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Background: Advantages offered by canine population substructure, combined with clinical presentations similar to human disorders, makes the dog an attractive system for studies of cancer genetics. Cancers that have been difficult to study in human families or populations are of particular interest. Histiocytic sarcoma is a rare and poorly understood neoplasm in humans that occurs in 15% to 25% of Bernese Mountain Dogs (BMD).Methods: Genomic DNA was collected from affected and unaffected BMD in North America and Europe. Both independent and combined genome-wide association studies (GWAS) were used to identify cancer-associated loci. Fine mapping and sequencing narrowed the primary locus to a single gene region.Results: Both populations shared the same primary locus, which features a single haplotype spanning MTAP and part of CDKN2A and is present in 96% of affected BMD. The haplotype is within the region homologous to human chromosome 9p21, which has been implicated in several types of cancer.Conclusions: We present the first GWAS for histiocytic sarcoma in any species. The data identify an associated haplotype in the highly cited tumor suppressor locus near CDKN2A. These data show the power of studying distinctive malignancies in highly predisposed dog breeds.Impact: Here, we establish a naturally occurring model of cancer susceptibility due to CDKN2 dysregulation, thus providing insight about this cancer-associated, complex, and poorly understood genomic region. Cancer Epidemiol Biomarkers Prev; 21(7); 1019–27. ©2012 AACR.

PDF file - 493K, The MTAP-CDKN2A Locus Confers Susceptibility to Cancer in a Naturally Occurring Canine Model

Histiocytic sarcoma is a rare, aggressive hematopoietic malignancy of mature tissue histiocytes with a poorly understood etiology in humans. A histologically and clinically similar counterpart in dogs affects two breeds at unusually high frequency, with 20-25% of Flatcoated Retrievers and Bernese Mountain Dogs developing histiocytic sarcoma, while the disease is extremely rare in other breeds. In this study, we leveraged the canine model system to investigate genetic risk factors for histiocytic sarcoma in the Flatcoated Retriever. Population bottlenecks and artificial selection during breed formation within last 200 years have created the unique breed structure in dogs, characterized by long linkage disequilibrium and low within-breed genetic diversity. This allows for the identification of disease-associated traits with relatively few markers and individuals. We conducted multiple genome-wide association studies in Flatcoated Retrievers in which we identified two loci conferring risk for histiocytic sarcoma (177 cases and 132 controls, total). The first locus, on canine chromosome (CFA) 5, also colocalizes with previously identified risk for hemangiosarcoma and B-cell lymphoma, two hematopoietic malignancies in the closely related golden retriever breed. The second locus is unique to the Flatcoated Retriever and increases risk for histiocytic sarcoma in combination with CFA5. There have been multiple reports of human histiocytic sarcoma diagnosis concurrent with other hematological cancers, including lymphoma, suggested to be the result of transdifferentiation or a common neoplastic progenitor. Here we link germline predisposition for histiocytic sarcoma and B-cell lymphoma to the same locus in retrievers. Subsequent whole genome, transcriptome, and ChIP sequencing, highlighted candidate genes at each locus with roles in cell migration, as well as candidate regulatory variants with predicted impact on transcription factor binding. This comprehensive, multi-omics approach has been previously largely unexplored in the canine model. Here we advance our knowledge of the biology of histiocytic sarcoma, which will have direct impact on canine health and elucidate the genetic mechanisms underlying a rare human cancer. Citation Format: Jacquelyn M. Evans, Heidi G. Parker, Jocelyn Plassais, Gerard R. Rutteman, Elaine A. Ostrander. Genetic risk factors for histiocytic sarcoma in a canine disease model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 829.

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.

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.

Histiocytic sarcoma (HS) is a rare human hematopoietic neoplasm that accounts for less than 1% of hematolymphoid cancers and occurs either concurrently with or subsequently to B- or T-lymphoblastic lymphoma and other B-cell cancers. As such, its genetic etiology has been difficult to establish. HS in dogs, by comparison, shows strong breed specificity with 20% of flat-coated retrievers and 25% of Bernese mountain dogs (BMD) estimated to develop the disease in their lifetime. These two affected breeds share no recent common ancestors and display differences in disease presentation, offering a unique system in which to investigate the genetic predisposition and progression of HS. Our lab has previously undertaken genome wide association study (GWAS) on both BMD and FCR collected in North America and Europe and identified four significantly associated loci, two in BMD and two in FCR. In each breed, only one locus was segregating with disease in the American population while both were present in the European populations. There were no loci in common between the two breeds. Fine mapping of the common BMD HS locus on canine chromosome (CFA) CFA11 revealed a single haplotype spanning the MTAP (methylthioadenosine phosphorylase) gene and part of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, which was present in 96% of BMD with HS. The European BMD also showed a significant locus on CFA14 that may be approaching fixation in the American dogs based on allele frequency calculations. We have done targeted resequencing of the CFA14 locus in a subset of European BMD and are in the process of filtering the identified mutations. Promising candidate genes in this region include GRM8, which inhibits the cyclic AMP cascade in the central nervous system and has shown to be involved in several of cancers; POT1, which is involved in telomere length maintenance and protection; and HYAL4, a hyaluronidase potentially involved in extracellular matrix degradation. In addition, the laboratory has generated 30x sequence from two HS cases and a control BMD, two FCR cases, and a control and low pass sequence from 16 dogs (8 cases and controls) in order to identify the likely causative mutations in each of the associated loci as well as breed specific mutations that may predispose the dogs to aggressive cancers. Citation Format: Heidi G. Parker, Edouard Cadieu, Abigail Shearin, Gerard Rutteman, Elaine A. Ostrander. Analysis of a histiocytic carcinoma in the dog: The canine's utility in fetching disease genes [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA08.

