Genetic variations of BRCA1 and BRCA2 genes in dogs with mammary tumours

Mammary tumours are the most common tumour type in female dogs. The formation of the mammary tumours is multifactorial but the high incidence of tumour disease in certain canine breeds suggests a strong genetic component. BRCA1 and BRCA2 are the most important genes significantly associated with mammary tumours. The aim of this study was to determine the association between the variations of these two genes and canine mammary tumours. 5′-untranslated region, intron 8 and exon 9 of BRCA1 and exons 12, 24, 27 of BRCA2 were sequenced in order to detect the genetic variations. In addition to six previously identified polymorphisms, six novel single nucleotide polymorphisms (SNPs) were detected. Five of the coding SNPs were synonymous and three of them were non-synonymous. The comparison of the sequences from 25 mammary tumour bearing and 10 tumour free dogs suggested that the two SNPs in intron 8 and exon 9 of BRCA1 and two SNPs in exon 24 and exon 27 of BRCA2, which are firstly identified in this study, might be associated with mammary tumour development in dogs. Especially one SNP in exon 9 of BRCA1 and one SNP in exon 24 of BRCA2 were found to be significantly associated with canine mammary tumours.     

Common germline haplotypes and genotypes identified in BRCA2 exon 11 of dogs with mammary tumours and histopathological analyses

The canine BRCA2 is a tumor supressor gene which encodes the BRCA2 protein, involved in DNA repair through interaction with the RAD51 recombinase. This process is mediated by eigth BRC repeats that are encoded by BRCA2 exon 11. Two variants corresponding to human mutations in human BRC3 repeat have been reported in canine BRC3 repeat. In addition, other variants have also been described in canine BRCA2 exon 11. Considering the importance of polymorphisms in human BRCA2 to breast cancer development, this study aimed to investigate the frequency of variants in BRCA2 exon 11 in 48 blood and tissue DNA samples from bitches with canine mammary tumors (CMT), as well as, to analyze tumor stage and histopathological features. Seven Single Nucleotide Polymorphisms (SNPs) were identified, three of which were evaluated as possibily or probably deleterious variant. Interestingly, almost all the 22 mammary tumors (except one) which presented a clinical staging equal to or greater than III carried at least one mutant allele of these three variants. Besides that, no statistically significant correlation was observed between any of the reported SNPs in heterozygosis or homozygosis and either dogs data (such as breed, age or disease stage) or mammary tumors histopathological characteristics. A total of 97.9% of bitches had one to three polymorphisms of the seven identified in this study, which suggests a possibly correlation between the canine BRCA2 exon 11 polymorphisms and mammary carcinogenesis.     

Expression of maspin in mammary gland tumors of the dog

Maspin is a serine protease inhibitor that inhibits tumor invasion and metastasis in human breast cancer and is consistently expressed by mammary myoepithelial cells (MECs). To analyze the value of maspin as a marker of the MEC layer of the normal and tumoral canine mammary gland, the immunohistochemical expression of maspin was studied in formalin-fixed tissues from 55 benign and malignant tumors (40 tumors also contained the surrounding normal mammary gland) using a commercially available monoclonal antibody. Periacinar and periductal MECs of all 40 normal mammary glands were stained by the anti-human maspin monoclonal antibody, and immunoreactivity was observed in the nucleus and cytoplasm of these cells. In addition, maspin was found in 53 (98%) of the tumors studied, reacting with the MECs in 100% of benign tumors and 93% of malignant tumors and to the epithelial cells of 16% of benign and 73% of malignant tumors. In the MEC compartment, immunoreactivity was observed in the cytoplasm of hypertrophic MECs, fusiform MECs, stellate MECs, rounded (myoepithelial) cells, and chondroblasts. In the epithelial cell compartment, immunoreactivity was observed in the cytoplasm of cells with and without squamous differentiation. Stromal myofibroblasts were unreactive. Maspin appears to be a very sensitive marker of the normal and neoplastic myoepithelium that, contrary to smooth muscle differentiation markers, does not stain stromal myofibroblasts. In addition, a subset of neoplastic epithelial cells reacted with the maspin antibody. The relationship between maspin expression in different cellular compartments of canine mammary carcinomas and the biologic aggressiveness of the disease remains to be elucidated.     

Expression patterns of the BRCA1 splicing variants in canine normal tissues and mammary gland tumors

Human BRCA1 is familial breast cancer susceptibility gene. Recently, decreased BRCA1 mRNA and protein expression has been identified in sporadic breast tumors. In the reported human BRCA1 splicing variants, delta11b lacks the majority of exon11 and is suspected to have a distinct function in normal tissues. The splicing variants display a variety of expression pattern in breast cancer samples. Although mammary gland tumor is important disease in dog, there are few reports for BRCA1 in the canine tumors. In this study, we examined the relative amounts of BRCA1 splicing variants mRNA in canine normal and mammary tumor samples by RT-PCR to investigate whether there is the altered expression of variant mRNAs in the canine tumor as reported in human. The exon11b-defecting RT-PCR products were observed in all the normal tissues examined and the nucleotide sequence was quite similar to that of human BRCA1 delta11b. In some tumor samples, we did not detect the products targeted for exon10-13 and exon14-15, while these products were observed in all the normal samples examined. Especially, the relative amounts of the exon11-defecting products were remarkably decreased in most of the tumors (11/16).     

