Intraperitoneal antineoplastic drug delivery: experience with a cyclophosphamide, vincristine and prednisolone protocol in cats with malignant lymphoma.

In this retrospective study, the efficacy and safety were examined for an intraperitoneal chemotherapy protocol-cyclophosphamide, vincristine and prednisolone (IP-COP) in 26 cats with malignant lymphoma. Certainly in cats fiercely resisting IV administration the IP route is a more practical method, safer for the administrator and less stressful for the cat. Complete remission (CR) rate was 76.9% (n = 20). Median duration of first remission was 421 days. Estimated 1- and 2-year disease free period were 67.1 and 48.0%, respectively. Median duration of survival was 388 days and estimated overall 1- and 2-year survival periods were 54.7 and 46.9% respectively. Young cats had a more favourable prognosis. Reaching CR was essential for long-term survival. No specific IP-related adverse events (AE) were seen. AE were generally scored as mild and were not excessively abundant. These results indicate that the IP route is a safe and effective alternative for the administration of COP protocol chemotherapeutics.

Prednisolone inclusion in a first-line multidrug cytostatic protocol for the treatment of canine lymphoma does not affect therapy results.

Chemotherapy protocols for canine lymphoma include the routine use of glucocorticoids for their lympholytic effect. However, glucocorticoids are associated with side effects (e.g. polyphagia, polyuria, and weight gain), limit the use of non-steroidal anti-inflammatory drugs, and can induce drug transporter expression that could lead to drug resistance. Despite these negative effects, there are no data to support the use of glucocorticoids as part of a multidrug chemotherapy protocol for the treatment of canine lymphoma. A prospective, randomized clinical trial was conducted in 81 dogs with multicentric lymphoma and no history of recent glucocorticoid use. All dogs were staged and treated with the same chemotherapy protocol (L-asparaginase, cyclophosphamide, doxorubicin, vincristine, and prednisolone) with half of the dogs receiving prednisolone. Both treatment groups were similar with respect to demographics, immunophenotype, and clinical stage, except for a higher number of substage b patients in the prednisolone group (5 vs. 14; P=0.015). Treatment results obtained with the initial treatment (complete response rate 75%, disease-free period 176 days) and rescue treatment (complete response rate 45%, disease-free period 133 days), overall survival (283 days) and adverse events (number and grade) were similar for both groups. In conclusion, prednisolone, as part of a multidrug chemotherapy protocol, has no additional effect on treatment results and can be omitted from first-line multidrug protocols used for the treatment of canine lymphoma.

Prognostic studies of canine and feline mammary tumours: the need for standardized procedures.

For several years, veterinary oncologists have been struggling with the prognosis of mammary tumours in dogs and cats. Translation of tumour characteristics into prognostic information is an invaluable tool for the use of the most appropriate therapies, as well as for planning innovative therapeutic trials. Moreover, canine and feline spontaneous mammary gland tumours are good models for the study of human breast cancer. Collecting and interpreting information regarding the prognosis of canine and feline mammary tumours is difficult due to the fact that different methods have been applied to study various components and characteristics. This review identifies some of the challenges of prognostic studies of spontaneous canine and feline mammary tumours and suggests standardized procedures to overcome these challenges and facilitate reproducibility and assessment of results.

Progesterone receptor isoforms in the mammary gland of cats and dogs.

Progesterone exerts its effect by binding to specific progesterone receptors (PR) within the cell. In dogs and cats, no data are available on PR isoforms as found in other species. We therefore investigated the sequence of the PR gene and encoded protein in dogs and cats, the expression of PR isoforms in mammary tissue using Western blots and the presence of PR in mammary tissue using immunohistochemistry. Comparison of the amino acid sequence of the canine and feline PR with human PR revealed major differences in the PR-B-specific upstream segment (BUS). However, the essential activation function 3 (AF3) domain was intact in the cat but mutated in the dog. The DNA and ligand-binding domains were highly similar among the species. In cats with fibroadenomatous hyperplasia (FAH), high expression of PR mRNA together with growth hormone (GH), GH receptor (GHR) and IGF-I mRNA was found in comparison with feline mammary carcinomas. Immunohistochemical analysis showed strong nuclear as well as cytoplasmic staining for PR in FAH. Western blot analysis revealed expression of the PR-A and PR-B isoforms in the feline mammary gland. In canine mammary tissue, the most abundant PR staining was found in proliferative zones of the mammary gland. Western blot analyses showed mainly staining for PR-A with lower PR-B staining. It is concluded that in dogs and cats both PR isoforms are expressed. The role of mutations found in the canine PR-B is discussed.