Extensive expression of craniofacial related homeobox genes in canine mammary sarcomas Journal Breast Cancer Research and Treatment Publisher Springer Netherlands ISSN 0167-6806 (Print) 1573-7217 (Online) Issue Volume 118, Number 2 / November, 2009 Category Preclinical Study DOI 10.1007/s10549-008-0243-7 Pages 333-343 Subject Collection Medicine SpringerLink Date Tuesday, December 02, 2008 Preclinical Study Extensive expression of craniofacial related homeobox genes in canine mammary sarcomas Helena Wensman1 , Hanna Göransson2, Karl-Johan Leuchowius3, Sara Strömberg3, Fredrik Pontén3, Anders Isaksson2, Gerard Roel Rutteman4, Nils-Erik Heldin3, Gunnar Pejler1 and Eva Hellmén1 (1) Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, P.O.Box 7011, 750 07 Uppsala, Sweden (2) Department of Medical Sciences, Uppsala University, Uppsala, Sweden (3) Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden (4) Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Received: 10 September 2008 Accepted: 30 October 2008 Published online: 2 December 2008 Abstract The global gene expression in three types of canine mammary tumors: carcinoma, fibrosarcoma and osteosarcoma were investigated by Affymetrix gene array technology. Unsupervised clustering analysis revealed a close clustering of the respective tumor types, with fibrosarcomas clustering close to the osteosarcomas and the carcinomas clustering closer to non-malignant mammary tissues (NMTs). A number of epithelial markers were expressed in both carcinomas and NMTs, whereas the sarcomas expressed genes related to mesenchymal differentiation. A comparison of the gene expression profile of the sarcomas versus carcinoma/NMTs revealed that the sarcomas, in particular the osteosarcomas, showed a striking upregulation of a panel of homeobox genes previously linked to craniofacial bone formation. In line with this finding, osteosarcomas showed an upregulation of bone morphogenetic proteins (BMPs) and of genes associated with retinoic acid signaling. Increased homeobox gene expression in sarcomas was also confirmed at the protein level by immunohistochemical analysis of tumor tissue, and in an osteosarcoma cell line after stimulation by BMP-2. These findings suggest that the development of mammary sarcomas specifically involves triggering of a set of homeobox genes related to neural crest and craniofacial bone development. Electronic supplementary material The online version of this article (doi:10.1007/s10549-008-0243-7) contains supplementary material, which is available to authorized users. Keywords Canine mammary tumors - Mammary sarcoma - Mammary osteosarcoma - Homeobox transcription factor - Gene expression profiling - Craniofacial

Objective— To investigate mutations of the TP53 gene in canine osteosarcoma (OS). Study Design— Clinical historic cohort study. Animals— Client-owned dogs. Methods— OS (n=59) were screened for mutations of the complete TP53 gene using polymerase chain reaction and the mutation was analyzed by single-strand conformational polymorphism. Clinical outcome of dogs with TP53-mutated OS were compared with dogs with OS without a mutation after complete surgical excision of the primary tumor. Results— TP53 gene mutations were observed in 24 of 59 (40.7%) OS; 3 mutated OS had 2 mutations. The alterations consisted mainly of point mutations (74%). Dogs with mutated OS had a significantly shorter survival time (ST) after surgery than dogs with normal tumor TP53 gene expression (P=.03). Other significant prognosticators for ST and disease-free interval included elevated serum alkaline phosphatase (P

Recent studies of canine malignant mammary tumours suggest that reduction of E-cadherin and/or beta-catenin correlates with invasive behaviour and lymph node metastasis. The aims of this study were to examine the interrelationships between the expression of E-cadherin and beta-catenin, and the relationship between the expression of E-cadherin and/or beta-catenin and the mode of growth and metastatic capacity of canine malignant mammary tumours. 90 spontaneous malignant tumours and local and regional lymph nodes were studied. A significant relationship was evidenced between membranous expression of E-cadherin and beta-catenin (p=0.0027), but not between E-cadherin and cytoplasmic beta-catenin. Only E-cadherin as a separate factor was significantly related to tumour invasion (p=0.0072) and lymph node metastasis (p=0.0001). Neither membranous nor cytoplasmic beta-catenin expression was significantly related to either of these phenomena.

Galectin-3 is implicated in tumor progression and metastasis. High levels of galectin-3 have been reported in intravasated cells in primary and metastatic tumor sites of canine malignant mammary tumors (CMMT). Nevertheless, it is still unknown whether this increase is limited to the site of the lesion or if it is a systemic feature. To better understand the pattern of the expression of galectin-3 and to investigate the possibility of using serum galectin-3 levels as a relevant biomarker in this disease, galectin-3 concentrations were determined in a series of sera from CMMT-bearing female dogs. None of the dogs included in the study had detectable metastases at the time of presentation. Animals were retrospectively divided into two groups dependent on whether or not they developed metastatic lesions during a 25-month follow-up period. Samples were collected from all dogs before surgery, 1 month after resection of the primary tumor and every 3 months during the postoperative period. Galectin-3 levels were significantly higher 1 month after than at the time of surgery (p=0.0058). Higher galectin-3 was found in samples collected 7 (p=0.0007), 10 (p=0.0061) and 13 months (p=0.0052) after surgery from dogs of the metastatic group when compared to those remaining free of development of detectable metastases. In conclusion, increased serum galectin-3 levels seem to be present in both metastatic and non-metastatic cases during the postoperative period, however, while in non-metastatic cases the values tend to return to baseline levels after surgery, in metastatic cases, levels remain persistently elevated.

