Effects of Obesity and Obesity-Related Molecules on Canine Mammary Gland Tumors.

Obesity can affect the clinical course of a number of diseases, including breast cancer in women and mammary gland tumors in female dogs, via the secretion of various cytokines and hormones. The objective of this study was to examine the expression patterns of obesity-related molecules such as aromatase, leptin, and insulin-like growth factor 1 receptor (IGF-1 R) in canine mammary carcinomas (CMCs) on the basis of the body condition score (BCS). Comparative analyses of the expression of these molecules, together with prognostic factors for CMCs, including hormone receptors (HRs; estrogen and progesterone receptors), lymphatic invasion, central necrosis of the tumor, and histologic grade, were performed on 56 CMCs. The mean age of CMC onset was lower in the overweight or obese group (8.7 ± 1.9 years) than in the lean or ideal body weight group (10.4 ± 2.7 years). The proportion of poorly differentiated (grade III) tumors was significantly higher in the overweight or obese female dogs. Aromatase expression was significantly higher in the overweight or obese group and was correlated with the expression of HRs (P = .025). These findings suggest that overweight or obese status might affect the development and behavior of CMCs by tumor-adipocyte interactions and increased HR-related tumor growth.

CD44+/CD24- Cancer Stem Cells Are Associated With Higher Grade of Canine Mammary Carcinomas.

The CD44+/CD24- phenotype identifies cancer stem cell (CSC) properties in canine mammary carcinoma (MC); however, the histopathological features associated with this phenotype remain to be elucidated. Here, we determined whether the CD44+/CD24- phenotype was associated with hormonal receptor (HR; estrogen receptor [ER] and/or progesterone receptor [PR]) status and/or triple (ER, PR, and human epithelial growth factor receptor 2)-negative (TN) subtype; conventional histological evaluation was also performed. We found that, as single markers, both CD44+ and CD24+ were associated with less aggressive histological types, low grade, and a non-TN subtype; both markers were associated with HR positivity. On the other hand, a CD44+/CD24- phenotype was associated with higher grade of carcinoma. Therefore, our results suggest that immunohistochemical phenotyping for CD44/CD24 is useful for the evaluation of tumor behavior as well as CSC-like properties in canine MCs.

Analysis of a new histological and molecular-based classification of canine mammary neoplasia.

Canine mammary tumors (CMTs) are morphologically and biologically heterogeneous, prompting several attempts to classify such tumors on the basis of their histopathological characteristics. Recently, molecular-based analysis methods borrowed from human breast cancer research have also been applied to the classification of CMTs. In this study, canine mammary neoplasms (n = 648) occurring in Korea from 2008 to 2011 were analyzed according to the histological classification and grading system proposed by Goldschmidt et al. Furthermore, randomly selected mammary carcinomas (n = 159) were classified according to the molecular subtype using immunohistochemical characteristics. Canine mammary neoplasia accounted for 52.6% (648/1250) of the tumors in female dogs, and 51.7% (340/648) of these were malignant. All of the carcinoma-anaplastic subtypes were grade III tumors (5/5, 100%), while most of the carcinoma-tubular subtypes (15/18, 83.3%) and carcinoma arising in a complex adenoma/mixed-tumor subtype (115/135, 85.2%) were grade I tumors. Tumor cell invasion into lymphatic vessels was most common in the comedocarcinoma, carcinoma-anaplastic, and inflammatory carcinoma subtypes. The most frequently occurring molecular subtype (70/159, 44%) was luminal A. However, the basal-like subtype was the most malignant and was frequently associated with grade III tumors and lymphatic invasion. The carcinoma-solid subtypes were also often of the basal-like subtype. Reclassification of CMTs using the newly proposed histopathological classification system and molecular subtyping could aid in determining the prognosis and the most suitable anticancer treatment for each case.

Infiltrating Foxp3+ regulatory T cells and histopathological features in canine classical and spermatocytic seminomas.