A randomized controlled study into the efficacy and toxicity of pegylated liposome encapsulated doxorubicin as an adjuvant therapy in dogs with splenic haemangiosarcoma.

Safety and efficacy of pegylated liposome encapsulated doxorubicin (PL-DOX) was compared with free doxorubicin as an adjuvant monotherapy in dogs with splenic haemangiosarcoma after splenectomy in a randomized prospective clinical trial. A total of 17 dogs in each group were treated. No significant difference in survival between the two treatments was found. The calculated median overall survival time for the 34 dogs was 166 days [95% confidence interval (CI) 148-184]. The ½ year and one-year survival was 41.2% (95% CI 24.8-56.9) and 22.7% (95% CI 9.9-37.4), respectively. In dogs treated with PL-DOX, a desquamating dermatitis like palmar-plantar erythrodysesthesia (PPES) was seen in two dogs, while three other dogs showed anaphylactic reactions. Cardiotoxicity was not seen in either treatment groups.

Immunohistochemical evaluation of p53 expression with different antibodies in malignant canine tumours with or without p53 gene mutation.

Six monoclonal antibodies and a polyclonal antibody (CM1) were used to investigate the overexpression of p53 protein by immunohistochemistry (IHC) in six sarcomas and 21 mammary carcinomas from 27 dogs. IHC was compared with p53 gene mutation analysis performed on the same samples. Only the monoclonal PAb240, PAb421 and the CM1 antibodies were able to detect expression of canine p53 protein. CM1 was found to give the highest concordance (8/11) between positive expression of the p53 protein by IHC and the presence of a gene mutation. In the samples that were negative for p53 expression by IHC, but contained a p53 gene mutation according to DNA analysis, the mutation often affected the epitopes that could have been recognized by these antibodies. Only one out of 16 tumours without a p53 gene mutation had a weakly positive IHC result. These findings indicate that in these two types of canine tumours, IHC - particularly with CM1 - can detect many alterations in p53 expression owing to a gene mutation. False-positive results were very infrequent.

Telomerase reverse transcriptase (TERT) expression and proliferation in canine brain tumours.

Telomerase is a ribonucleoprotein enzyme complex that synthesizes telomere DNA. It is detected in 85-90% of malignant tumours in humans, but not in most somatic cells. Because telomerase plays a critical role in cell immortality, it represents an important target for anticancer therapies. We have previously shown that the dog is a potentially useful model for evaluating telomerase-based therapeutics. In this present study we analysed 93 canine brain tumours for telomerase reverse transcriptase (TERT) expression by immunohistochemistry. TERT immunoreactivity was detected in 16 of 50 grade 1 (32%) and 29 of 43 grade 2 tumours (67.4%), demonstrating a statistically significant association with histological grade (P = 0.00012). A subset of 51 tumours was also assessed for MIB-1 expression. The MIB-1 labelling index (LI) was found to correlate significantly with tumour grade, with a mean MIB-1 LI of 1.5% for grade 1 tumours, as compared with a mean MIB-1 LI of 21.7% for grade 2 tumours (P << 0.001). The MIB-1 LI was also significantly associated with TERT expression in all brain tumours (P << 0.001). These data further support the dog as a model for the preclinical development of telomerase-based therapeutics in brain tumours.

Detection of lymph node micrometastases in malignant mammary tumours in dogs by cytokeratin immunostaining.