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 I, 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.

This retrospective study in 61 cats with malignant lymphomas examined the efficacy of a well-established chemotherapy protocol (cyclophosphamide, vincristine, and prednisolone [COP]) in the Netherlands, a country with a low prevalence of feline leukemia virus (FeLV). Twenty-two cats (36.1%) had mediastinal lymphoma, 11 (18.0%) had alimentary lymphoma, 7 (11.5%) had peripheral lymphoma, 8 (13.1%) had nasal lymphoma, and 13 (21.3%) had miscellaneous lymphoma (including renal lymphoma in 2 [3.3%]). Of the 54 cats that were tested, only 4 (7.4%) were FeLV positive. Complete remission (CR) was achieved in 46 of the 61 cats (75.4%). The estimated 1- and 2-year disease-free periods (DFPs) in the 46 cats with CR were 51.4 and 37.8%, respectively, whereas the median duration of remission was 251 days. The overall estimated 1-year survival rate in all cats was 48.7%, and the 2-year survival rate was 39.9%, with a median survival of 266 days. The median survival time and the 1-year survival rate for mediastinal lymphoma were 262 days and 49.4%. respectively. Siamese cats had a more favorable prognosis for survival and remission than other breeds. Response to therapy in this study was shown to be a significant prognostic indicator. CR is necessary for long-term survival. Cats that did not achieve CR had little chance of survival for longer than I year. Young Siamese cats in this study had a greater tendency to develop mediastinal malignant lymphoma at a young age, and all were FeLV negative. In comparison with results reported in other studies with different combination chemotherapy protocols, these are among the highest percentages of remission and the longest survival rates for cats with malignant lymphoma.

Galectin-1 and galectin-3 are carbohydrate-binding proteins that have been implicated in the pathobiology of several types of cancer. The aim of the present study was to investigate the expression pattern of both these galectins in canine non-neoplastic mammary tissues and mammary tumors (CMT).Protein and mRNA expression of galectin-1 and -3 were assessed in 12 benign and 41 malignant CMT.Galectin-1 was overexpressed in the majority of malignant CMT cases in tumor cells and stroma. Its expression in malignant tumor cells was associated with smaller-sized tumours. Distant metastases presented a strong intensity of galectin-1 and reduced galectin-3 expression, while the opposite was observed in circulating tumor cells. Interestingly intravascular tumor cells presented galectin-3 up-regulation at the mRNA level. Double-labelling further made it clear that galectin-3 and galectin-1 expression did not overlap in normal-adjacent mammary and CMT cells.Taken together, our data suggest that malignant CMT cell sub-populations have alternating expression of galectin-1 or -3. This might confer survival advantage to tumour cells in different phases of tumour progression.

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. © 1999 Cancer Research Campaign

Clinical, macroscopic, and histologic features of 12 lymphangiosarcomas in cats are described. Nine tumors were located in the subcutaneous tissue at the caudoventral abdominal wall (eight cats) or in the neck (one cat). The remaining three cats had lymphangiosarcomas around the cranial mesenteric artery (two cats) or precardial in the mediastinum (one cat). Macroscopically, the tumors were noncircumscribed, white, edematous, and intermixed with fat tissue. Histologic features varied from cleft-forming and cavernous growth to papilliform and solid patterns. Follow-up data were available for seven cats with subcutaneous lymphangiosarcomas. All these cats died or were euthanatized within 6 months after surgery because of poor wound healing, local recurrence, or distant metastases. The cats with abdominal or thoracic masses were either euthanatized at surgery or within 6 months after the first surgery because of recurrent chylothorax, chyloperitoneum, or distant metastases.

In the present case-control study several dietary and nutritional factors were investigated to determine if a relationship exists between diet and development of mammary tumors in female dogs. Control female dogs (n = 86) were compared with a case group of dogs (n = 102) with dysplasias or tumors of the mammary gland. A questionnaire providing information on the dog's body conformation and dietary and reproductive histories was answered by the owners. Serum selenium and retinol concentrations and the fatty acid profile in subcutaneous adipose tissue were analyzed as indicators of nutritional status. Obesity at 1 year of age and 1 year before the diagnosis of mammary nodules was found to be significantly related to a higher prevalence of mammary tumors and dysplasias. The intake of homemade meals (compared to that of commercial foods) was also significantly related to a higher incidence of tumors and dysplasias. Other significant risk factors were a high intake of red meat, especially beef and pork, and a low intake of chicken. The subcutaneous fatty acid profile and the serum selenium concentration were not significantly different in the cases and the controls, with the exception of C18:1 fatty acid (oleic acid) content, which was significantly higher in the cases than in healthy controls. Serum retinol concentration was significantly lower in the cases than in the controls. In the multivariate analysis, older age, obesity at 1 year of age, and a high red meat intake were independently and significantly associated with the risk of developing mammary tumor and dysplasias.

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.