In humans, regulatory T (T reg) cells are known to play a critical role in both the regulation of immune homoeostasis and the progression of cancer. However, there is little information about the identification, characterization and the function of T reg cells in canine tumours. We identified T reg cells in 28 canine seminoma samples using a Forkhead box P3 (Foxp3) antibody and investigated the relationship between T reg cell infiltration and histopathological features of classical and spermatocytic seminomas (SE and SS, respectively). The Foxp3 protein showed nuclear immunostaining in infiltrating lymphocytes, and Foxp3+ cells were diffused or focally distributed in seminoma tissues. Foxp3+ cells were frequently present in the SS histotype, in seminomas that showed no evidence of tumour cell invasion into the vessels and in seminomas showing a diffuse growth pattern with three cell types. Neither the SE/SS histotype nor the histopathological features of the tumour correlated with Foxp3+ cell counts. These results indicate that Foxp3+ T reg cells may be associated with a less malignant histological phenotype or may not play a critical role in the immune response of canine seminomas. Moreover, Foxp3+ T reg cells may be associated with SS seminoma, but further studies, involving a larger number of samples, are required to better understand whether these cells play a critical role in the immune response in canine seminomas. This is the first report to demonstrate the characteristics of T reg cell infiltration in canine seminoma.

Overexpression and Mutation of p53 Exons 4-8 in Canine Intestinal Adenocarcinoma.

p53 is mutated and overexpressed during malignant transformation, including in human colorectal cancer. This study investigated the overexpression of p53 protein and mutations in the p53 gene in canine intestinal neoplasia (CIN). Immunohistochemical analysis of p53 was carried out in formalin-fixed and paraffin wax-embedded (FFPE) sections of intestinal tissues from 35 dogs with CIN by the standard peroxidase-anti-peroxidase method. Expression of p53 protein in malignant (adenocarcinoma, n = 20) and benign (adenoma and polyp, n = 8) CINs was compared with tissue from negative controls (samples with no proliferation, n = 7). DNA was extracted from FFPE tissue from one control and 13 cases with overexpression of p53, and exons 4-8 were sequenced. p53 expression was higher in malignant than in benign tissues (P = 0.001). Sequencing was successfully performed in nine cases and mutations were confirmed in three of these cases. One non-sense mutation, one missense mutation and one germline mutation were confirmed for the three cases. This study suggests that p53 overexpression can be a prognostic factor for CIN; however, p53 overexpression in CIN may occur through a mechanism distinct from mutations within the p53 exon 4-8 region.

Pericardial effusion in a dog concurrent with carcinoma of unknown primary origin.

BACKGROUND: Pericardial effusion (PE) due to secondary metastasis has rarely been reported in dogs. CASE DESCRIPTION: This case describes clinical signs and further diagnostics regarding metastatic carcinoma of unknown primary origin (CUP) in refractory PE of a dog. FINDINGS/TREATMENT AND OUTCOME: A nine-year-old, castrated male Shih Tzu dog was referred for evaluation of cough and dyspnea. On presentation, tachypnea, intermittent cough, and muffled heart sounds were noted. Thoracic radiography, electrocardiography, and echocardiography confirmed a PE. No mass lesion was detected at the heart base, aorta, or right atrium (RA). Analysis of the PE showed hemorrhagic cytology, and an idiopathic hemorrhagic PE was tentatively diagnosed. The dog responded to conservative treatment with steroid and diuretics, but the clinical sign recurred. Further evaluation with multi-detector computed tomography (MDCT) was non-diagnostic. The dog died 457 days after initial presentation. Necropsy and histopathology revealed metastatic CUP origin. CONCLUSION: This case illustrated a rare cause of recurrent PE in dogs.

Immunohistochemical application of an antibody specific for human CD1a to the diagnosis of canine mast cell tumour.

Canine mast cell tumours (MCTs) may be graded microscopically for prognostic purposes. Grade I (well-differentiated) and grade II (intermediate differentiation) tumours have an abundance of metachromatic granules within the cytoplasm; however, grade III (poorly differentiated) MCTs may be difficult to diagnose as they frequently have fewer discernable granules. Herein we report that a cross-reactive anti-human CD1a monoclonal antibody (clone O10) may be used in immunohistochemistry to identify canine MCTs of all grades. The antibody was applied to tissue sections from 48 canine MCTs of different histological grades. Serial sections from each tumour were stained with toluidine blue and safranin O to compare diagnostic sensitivity. All MCTs were labelled positively by the CD1a antibody, but histochemical staining was often equivocal and identification of mast cells was extremely difficult in some cases. This antibody did not label neoplastic cells in cases of canine histiocytoma, plasmacytoma or amelanotic melanoma; therefore, the reagent may be a valuable marker for the diagnosis of canine MCTs, especially those tumours of histological grade III.