A series of 131 local and regional lymph nodes from 40 dogs with malignant mammary tumours were evaluated by staining with haematoxylin and eosin and immunohistochemically for antibodies to pancytokeratin (AE1/AE3) and cytokeratin 14. The immunohistochemical tests detected occult micrometastases in 9.2 per cent of the lymph nodes that were negative by haematoxylin and eosin staining. Under the modified TNM classification of canine mammary tumours, these results raised the clinical stage of 12.5 per cent of the affected dogs. However, if the latest TNM classification of human breast cancer had been applied, none of the animals would have been reclassified.

E-cadherin expression in canine malignant mammary tumours: relationship to other clinico-pathological variables.

The relationship between E-cadherin epithelial expression, as detected by immunohistochemical methods, and other clinico-pathological characteristics of canine malignant mammary tumours was studied in 77 tumours surgically removed from 45 female dogs. The immunohistochemical assessment was based on the estimated percentage of epithelial cells with membranous labelling. Reduction of E-cadherin expression was significantly related to size and ulceration of tumours but not to fixation to skin or underlying tissue; it was also related to lymph node metastasis, necrosis and infiltrative growth. Histological type (but not histological grade) was related to E-cadherin expression, with solid tumours more frequently lacking expression and tubulopapillary tumours showing increased expression as compared with the other types. The significant relationship between E-cadherin and other known factors of poor prognosis suggests that the loss of E-cadherin expression may have prognostic value in canine malignant mammary tumours.

Validation of the use of proliferation markers in canine neoplastic and non-neoplastic tissues: comparison of KI-67 and proliferating cell nuclear antigen (PCNA) expression versus in vivo bromodeoxyuridine labelling by immunohistochemistry.

In order to evaluate the suitability of Ki-67 and proliferating cell nuclear antigen (PCNA) for determination of proliferative activity, the immunohistochemically determined nuclear expression of these antigens in canine non-neoplastic and neoplastic tissues was compared with the results of in vivo bromodeoxyuridine (BrdU) labelling, which - by measurement of the fraction of S-phase cells - is considered as the standard in the analysis of proliferative activity. The samples investigated consisted of non-neoplastic mammary and lymphoid tissues, and of benign and malignant (primary/metastatic) mammary tumours, and malignant lymphomas. Great regional heterogeneity prevented determination of an overall labelling index (LI) in normal lymphoid tissues. In the remaining combined group of samples, LI values were significantly ranked in the order PCNA>Ki-67>BrdU. However, the correlation of Ki-67 or PCNA as compared to BrdU LI values was only moderate in the combined group [approximately 0.5, Spearman rank test] as well as in most subgroups, whilst it was very poor in the group of primary mammary cancers. These observations indicate that Ki-67 or PCNA LIs as markers of proliferation do not evenly match in vivo BrdU labelling.

Study of dog and cat owners' perceptions of medical treatment for cancer.

Between April 10 and June 9, 2000, 91 owners of cats and dogs that were being or had been treated with anticancer chemotherapy were given a questionnaire designed to obtain information about their experiences as a result of the discovery and treatment of the disease, the efficacy and side effects of the treatment and their perceptions of the procedures associated with the administration of the drugs. Nearly all of the owners felt that the treatment was worthwhile. The level of observed side effects was low. Well over half of the owners believed that their animal had lived longer than it would have if it had not been treated and that its general wellbeing had improved. In general, they felt that the treatment had been rewarding and that any adverse side effects had been outweighed by the positive experiences during the treatment; they felt that they had been well informed and that their animals had benefited from the treatment.

Prognostic significance of a new histologic grading system for canine osteosarcoma.

Histologic grade is an important determinant in clinical outcome of human osteosarcoma (OS). In this study, the histologic characteristics of primary and metastatic canine OS were evaluated using a new classification system. Histologic characteristics were classified in 166 primary and 34 metastatic canine OS. Prognostic variables for clinical outcome were determined using multivariate analysis. Most OS were histologically characterized by severe to extreme cellular pleomorphism, a variable number of mitoses, small to moderate amounts of matrix, a high percentage of tumor cells, and minimal to moderate amounts of necrosis. Tumor invasion into vessels was present in 117/152 (71%) tumors, and 12/50 (24%) of the regional lymph nodes had evidence of metastasis. Classification of the 166 tumors resulted in seven (4%) grade 1, 34 (21%) grade II, and 125 (75%) grade III OS. In the multivariate analysis, histologic grade III OS and elevated pretreatment plasma alkaline phosphatase (AP) levels were independent predictors of clinical outcome. Dogs with high-grade tumors and elevated AP should be carefully evaluated for the presence of metastatic disease before starting adjunctive therapy protocols.