BackgroundThe determination of altered expression of genes in specific tumor types and their effect upon cellular processes may create insight in tumorigenesis and help to design better treatments. The Flatcoated retriever is a dog breed with an exceptionally high incidence of histiocytic sarcomas. The breed develops two distinct entities of histiocytic neoplasia, a soft tissue form and a visceral form. Gene expression studies of these tumors have value for comparable human diseases such as histiocytic/dendritic cell sarcoma for which knowledge is difficult to accrue due to their rare occurrence. In addition, such studies may help in the search for genetic aberrations underlying the genetic predisposition in this dog breed.MethodsMicroarray analysis and pathway analyses were performed on fresh-frozen tissues obtained from Flatcoated retrievers with localized, soft tissue histiocytic sarcomas (STHS) and disseminated, visceral histiocytic sarcomas (VHS) and on normal canine spleens from various breeds. Expression differences of nine genes were validated with quantitative real-time PCR (qPCR) analyses.ResultsQPCR analyses identified the significantly altered expression of nine genes; PPBP, SpiC, VCAM1, ENPEP, ITGAD (down-regulated), and GTSF1, Col3a1, CD90 and LUM (up-regulated) in the comparison of both the soft tissue and the visceral form with healthy spleen. DAVID pathway analyses revealed 24 pathways that were significantly involved in the development of HS in general, most of which were involved in the DNA repair and replication process.ConclusionsThis study identified altered expression of nine genes not yet implicated in histiocytic sarcoma manifestations in the dog nor in comparable human histiocytic/dendritic sarcomas. Exploration of the downside effect of canine inbreeding strategies for the study of similar sarcomas in humans might also lead to the identification of genes related to these rare malignancies in the human.

In contrast to the protective, anti-proliferative, action of progestins on the development of endometrium cancer, progestins may have local stimulatory and inhibitory effects on the proliferation of mammary epithelium. Until now there was no final molecular explanation of this discrepancy. Prolonged treatment of dogs with depot medroxyprogesterone acetate (DPMA) or with proligestone (PROL) results in enhanced plasma concentrations of growth hormone (GH), insulin-like growth factor (IGF)-I, IGF-II and IGF-binding proteins, together with the development of benign mammary tumours. The stimulated plasma GH levels do not have the typical pulsatile secretion pattern, and are not sensitive to stimulation with GHRH or to inhibition with somatostatin. The autonomous secretion can be inhibited by the anti-progestin RUU-486. The source of progestin-induced plasma GH levels has been demonstrated to be the canine mammary gland where progestins induce the expression of the gene encoding GH. The expression of the GH gene is restricted to focal areas of hyperplastic epithelium as shown by immunohistochemistry, and is predominantly located in single positive epithelial cells with an intermediate position between luminal- and myo-epithelium. Progestin-induced fibroadenomatous changes in the mammary gland of cats are also associated with locally enhanced GH expression. In both normal, benign and malignant mammary tumours of humans GH mRNA expression has been demonstrated by RT-PCR. The presence of GH mRNA is associated with the presence of immunoreactive GH as shown by immunohistochemistry. Sequence analysis revealed 100% homology to the pituitary expressed GH gene. In malignant mammary tumours of humans and dogs GH expression is also found in specimens negative for progesterone receptors as measured by ligand binding. It is concluded that the gene encoding GH is expressed in the mammary gland of a variety of species, including man. This appears to represent a contribution to the molecular explanation of the action of progestins on proliferation of mammary epithelium. It needs, however, to be proven whether this local biosynthesis of GH in the mammary gland is the cause of the local stimulatory effect of progestins on the proliferation of mammary epithelium.

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

We have shown recently that in the dog progestin administration results in mammary production of immunoreactive growth hormone (GH). At present we demonstrate the expression of the gene encoding GH in the mammary gland of dogs and cats using reverse-transcriptase PCR. GH mRNA was found in the great majority of normal mammary tissues as well as benign and malignant mammary tumors of the dog and was associated with the presence of immunoreactive GH in cryostat sections. The mammary PCR product proved to be identical to that of the pituitary. The highest expression levels were found after prolonged treatment with progestins. In carcinomas GH mRNA was also found in progesterone receptor-negative tissue samples, indicating that after malignant transformation GH gene expression may become progestin independent. GH mRNA was also present in mammary tissues of cats with progestin-induced fibroadenomatous changes. It is concluded that GH gene expression occurs in normal, hyperplastic, and neoplastic mammary tissue of the dog. The expression in normal tissue is stimulated by progestins and might mediate the progestin-stimulated development of canine mammary tumors. The demonstration of progestin-stimulated GH expression in mammary tissue of cats indicates that the phenomenon is more generalized among mammals.