Expression of Oestrogen Receptor, Progesterone Receptor and Akt in Canine Circumanal Gland Tumours.

We investigated the expression of oestrogen receptor alpha (OR-α), progesterone receptor (PR) and Akt in canine circumanal gland tumours. Immunohistochemistry was conducted on seven normal circumanal glands, 30 circumanal gland adenomas and 40 circumanal gland carcinomas. The expression of OR-α and PR was significantly lower in circumanal gland carcinomas than in circumanal gland adenomas. In contrast, the expression of Akt was markedly higher in circumanal gland carcinomas than in circumanal gland adenomas. These results indicate that the progression of canine circumanal gland tumours is influenced by changes in the expression levels of OR-α, PR and Akt. Identifying the molecular mechanisms of canine circumanal gland tumours requires further study.

Analysis of Obesity-Related Factors and their Association with Aromatase Expression in Canine Malignant Mammary Tumours.

This study was designed to investigate the role of obesity in canine malignant mammary tumours (CMMTs), by assessing aromatase expression and the regulatory roles of immune mediators such as cyclo-oxygenase-2 (COX2), prostaglandin E2 (PGE2), nuclear factor kappa beta (NF-κB), hypoxia inducible factor-1α (HIF-1α) and adipokines (i.e. leptin) in lean, optimal body weight, overweight and obese animals. Clinicopathological data, including the breed, body weight, body condition score and age and neutering status, were collected, together with histopathological characteristics (i.e. histological types, grading and lymphatic invasion). To determine the expression of each factor, immunohistochemistry was conducted with 60 samples of malignant CMMTs. CMMTs from overweight and obese animals had significantly elevated levels of PGE2, and aromatase expression correlated significantly with PGE2, NF-κB and leptin expression. However, no significant difference was observed in terms of histopathological characteristics. The results suggest that PGE2, a known obesity-related immune mediator, could be upregulated in CMMTs from overweight and obese animals. In addition, PGE2, NF-κB and leptin influenced the expression of aromatase, as observed in women.

A simple and rapid chromatographic strip test for detection of antibody to porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a major economic problem for swine industries worldwide despite several disease-reduction strategies such as age-segregated early weaning and all-in-all-out pig movement. Routine diagnosis of PRRSV is carried out by the combined use of an antibody-detecting enzyme-linked immunosorbent assay (ELISA), immunofluorescence, reverse transcription-polymerase chain reaction, and virus isolation. These assays require specialized laboratory equipment in addition to multistep sample handling and sample preparation. The objective of this study was to evaluate a simple pen-side assay (BioSign PRRSV) for rapid detection of PRRSV antibody based on a lateral flow chromatographic strip immunoassay system. This assay uses Escherichia coli-expressed viral nucleocapsid protein antigen for detecting antibodies against PRRSV in swine sera. In this report, the authors describe the evaluation of this assay using sera from both clinical samples and experimentally infected piglets. The results were compared with those of a standard, commercially available antibody ELISA (HerdChek PRRS ELISA) and an indirect immunofluorescence assay using the same serum samples. The BioSign PRRSV assay was capable of detecting antibodies in sera known to contain antibodies to PRRSV, resulting in 93.2% sensitivity for samples from experimentally infected pigs and 98.7% sensitivity for clinical serum samples. For sera that did not contain antibodies to PRRSV, the specificity was found to be 98.5% and 99.2% for clinical and experimental serum samples, respectively.

Analysis of Hypoxia-Inducible Factor-1α Expression Relative to Other Key Factors in Malignant Canine Mammary Tumours.