Lobaplatin as an adjuvant chemotherapy to surgery in canine appendicular osteosarcoma: a phase II evaluation.

Canine osteosarcoma, the most common bone tumor in dogs, is a well-established, naturally-occurring animal model for human OS. The aim of this study was to evaluate the clinical and hematological side-effects and to assess the efficacy of lobaplatin chemotherapy in dogs with appendicular osteosarcoma as an adjuvant therapy to surgical resection. Twenty-eight dogs without systemic signs of disease were treated with surgical resection of the tumor and adjuvant lobaplatin chemotherapy at a dose of 35 mg/m2, i.v., once every three weeks, for a maximum of 4 doses. Clinical signs of toxicosis were uncommon and consisted mainly of vomiting and depression. Hematological signs of toxicoses were common 7 to 10 days after lobaplatin chemotherapy and consisted of thrombocytopenia, leukopenia and neutropenia. All the signs were transient and most disappeared within three weeks of lobaplatin administration. A one-year disease-free fraction of 21.8% and a one-year survival fraction of 31.8% were calculated. Multivariate Cox regression analyses showed that a high histological tumor grade and presence of metastasis in the tumor vessels were associated with significantly shorter disease-free interval and survival time. Also, an increased pretreatment plasma alkaline phosphatase level at first presentation and a high histological level of tumor necrosis were associated with a shorter survival interval. Lobaplatin was easy to administer as an i.v. bolus injection at a three-week interval in dogs without the need for pretreatment infusions.

Analysis of p53 mutational events and MDM2 amplification in canine soft-tissue sarcomas.

Canine cancer is of major significance in terms of animal health and welfare and soft tissue sarcomas are an important group of tumours accounting for approximately 15% of all canine tumours presented. Abnormal p53 protein expression and gene mutations have been identified in a number of different canine tumour types. However, mdm2 gene amplification has only been investigated in a limited number of canine osteosarcomas. In this present study a series of canine soft-tissue sarcomas (STS) were examined for p53 mutations and/or mdm2 amplification. For p53 mutational studies polymerase chain reaction and direct DNA sequencing was used. Gene mutations were identified in 6 of 30 (20%) primary tumour cases including MPNST (n=3) leiomysarcoma (n=1), heamangiosarcoma (n=1) and sarcoma (n=1). mdm2 gene amplification was assessed by Southern Blot. Although there was no evidence for major gene rearrangements, gene amplification was detected in 4 of 35 (11.4 %) primary tumours including MPNST (n=2), rhabdomyosarcoma (n=2). A total of 33 cases were examined for both p53 mutations and mdm2 amplification. Seven of the tumours were positive for p53 mutations, while five were positive for mdm2 amplification. With the exception of one case, a reciprocal relationship between the presence of a p53 mutation and mdm2 gene amplification was demonstrated.

Cloning and cellular localization of the canine progesterone receptor: co-localization with growth hormone in the mammary gland.

The mammary gland has been found to express the gene encoding growth hormone (GH) in several species. Within the mammary gland, it may act as an autocrine/paracrine growth factor for cyclic epithelial changes, and may be a determinant in mammary carcinogenesis. In the dog, progestins enhance mammary GH expression. To elucidate the mechanism of progestin-induced mammary GH expression, the canine progesterone receptor (PR) is characterized and the cellular localization of the PR in normal and tumorous mammary tissues is examined. Sequence analysis of the canine PR revealed two in-frame ATG codons, encoding a putative PR-B protein of 939 amino acids and a putative PR-A protein of 765 amino acids. Western blot analysis indicated that both isoforms occur in uterus and mammary gland issues. Immunohistochemical analysis of the PR revealed that the PR was differentially expressed in mammary tissue, with many PR-positive epithelial cells in the proliferation phase of the glandular tissue and a low number of PR-positive cells in differentiated mammary tissue. Stromal and myoepithelial cells had no specific PR staining. Mammary tumours had a variety of staining patterns, including no staining, normal nuclear staining, marked heterogeneous immunoreactivity and perinuclear staining of tumorous epithelial cells and cytoplasmic-staining of spindle cells. Double staining showed that all GH-producing cells were positive for PR, whereas not all PR containing cells stained for GH. It is concluded that the activated PR may transactivate GH expression in the mammary gland within the same cell and functions as a pre-requisite transcription factor. However, during malignant transformation this regulation may be lost.