Selman PJ, Mol JA, Rutteman GR, Rijnberk A. Progestin treatment in the dog. I. Effects on growth hormone, insulin-like growth factor I and glucose homeostasis. Eur J Endocrinol 1994;131:413–21. ISSN 0804–4643 The effects of two synthetic progestins, medroxyprogesterone acetate (MPA) and proligestone (PROL), on the release of growth hormone (GH) and glucose metabolism were studied in two groups of eight ovariohysterectomized dogs. Eight injections of long-acting progestins were administered at 3-week intervals. Recovery was studied in four dogs of each treatment group in the 6 months following cessation of progestin administration. Treatment with both MPA and PROL resulted in similar increases in plasma levels of GH and insulin-like growth factor I (IGF-I). The GH responses to both clonidine and growth hormone-releasing hormone became impaired. In neither treatment group did the elevated plasma GH levels decrease after administration of the synthetic somatostatin analogue SMS 201-995. The size and shape of the pituitary gland were not changed by progestin treatment. After cessation of progestin administration, basal plasma levels of GH and IGF-I did not return to pretreatment values. The GH response to growth hormone-releasing hormone remained impaired for at least 6 months after the last progestin administration. In both treatment groups, glucose homeostasis was sustained initially by increased insulin production. Prolonged treatment with MPA and PROL resulted in glucose intolerance. No amelioration was observed during the recovery period in either group. A small number of dogs developed diabetes mellitus. In more than 50% of the dogs in both treatment groups small mammary tumours developed. The recently discovered local production of GH probably played a role in mammary tumorigenesis. It is concluded that treatment with MPA and PROL results in similar increases in plasma levels of GH and IGF-I and leads to a similar degree of insulin resistance. The elevated GH levels can be neither stimulated nor inhibited, which is a feature compatible with autonomous secretion. These results are consistent with the recent finding of a progestin-induced ectopic production of GH in the mammary gland of the dog. JA Mol, Department of Clinical Sciences of Companion Animals, Utrecht University, PO Box 80. 154, 3508 TD Utrecht, The Netherlands

Galectin-3 is involved both in facilitating detachment of cells from primary tumour sites and favouring cancer cell adhesion and survival to anoikis in the blood stream. The mechanisms behind these apparently contradictory roles of the lectin have not yet been resolved. In order to investigate possible interplays between galectin-3 and its ligands underlying their role in the metastatic process, we examined mucin-1 (MUC1) and epidermal growth factor receptor (EGFR), well-known galectin-3 ligands, as well as galectin-3-binding site expression in a series of spontaneous canine malignant mammary tumours (CMMT) and a metastatic CMMT cell line. Despite the fact that CMMT cells expressed MUC1 and EGFR homogeneously over their plasma membrane, intravascular tumour cells, positive for galectin-3, expressed MUC1 and EGFR in a more focal membrane localization. Moreover, MUC1 overexpression in primary CMMT was present in parallel with down-regulation of galectin-3. Furthermore, in the CMT-U27 cell line, galectin-3 knock-down led to increased MUC1 expression, while MUC1 knock-down led to down-regulation of the lectin. Finally, removal of sialic acid from both CMMT and CMT-U27 xenograft samples exposed galectin-3-ligands throughout the tumour tissue, whereas these ligands were only present in galectin-3-positive invading cells in untreated samples. Interestingly indeed, we show that in vessel-invading cells, there is interaction between galectin-3 and the T antigen in vivo. We therefore hypothesized that loss of galectin-3 and sialylation-related masking of its ligands, in conjunction with their overexpression in specific tumour cell subpopulations, are crucial in regulating adhesive/de-adhesive events in the progression and invasive capacity of metastatic cells.

Overexpression of mucins is known to decrease cell-to-cell adhesion and thus to facilitate the invasion of cancer cells through the extracellular matrix. Mucin 6 (MUC6) is overexpressed and aberrantly O-glycosylated in human breast cancer, serving as a carrier for one of the most specific cancer-associated antigens, Tn antigen. Despite its relevance in breast cancer, MUC6 expression has not yet been characterized in canine mammary tumours (CMTs). The aims of this study were to assess the expression of MUC6 and Tn antigen in 55 benign and 77 malignant CMTs of different histological types and to investigate possible correlations with pathological features. MUC6 and Tn antigen were found to be significantly overexpressed in malignant compared with benign CMTs. MUC6 was significantly overexpressed in simple and complex carcinomas compared with simple and complex adenomas, respectively. When considering only the epithelial population, significant MUC6 overexpression was observed in carcinosarcomas when compared with benign mixed tumours. In addition, MUC6 was significantly overexpressed in simple compared with complex carcinomas. Finally, double-labelling immunofluorescence performed on seven malignant CMTs showed MUC6 and Tn co-expression. Therefore, MUC6 and Tn antigen overexpression is associated with malignant phenotypes of CMTs.

Summary The results of clinical and radiographic examinations of 15 dogs with confirmed malignant histiocytosis (MH) were reviewed. The most common clinical signs were anorexia (14 dogs), weight loss (13 dogs), lethargy (13 dogs), anaemia (11 dogs), and dyspnoea and\or coughing (8 dogs). Radiographs revealed abnormalities in all dogs, either intrathoracic (pulmonary nodules or consolidation [7 dogs], mediastinal masses [10 dogs], and incidentally pleural effusion [3 dogs] or abdominal (hepatomegaly [6 dogs] and splenomegaly [2 dogs]), or both. MH occurs relatively frequently in Bernese Mountain dogs. Both clinical and radiographic signs are non‐specific, but when they are present in a middle‐aged Bernese Mountain dog, MH should be included in the differential diagnosis.

