml Adriamycin, both in vitro. Tumors recurring after treatment showed acquired resistance in vitro.

Prolactin binding in benign and malignant mammary tissue of female dogs.

Prolactin receptor (PRL-R) concentrations were determined in membrane preparations of canine mammary tumours and of non-affected mammary tissues by a radioreceptor-assay using ovine prolactin (oPRL) both for 125I-labelling and for displacement. Receptor levels greater than or equal to 3 fmol/mg membrane protein were considered positive. Histologically non-affected samples of mammary tissue from 6 dogs were PRL-R positive (12-195 fmol/mg protein). These levels were positively correlated with epithelium content (based on surface area in microscopic sections; r = 0.943, P less than 0.02). In tumour samples where pre-existing mammary epithelium (PME) was present (3 non-malignant and 6 malignant tumour samples; PME content 5-10%), the cut-off limit for PRL-R positivity was increased to 50 fmol/mg protein to forestall false positives due to non-affected tissue. If no PME was present the general limit of 3 fmol/mg protein was maintained. All 18 non-malignant tumours showed PRL-R (18-162 fmol/mg protein). The PRL-R levels were positively correlated with levels of oestrogen-(ER; r = 0.735, P less than 0.002) and progesterone receptors (PgR; r = 0.556, P less than 0.02) as measured by a multi-concentration dextran-coated charcoal method. ER and PgR levels were also proportional (r = 0.660, P less than 0.01). In 6 dogs bearing primary cancers with 5-10% PME, 1 out of a total of 6 tumours was PRL-R positive. In 9 dogs bearing primary or locally recurrent cancers without PME, significant PRL-R levels were measured in 2 out of a total of 10 tumours. Three metastatic sites in 2 other dogs were PRL-R positive. In 2 dogs (1 with a PRL-R negative local recurrence) the metastatic lesions were PRL-R negative. Thus 5 dogs of a total of 18, had PRL-R positive mammary cancers (3-377 fmol/mg protein). Unlike in non-malignant lesions the ER, PgR, and PRL-R levels were not related. In mammary cancer the presence of PRL-R was less common (P less than 0.001), and the ultimate levels less high (P less than 0.001) than in non-malignant tumours. In comparative studies using pooled membrane preparations from benign mammary tissues, oPRL was far more effective than canine prolactin (cPRL) in displacing 125I-oPRL; canine growth hormone (cGH) in this respect was ineffective. It is concluded that non-malignant mammary tissue in the dog generally is PRL-R positive; only some mammary cancers retain the PRL receptors.

Feline mammary carcinomas as a model for human breast cancer. I. Sensitivity of mammary tumor cells in culture to cytostatic drugs. A preliminary investigation of a predictive test.

Feline mammary carcinomas were found to maintain well in short-term cultures. Principally the same types of nuclear DNA frequency distribution histograms were recognized in feline mammary carcinomas as in human mammary carcinomas. However, the more abnormal histograms are less frequent in feline than in human mammary carcinomas. Feline mammary carcinomas appeared, at least in vitro, most sensitive to Doxorubicin and 5-fluorouracil. Preliminary, thymidine incorporation studies indicate both cytotoxic and cytostatic effects of Doxorubicin and 5-FU. Methotrexate was found to stimulate thymidine incorporation.