Expression and molecular characterization of the growth hormone receptor in canine mammary tissue and mammary tumors.

GH synthesis has been documented in canine mammary tissue and mammary tumors. In the present report, the characteristics of the GH receptor (GHR) are studied in these tissues. First, using immunohistochemistry, GHR was found to be present throughout normal and tumorous mammary tissues, being localized in epithelial and myoepithelial/spindle cell components and in the activated fibroblasts of desmoplastic tumor stroma. GHR expression seemed to be down-regulated only in terminally differentiated alveoli in normal tissue. GHR immunoreactivity in particular mammary (adeno)carcinomas was heterogenous. Second, the canine GHR was characterized at the molecular level. Northern blot analysis revealed a major GHR transcript of approximately 4.2 kb. The coding sequence of the canine GHR shows extensive homology with the GHR of several species. Seminested RT-PCR (using primers annealing in exons 4-5, exon 6, and exon 9) generated, next to the primary product, four different products in mammary tissues and the canine mammary tumor cell line CMT-U335, which seemed to be alternative GHR transcripts. These alternative GHR transcripts were characterized by exon 8 skipping, exon 7 skipping, and use of alternative splice donor and acceptor sites. Especially, the transcript that is missing exon 8 may encode a GH binding protein. In most malignant mammary samples, only the primary transcript was present; and alternative transcripts could not be detected. The absence of alternative GHR transcripts in mammary carcinomas, and thus putative inhibitors of GH-induced signal transduction, may contribute to enhanced sensitivity of malignant tumors to GH.

Stromal accumulation of chondroitin sulphate in mammary tumours of dogs.

To contribute to the investigation of the composition of the extracellular matrix in epithelial tumours, mammary gland tissues of dogs (including tumours, hyperplasias and normal tissue as well as metastatic lesions in lymph nodes and lung) were studied histochemically and immunohistochemically for distribution of sulphated glycosaminoglycans (s-GAGs). The formaline-fixed tissue was stained by alcian blue at pH 5.8, using the 'critical electrolyte concentration' to study the degree of sulphation of s-GAGs. s-GAGs were characterized by degradation with enzymes and nitrous acid and by immunohistochemistry with two anti-chondroitin sulphate monoclonal antibodies. The light microscopic investigation of s-GAG deposits revealed a limited number of patterns of their distribution. The main s-GAGs found in the mammary gland tumours of dogs and in metastatic lesions were chondroitin sulphate (CS) and heparin/heparan sulphate (HEP/HS). CS accumulated in diffuse structures between epithelial cells as well as around clusters of tumour cells. The latter pattern, possibly representing a mesenchymal reaction to the tumour, was present in 74% of the tumours, and in 67% of these, highly sulphated CS was present. A diffuse accumulation of CS was present almost exclusively in complex and mixed tumours; because of the expression of the 3B3 epitope for CS in immature cartilage the spindle cells of complex tumours are argued to be the precursors of the cartilage in mixed tumours. HEP/HS was stored mainly in mast cells that were found in increased numbers in hyperplasias and tumours. By pretreatment of microscopic slides with chondroitinase AC or ABC immunostaining of fibronectin could be made possible in areas in which CS was abundantly present, suggesting that CS may mask fibronectin epitopes. It is concluded that CS with different degrees of sulphation is the most important s-GAG in the extracellular matrix of mammary tumours of dogs. CS and other s-GAGs accumulate at different sites and may have a different pathogenetic significance.