Galectin-3 is a glycan-binding protein that mediates cell-cell and/or cell-extracellular matrix (ECM) interactions. Although galectin-3 is implicated in the progression of various types of cancers, the mechanisms by which galectin-3 enhances metastasis remain unclear. In order to elucidate the role of galectin-3 in the complex multistage process of cancer metastasis, we examined galectin-3 and galectin-3-binding site expression in a series of 82 spontaneous canine mammary tumors (CMT) and two CMT cell lines. Benign CMT tumors exhibited strong nuclear/cytoplasmic galectin-3 immunostaining, whereas malignant CMT tumors and metastases exhibited dramatically decreased galectin-3 expression with the majority of the immunostaining confined to the cytoplasm. Interestingly, intravascular tumor cells overexpressed galectin-3 regardless of their location. CMT-U27 xenografts displayed the same pattern of galectin-3 expression found in spontaneous malignant CMT. In parallel with the downregulation of galectin-3, malignant CMT displayed an overall loss of galectin-3-binding sites in the ECM and focal expression of galectin-3-binding sites mainly detected in intravascular tumor cells and endothelium. Furthermore, loss of galectin-3-binding sites was correlated with the downregulation of GLT25D1, a β (1-O) galactosyltransferase that modifies collagen, and upregulation of stromal galectin-1. Finally, GLT25D1 mRNA expression was strikingly downregulated in malignant CMT-U27 compared with the benign cell line, and its expression was further decreased in a galectin-3 knockdown CMT-U27 cell line. We therefore hypothesized that the loss of galectin-3-binding sites in the ECM in conjunction with the overexpression of galectin-3 in specific tumor cell subpopulations are crucial events for the development of mammary tumor metastases.

Thyrotrophin (TSH) is the conditional growth factor of thyroid epithelial cells. Abnormalities in TSH-receptor binding such as a low receptor number or low binding affinity may be a marker of thyroid carcinoma or metastases, or may exhibit a relationship with the functional variability of such tissues. The dog was used as a model to characterize TSH-receptor binding in normal thyroid tissues, naturally occurring thyroid neoplasms and distant metastases. In normal dog thyroid tissues, specific 125I-labelled TSH binding ranged from 2·7 to 15·5%, and low cross-reactivity with bovine LH (0·023%) was observed. One class of TSH-binding sites was found in eight normal thyroid tissues and 22 thyroid carcinomas; two normal thyroid tissues and one tumour exhibited two classes of binding sites. The concentration of binding sites was lower in the five carcinomas with reduced pertechnetate uptake (0·09 pmol/mg protein) than in the five thyroid neoplasms with increased uptake (0·19 pmol/mg) (P= 0·055). Compared with the original carcinoma tissues, TSH binding revealed a reduced binding affinity in eight out of eleven metastases. Two metastases showed a complete absence of TSH binding, suggesting that they were not dependent on TSH for growth. We conclude that one class of TSH-binding site is predominant in normal dog thyroid tissues and dog thyroid carcinomas. TSH could therefore contribute, at least in theory, to further growth of primary dog thyroid carcinomas. Secondly, assays measuring TSH binding may not be able to discriminate between malignant and benign dog thyroid tumours. TSH receptor number or affinity may be related to the functional variability of thyroid neoplasms. The absence of TSH binding in some metastases demonstrated that this characteristic can be acquired during the natural history of a differentiated thyroid carcinoma. Journal of Endocrinology (1992) 132, 461–468

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.

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.

For a proper determination of relative mRNA expression levels with real-time quantitative PCR (Q-PCR) internal standards, such as the expression of reference genes, are of utmost importance. For cats, in contrast to dogs, no validation of reference genes has been published. Our goal was to evaluate frequently used reference genes for the analysis of relative mRNA levels from feline tissues in a SYBR Green-based Q-PCR protocol. First, primers were optimized on mRNA-derived cDNA from liver and kidney tissues of randomly chosen (healthy and diseased) cats. Then, the expression variation and stability of each reference gene within a specific tissue was determined. Dental roots and crowns, heart (left ventricle), renal, liver, lung, and mammary gland tissues from 3 to 11 cats of different breeds, sexes, ages, and disease status were included in this study. Averaging relative stabilities over these six tissues revealed the usefulness of each tested gene as reference gene. In order to compensate for the expression variation of a reference gene within a specific tissue, as much as six reference genes (e.g. RPL17, RPL30, RPS7, YWHAZ, and HPRT) were required to obtain highly reliable data in cat tissues. The optimal set of reference genes depended on the tissue analyzed and should, ideally, be selected and evaluated at the start of each experimental condition. A comparison with a similar evaluation in dogs revealed three issues: (i) most ribosomal genes are suitable in both species; (ii) good non-ribosomal reference genes differ; (iii) more feline than canine reference genes are required for proper analysis.

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.

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.

(1995). Cytostatic treatment in veterinary medicine. Veterinary Quarterly: Vol. 17, No. sup1, pp. 25-27.

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.

The indirect growth-promoting action of pituitary-derived growth hormone (GH) on skeletal growth is thought to be mediated by systemically released insulin-like growth factor-I (IGF-I) and by locally produced IGF-I. The aim of the present study was to document whether GH is expressed locally in canine bone and spontaneous osteosarcoma. Using RT-PCR the expression of GH mRNA was demonstrated in the metaphyseal, but not in the majority of epiphyseal ends of the canine growth plate. GH mRNA was also present in 25% of the canine osteosarcoma specimens. The expression of GH mRNA in predominantly active osteoid forming areas was associated with the presence of immunoreactive GH in osteoblasts, as shown by immunohistochemistry. It is concluded that locally produced GH is involved in osteoid formation and may play a role in the growth of neoplastic bone lesions in the dog.

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.

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.