Breed-related differences in altered BRCA1 expression,phenotype and subtype in malignant canine mammary tumors

BRCA1 is a high-penetrance breast cancer susceptibility gene and BRCA1-associated breast cancer has a high familial prevalence that is more common among certain populations of humans. A similar high prevalence also exists for canine mammary tumors (CMTs) and the objective of this study was to determine the breed-related differences in malignant CMTs. Comparative analyses of the expression of various prognostic factors for CMTs, including BRCA1, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2) were conducted on 139 malignant CMT cases from five breeds with the highest prevalence of CMTs in Korea.Significant breed-related differences were observed in the expression of BRCA1 (P = 0.003), histological grade (P = 0.038), and extensive lymphatic invasion (P = 0.042). The Shih Tzu breed had the highest proportion of dogs with malignant CMT and strong overexpression of BRCA1. Cytoplasmic and membranous expression of BRCA1 was associated with the ER negative (P = 0.004), PR negative (P = 0.046), and triple negative (ER, PR, and HER-2 negative; P = 0.016) phenotype and the basal-like molecular subtype (P = 0.019) in Shih Tzu dogs. Since these features are similar to BRCA1-related human breast cancer, dogs with BRCA1-associated CMT, particularly Shih Tzu dogs, may serve as a suitable spontaneous model, although additional molecular studies are needed.     

Paraneoplastic hypoglycemia in a diabetic dog with an insulin growth factor-2-producing mammary carcinoma

A 6-year-old intact female Labrador Retriever had diabetes mellitus, which had been difficult to control with insulin. The dog also had a solid ductal mammary carcinoma with very rapid growth, which was temporally related to onset of hypoglycemia. Eight months after initial diagnosis of diabetes, the dog had a hypoglycemic crisis. Insulin administration was stopped and serum glucose concentration returned to normal. Three months after discontinuing insulin, another hypoglycemic crisis occurred. During subsequent months, serum glucose concentrations remained at life-threatening levels (1.64-2.12 mmol/L, reference interval 4.44-6.66 mmol/L) simultaneously with an increase in the size of the mammary tumor, which reached a diameter of about 16 cm. At the time of surgery for removal of the tumor serum glucose concentration was 2.20 mmol/L and was then monitored every 3 hours after excision of the tumor. The glucose concentration continued to rise and reached 9.99 mmol/L 12 hours after the removal of the mammary tumor. Immunohistochemical staining demonstrated expression of insulin growth factor-2 by tumor cells, which apparently had caused the hypoglycemia during tumor growth even in a diabetic dog. Hyperglycemia associated with diabetes was pronounced after excision of the tumor and had been masked by the paraneoplastic effect of the tumor.     

Cyclooxygenase-2 expression in canine mammary tumors

Mammary tumors are the most common neoplasms in female dogs. Induction of cyclooxygenase-2 (COX-2) has been implicated in various cancers in humans. However, expression of COX-2 has not been investigated in canine mammary tumors. Normal mammary gland (n = 4), simple or complex adenomas (n = 63), and simple or complex adenocarcinomas (n = 84) were studied by immunohistochemistry. Results showed that COX-2 was not expressed in the normal gland but was detected in 24% of adenomas and in 56% of adenocarcinomas (P < 0.001). The incidence of COX-2 expression and the intensity of the COX-2 signal were higher in adenocarcinomas than in adenomas (P < 0.001). These results demonstrate for the first time that COX-2 is induced in a proportion of canine mammary tumors and that COX-2 expression is more frequent and more intense in malignant than in benign tumors, suggesting a potential role for COX-2 in canine mammary tumorigenesis.     

Somatic SNPs of the BRCA2 gene at the fragments encoding RAD51 binding sites of canine mammary tumors

Mammary tumors are the most common tumor type both in women and in female dogs. In women, heritable breast cancers have been linked mutations in the breast cancer susceptibility gene BRCA2 and it contains eight BRC repeats in exon 11 that bind to RAD51. In this study, we investigated the sequence variations of BRC1‐BRC8 and C‐terminus of canine BRCA2 gene. From a total of 64 canine patients with mammary tumors, 31 mammary tumors with benign and malign carcinomas and the 3 normal mammary glands were used for the study. In this study, 19 SNPs of exon 11 of BRCA2 in canine mammary tumors were detected for the first time. The c.2383A>C (T1425P) SNP was found to be the most probable disease‐associated nsSNP. Our findings suggest that T1425P variation in BRC3 to be the most probable disease‐associated nsSNP and may affect RAD51 binding strength.     