Hypoxia-inducible factor (HIF)-1α plays important physiological roles, but is also of significance in carcinogenesis in man and animals. This study aimed to identify HIF-1α expression in malignant canine mammary tumours (CMTs) and to find correlations with other key factors by using immunohistochemistry (IHC). The histological classification, grading and evaluation of lymphatic invasion were achieved by examining sections stained by haematoxylin and eosin. Determination of molecular subtype, expression of HIF-1α, oestrogen receptor (OR), progesterone receptor (PR), human epidermal growth factor receptor (HER)-2, Ki67, vascular endothelial growth factor (VEGF) and E-cadherin was evaluated by IHC in 87 samples of malignant CMTs. HIF-1α expression correlated significantly with histological type, grade of cancer, negativity for OR and expression of Ki67 and VEGF. Lymphatic invasion, molecular subtype, PR, HER-2 and E-cadherin levels did not significantly correlate with HIF-1α expression. The results of this study imply that HIF-1α may potentially play a role in increased malignancy of CMTs, as it does in human breast cancer.

Obesity, expression of adipocytokines, and macrophage infiltration in canine mammary tumors.

Obesity influences the development, progression and prognosis of human breast cancer and canine mammary cancer (MC) but the precise underlying mechanism is not well-documented in the fields of either human or veterinary oncology. In the present study, the expression of major adipocytokines, including leptin, adiponectin, and leptin receptor (ObR) in benign (n = 28) and malignant (n = 70) canine mammary tumors was investigated by immunohistochemistry and on the basis of the subject's body condition score (BCS). To evaluate the relationship between obesity and chronic inflammation of the mammary gland, macrophages infiltrating within and around tumoral areas were counted. The mean age of MC development was lower in overweight or obese dogs (9.0 ± 1.8 years) than in lean dogs or optimal bodyweight (10.2 ± 2.9 years), and the evidence of lymphatic invasion of carcinoma cells was found more frequently in overweight or obese group than in lean or optimal groups. Decreased adiponectin expression and increased macrophage numbers in overweight or obese subjects were significantly correlated with factors related to a poor prognosis, such as high histological grade and lymphatic invasion. Leptin expression was correlated with progesterone receptor status, and ObR expression was correlated with estrogen receptor status of MCs, regardless of BCS. Macrophage infiltration within and around the tumor may play an important role in tumor progression and metastasis in obese female dogs and may represent a prognostic factor for canine MCs.

Bovine herpesvirus 1 downregulates the expression of bovine MHC class I molecules.

The mechanism of immunosuppression induced by bovine herpesvirus 1 (BHV-1) was investigated by studying the effects of the virus on the expression of major histocompatibility complex (MHC) class I molecules. After infection with the virus, the expression of class I molecules was detected by flow cytometry and pulse-chase analysis. A selective downregulation of expression of class I molecules was seen in the infected cells, while the class II expression remained unaffected. The reduction in surface expression was evident as early as 8 hours postinfection, reaching significant levels by 12 hours. The downregulation was seen with a multiplicity of infection as low as 0.1. A modified live vaccine strain of BHV-1 also induced the downregulation of class I expression. Analysis of the viral proteins(s) involved in this downregulation with metabolic inhibitors (cycloheximide or phosphonoacetic acid), suggested that the immediate early and/or early proteins of the virus mediate this effect. Pulse-chase analysis revealed that the synthesis of the class I heavy chain, and the assembly/transport of class I molecules were affected by the virus infection. These results suggest that BHV-1 interferes with the molecular mechanisms involved in the synthesis, and assembly/transport of MHC-class I molecules. This interference with the class I antigen processing pathway might help the virus to evade the cytotoxic T-lymphocyte response of the host.

Analysis of bovine trigeminal ganglia following infection with bovine herpesvirus 1.