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 downregulated 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 RTPCR (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 CMTU335, 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. (Endocrinology 140: 5907‐5914, 1999)

To determine whether canine malignancies share common genetic lesions with their human counterparts, and are thus potentially interesting model systems in which to pose questions regarding tumor etiology and progression, we have elucidated the entire exon/intron structure of the canine p53 gene. A search for p53 gene abnormalities in mammary tumor tissue was undertaken utilizing single strand conformation polymorphism analysis. Mutations were detected in exons 4, 5, 6, and 7 of the p53 gene and consisted of nonsense, splicing, and frameshift mutations. None of 11 benign tumors and 6 of 40 primary carcinomas (15%) were found to harbor subtle p53 mutations. In 14 carcinomas examined the results in primary tumors and metastases were the same. These findings implicate involvement of this gene in the genesis of some malignant canine tumors, in a fashion similar to their human counterparts.

Seventeen dogs with mast cell tumours received chemotherapy. Fifteen dogs were treated with a vincristine, cyclophosphamide, hydroxyurea, and prednisolone (VCHP) regimen. Seven of these were later switched to doxyrubicin and prednisolone either because they stopped responding or because they did not respond from the start of the treatment. Two dogs received the latter regimen as the primary therapy. All dogs were treated with cimitidine and metoclopramide to minimize the effect of paraneoplastic syndrome associated with histamine release. Ten of the 17 dogs were found to respond (4/17 complete response (CR), 6/17 partial response (PR)). Response duration varied from 39 to 910 days (median 53 days), including 3 dogs with a CR that lasted more than 2 years. Survival time in responders varied from 41 to 910 days (median 97 days) and from 30 to 126 (median 39) in the other 7 dogs. Dogs that became refractory to VCHP did not respond to doxyrubicin and prednisolone. It is concluded that multi-agent chemotherapy has anti-tumour activity in a considerable proportion of dogs with mast cell tumours, but its efficacy is variable. The multivariate analyses showed that significant factors predicting survival in dogs with mast cell tumours were sex (P = 0.009), absence or presence of non-abdominal distant metastases, or abdominal metastases, respectively (P = 0.023), and malignancy grade of the tumours (P = 0.053).

Epidermal growth factor (EGFR), oestrogen (ER), and progestin (PR) receptor concentrations were determined by radioligand binding assay in non-affected mammary tissues (n = 13) and benign (n = 11) and primary/locally recurrent malignant proliferative mammary lesions (n = 45) and metastases (n = 19) in 65 female dogs. The number of specimens expressing EGFR was not significantly different among these tissues, but EGFR concentration was lower in metastases (P = 0.02) than in benign or primary/locally recurrent malignant lesions not mixed with non-affected mammary tissue. The presence of non-affected mammary tissue in primary cancer specimens was noticed as a factor that may influence results of receptor measurements. No relation was found between the expression of EGFR and that of ER or PR in non-affected or in tumorous mammary tissues. It was concluded that in the dog mammary gland EGFR expression is not associated with conditions of steroid receptor absence of biological agressiveness of neoplastic growth.

In the dog endogenous progesterone and synthetic progestins may incite overproduction of GH, resulting in acromegaly and insulin resistance. This progrestin-induced excessive GH secretion is characterized by disappearance of the pulsatile secretion pattern and insensitivity to both stimulation with GHRH and inhibition with a somatostatin analog. This progestin-induced GH hypersecretion is not associated with neoplastic transformation at the pituitary level. These observations were the impetus for a search of a possible extrapituitary site of GH production. In four ovariohysterectomized dogs elevated plasma GH levels (46.5 +/- 7.7 micrograms/liter; mean +/- SEM) were induced by administration of synthetic progestins. In these dogs hypophysectomy did not led to a significant decrease in plasma GH levels. Analysis of the GH content of various tissue homogenates revealed that the highest GH immunoreactivity was found in extracts of the mammary gland. Ectopic production of GH in the mammary gland was confirmed by lowering of plasma GH concentration to values within the reference range within 2 h after complete mammectomy in two dogs with progestin-induced elevations of plasma GH levels. In one of these dogs the arterial and elevations of plasma GH levels. In one of these dogs the arterial and venous GH concentrations across the mammary gland were measured and an arterio-venous GH gradient was demonstrated. Displacement studies in the RIA and analysis by reversed-phase HPLC revealed that mammary-derived GH is highly similar to pituitary-derived GH. Immuno-histochemical staining revealed that GH immunoreactivity was localized in focal areas of hyperplastic ductular epithelium. In mammary tissue of healthy untreated female dogs no GH immunoreactivity was found. It is concluded that treatment of dogs with synthetic progestins can induce the overproduction of GH in the mammary gland. This GH is biologically active, highly similar to pituitary derived GH, and originates from foci of hyperplastic ductular epithelium of the mammary gland.

An acquired defect in growth hormone secretion in mature dogs has been associated with some forms of generalised alopecia. In an attempt to elucidate the pathogenesis of the disturbance in growth hormone release, the plasma concentrations of growth hormone and insulin-like growth factor I (IGF-I) were measured in two seven-year-old poodles with alopecia and, for comparison, in two young German sheperd dogs with congenital hyposomatotropism (pituitary dwarfism). In the poodles the basal concentrations of growth hormone were low, although often above the detection limit of the assay. The concentrations of IGF-I were in the reference range for healthy poodles. No growth hormone could be detected in the plasma of the German sheperd dogs and the concentrations of IGF-I were very low. Stimulation with clonidine and growth hormone releasing hormone (GHRH) before and after repeated injections of GHRH did not result in significant increases in growth hormone concentrations in plasma. The concentrations of growth hormone in the poodles fluctuated at low levels during the test period. In the German sheperd dogs the levels of growth hormone remained unmeasurable during the stimulation tests. It was concluded that in the two poodles the basal concentrations of growth hormone were sufficient to maintain normal IGF-I concentrations, and thus the release of growth hormone was considered appropriate. Based upon measurements of urinary corticoids and a review of the literature it is suggested that the lack of a growth hormone response to stimulation was due to the enhanced release of somatostatin as a result of mild and fluctuating hyperadrenocorticism.(ABSTRACT TRUNCATED AT 250 WORDS)