Neoplastic pleocytosis in a dog with metastatic mammary carcinoma and meningeal carcinomatosis

Abstract: A 12‐year‐old female spayed Labrador Retriever was presented with a history of seizures and abnormal vocalization. Approximately 1 year before presentation, multiple mammary cysts had been surgically excised. A mammary mass was noted on physical examination, and 2 separate parenchymal brain lesions were found on imaging studies. Cerebrospinal fluid (CSF) collected from the cisterna magna was analyzed, and abnormalities included moderate pleocytosis with atypical discrete round cells that occasionally formed loose clusters. The dog was euthanized, and on necropsy a primary solid mammary carcinoma was identified as well as multiple metastatic foci in the brain with diffuse meningeal involvement. The cells in the CSF had a morphologic appearance similar to the cells in the primary mammary tumor and in the metastatic tumors in the brain. On immunostaining, cells from the primary mammary tumor, the brain tumors, and the CSF expressed cytokeratin. The CSF cells did not express CD18, CD3, or CD79a. A final diagnosis of mammary carcinoma with brain metastasis and meningeal carcinomatosis was made.     

p53 gene mutations occurring in spontaneous benign and malignant mammary tumors of the dog

Sixty-three cases of benign and malignant canine mammary tumors were analyzed to define the alteration of exons 5-8 for the p53 tumor suppressor gene using polymerase chain reaction direct sequence analysis with paraffin-embedded tissues. Four missense mutations were found in 38 benign mammary tumors (11%), and five missense (one tumor had two missense mutations) and one nonsense mutations were found in 25 mammary carcinomas (20%). These data suggest that the p53 gene alterations might be initiated at an early stage of canine mammary carcinogenesis and p53 mutations might be associated with malignancy. However, there was no evidence of any relationship between the p53 alterations and the histologic types of tumors or breeds of dogs.     

Haemangiosarcoma with a metastasis of a malignant mixed mammary gland tumour in a dog

An 11-year-old dog with a history of weight loss, anorexia, anaemia, epistaxis and a nodule in the mammary gland showed multifocal lung masses during radiography. At necropsy as well as in the histological examination two different tumours were identified in several organs including the right heart auricle. By immunohistochemistry, using a double-staining DAKO kit (DAKO Diagnostics, Zug, Switzerland), it was possible to identify the two components as haemangiosarcoma and metastasizing malignant mixed mammary gland tumour.     

Hypercalcemia and high parathyroid hormone-related peptide concentration in a dog with a complex mammary carcinoma

A 10-year-old female Dachshund was presented with a history of mammary masses, slight lethargy, polyuria, and polydipsia. Physical examination findings included masses involving the first, second, and fourth mammary glands of the left side. The mandibular, axillary, and right popliteal lymph nodes were mildly enlarged. Serum chemistry results included hypercalcemia (13.9 mg/dL, reference interval 8.0-11.5 mg/dL). Although intact parathyroid hormone (PTH) concentration (1.05 pmol/L) was below the reference interval (2-13 pmol/L), PTH-related protein (PTHrP) concentration was markedly increased (9.40 pmol/L, reference value < 2 pmol/L). The masses were surgically removed, and the histopathologic diagnosis was complex mammary carcinoma. Three weeks after surgery, serum total calcium concentration had decreased to 10.5 mg/dL. Resolution of the hypercalcemia and clinical signs supported a diagnosis of humoral hypercalcemia of malignancy associated with mammary gland carcinoma.     

Metastatic pericardial tumors in a dog with equivocal pericardial cytological findings

A metastatic tumor associated with pericardial effusion was diagnosed in a 6-year-old, female, mixed-breed dog. Echocardiography identified multiple echogenic masses adherent to both visceral and parietal pericardium, while results of pericardial fluid cytology were non-diagnostic. The distribution pattern of the masses is remarkable in that they protruded from both pericardial surfaces, rather than one, and demonstrated an oscillatory motion during the cardiac cycle. Pathological examination confirmed the diagnosis of multiple metastatic tumors of the pericardium, with the primary tumor being an anaplastic gastric adenocarcinoma.     

GH-producing mammary tumors in two dogs with acromegaly

Two intact female dogs were admitted for growing mammary tumors. They had symptoms of acromegaly including weight gain, enlargement of the head, excessive skin folds, and inspiratory stridor. Serum concentrations of growth hormone (GH), insulin-like growth factor-I (IGF-I), and insulin were elevated in the two cases. From these findings, both dogs were diagnosed with acromegaly. In case 1, the GH, IGF-I, and insulin levels subsided after removal of the focal benign mammary tumors and ovariohysterectomy. In case 2, those levels subsided after removal of only focal mammary carcinoma. In both cases, immunohistochemical investigations for GH were positive in the mammary tumor cells but not in the normal mammary glands. We concluded that GH-producing mammary tumors caused the present acromegaly.