Ten malignant canine mammary gland tumours and five metastases from three of these tumours were studied immunohistochemically with monoclonal antibodies (MoAbs) directed against different human keratin types (K), alpha-smooth muscle actin, vimentin, and desmin. In all tumours the neoplastic epithelium was rather homogeneously labelled with the keratin MoAbs RCK 102 (K 5 and 8) and CAM 5.2 (K 8). The adenocarcinomas (n = 5), the solid carcinomas (n = 2), and the carcinosarcoma (n = 1) showed heterogeneous labelling with the MoAbs specific for luminal cell antigens in the normal canine mammary gland, i.e., K 18, K 7 and K 19 MoAbs. These cells were also immunoreactive with K 4 and K 10 MoAbs. The spindle cell carcinomas (n = 2), however, did not react with these MoAbs. All tumours except one adenocarcinoma were characterized by the absence of immunoreactive labelling with the alpha-smooth muscle actin MoAb. In the solid carcinomas this was associated with the absence of labelling with one or both basal cell specific keratin MoAbs, i.e., 8.7 (K 14 and 17) and RCK 107 (K 14), respectively. In contrast, the other malignant tumours showed marked labelling of neoplastic epithelium with these MoAbs. Another remarkable finding was the labelling of a limited to moderate number of neoplastic epithelial cells with the vimentin MoAb. The presence of such labelling patterns in canine mammary gland tumours may be indicative of malignancy. Metastatic tumour tissues had a labelling pattern largely similar to that of the primary tumour, although also loss of reactivity for some keratin MoAbs was seen.

Duct ectasias (n = 2) and different types of benign canine mammary tumours (n = 19) were studied immunohistochemically with monoclonal antibodies (MoAbs) directed against various human keratin types (K), alpha-smooth muscle actin, vimentin, and desmin. In the duct ectasias and in most tumours the epithelial structures revealed an inner and outer cell layer. The inner cell layer was characterized by labelling with K 7, 8, 18, 19 and mostly also with K 4 and/or K 10 MoAbs. The outer cell layer was almost invariably labelled by K 14, K 14 and 17, and a-smooth muscle actin MoAbs. The labelling patterns of both duct ectasias and tumours corresponded largely to the patterns observed in normal mammary gland tissue, although a more distinct heterogeneity was seen. Tumours histomorphologically assumed to be of a myoepithelial origin did not show immunohistochemical features of myoepithelial cells. The myoepithelial nature of the vast majority of spindle-shaped cells present in the adenomas of the complex type and in the fibroadenomas of the benign mixed type could not be confirmed immunohistochemically. These cells, however, unequivocally expressed vimentin, suggesting proliferation of stromal cells in these tumours, which in the fibroadenomas of the benign mixed type may show metaplasia to bone or cartilage. In the duct ectasias and in some tumours, a fraction of elongated stromal cells, probably representing myofibroblasts, was labelled with the alpha-smooth muscle actin MoAb.

The safety of synthetic steroid hormones to be used for contraception in the human female is tested in rats, beagle dogs, and (once marketing starts) in monkeys. Because early studies did not show a mammary tumor stimulating effect in the human, in contrast to findings in the dog, many objections have been raised to the use of the dog for these toxicity studies. It has been claimed that the dog is unique in its sensitivity to the mammary tumor promoting effect of progestins and that this tumorigenic effect results from progestin-induced growth hormone (GH) induction. A thorough review of the literature does not support these claims. Tumor stimulatory effects of progesterone or synthetic progestins can be observed under some conditions in rodents as well as in cats and monkeys. In addition, recent evidence suggests a role for progesterone in mammary tumorigenesis in the human, and contraceptive steroids may also not be completely without risk. While the suggested role for GH in dog mammary tumorigenesis is far from proven, such a role cannot be excluded in the other species. Whether tumor stimulatory effects of sex steroids are based upon induction of proliferation in target cells or upon genotoxic effects or both is not yet certain.

Naturally occurring cancer in companion animals parallels cancer in man more closely than does experimentally induced cancer in inbred laboratory animals. In dogs and cats, as in man, a role for immune responses is indicated in the development of tumors. A survey is presented based on the literature and our own studies concerning the immunological and immunotherapeutic aspects of canine and feline mammary neoplasia. In dogs bearing mammary neoplasms, circulating immune complexes appear to play a negative role in the generation of effective antitumor immune responses. The functional role of peripheral blood lymphocytes and tumor-infiltrating lymphocytes in dogs and cats with mammary tumors is not yet fully established. No tumor antigen responsible for humoral or cellular responses has yet been identified. Extracorporeal perfusion of serum of dogs with mammary tumors and subcutaneous administration of mitomycin- and neuraminidase-treated autologous tumor cells are associated with improved prognosis. The opposite was true for i.v. treatment with BCG or Corynebacterium parvum vaccine in our study, in contrast to a previous report. A number of other treatment modalities in cats and dogs with mammary carcinomas failed to induce tumor regression. Canine and feline mammary carcinomas are good candidates for modern immunotherapeutic approaches.