Following primary infection of the eye, oral cavity, and/or nasal cavity, bovine herpesvirus 1 (BHV-1) establishes latency in trigeminal ganglionic (TG) neurons. Virus reactivation and spread to other susceptible animals occur after natural or corticosteroid-induced stress. Infection of calves with BHV-1 leads to infiltration of lymphocytes in TG and expression of IFN-gamma (interferon-gamma), even in latently infected calves. During latency, virus antigen and nucleic acid positive non-neural cells were occasionally detected in TG suggesting there is a low level of spontaneous reactivation. Since we could not detect virus in ocular or nasal swabs, these rare cells do not support high levels of productive infection and virus release or they do not support virus production at all. Dexamethasone (DEX) was used to initiate reactivation in latently infected calves. Foci of mononuclear or satellite cells undergoing apoptosis were detected 6h after DEX treatment, as judged by the appearance of TUNEL+ cells (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling). BHV-1 antigen expression was initially detected in lymphocytes and other non-neural cells in latently infected calves following DEX treatment. At 24h after DEX treatment, viral antigen expression and nucleic acid were readily detected in neurons. Our data suggest that persistent lymphocyte infiltration and cytokine expression occur during latency because a low number of cells in TG express BHV-1 proteins. Induction of apoptosis and changes in cytokine expression following DEX treatment correlates with reactivation from latency. We hypothesize that inflammatory infiltration of lymphoid cells in TG plays a role in regulating latency.

Latent infection by bovine herpesvirus type-5 in experimentally infected rabbits: virus reactivation, shedding and recrudescence of neurological disease.

Latent infection with bovine herpesvirus type-5 (BHV-5) was established in rabbits inoculated with two South American isolates (EVI-88 and 613) by intranasal or conjunctival routes. Nine rabbits (613, 8/27; EVI-88, 1/34) developed neurological disease and died during acute infection and other three (613, n=2; EVI-88, n=1) developed a delayed neurological disease, at days 34, 41 and 56 post-inoculation (p.i.). Between days 56 and 62 p.i., the remaining rabbits were submitted to five daily administrations of dexamethasone (Dx) to reactivate the infection. Twenty-five out of 44 rabbits (56.8%) shed virus in nasal or ocular secretions after Dx treatment. Virus shedding was first detected at day two post-Dx and lasted from one to 11 days. The highest frequencies of virus reactivation were observed in rabbits inoculated conjunctivally (10/15 versus 15/29); and among rabbits infected with isolate 613 (12/16 versus 13/28). Virus reactivation upon Dx treatment was accompanied by neurological disease in nine rabbits (20.4%), resulting in six deaths (13.6%). Virus in moderate titers and mild to moderate non-suppurative inflammatory changes in the brain characterized the neurological infection. Three other rabbits showed severe neurological signs followed by death after 31 to 54 days of Dx treatment. Virus, viral nucleic acids and inflammatory changes were detected in their brains. The late-onset neurological disease, after acute infection or Dx treatment, was probably a consequence of spontaneous virus reactivation. These results demonstrate that BHV-5 does establish a latent infection in rabbits and that clinical recrudescence may occur upon reactivation.

Detection of canine parvovirus in naturally infected dogs with enteritis and myocarditis by in situ hybridization.

An improved method for the diagnosis of canine parvovirus using in situ hybridization in standard formalin-fixed, paraffin-embedded tissue sections was developed. A digoxigenin-labeled probe complementary to DNA sequences that code for the entire sequence of the capsid protein VP-1 and the middle part of the sequence of the capsid protein VP-2 was designed. Specific histologic localization of canine parvovirus-infected cells was demonstrated in small intestine, tonsil, lymph node, thymus, spleen, heart, liver, and kidney from dogs diagnosed at necropsy with canine parvovirus infection. The in situ hybridization accurately pinpointed the specific sites of viral infection. The detection of canine parvovirus in liver, kidney, and heart tissues together in the same pups could represent an enhanced virulence of this strain of canine parvovirus and suggests a broadened tissue tropism not seen before in Korean strains of canine parvovirus.

Neuropathology of bovine herpesvirus type 5 (BHV-5) meningo-encephalitis in a rabbit seizure model.

The suitability of a rabbit seizure model for studying the neuropathogenesis of bovine herpesvirus type 5 (BHV-5) encephalitis was evaluated. Intranasal administration of BHV-5 (strain TX89) together with intramuscular administration of dexamethasone produced seizures in 70% of rabbits tested and meningo-encephalitis in 100%. Infectious BHV-5 was consistently isolated from the following sites: olfactory bulb; anterior cortex, containing the frontal cortex, olfactory tract and anterior portion of the olfactory cortex; posterior cortex, containing the temporal, parietal, piriform, entorhinal and occipital cortices; amygdala; hippocampus. Less frequently, BHV-5 was isolated from the midbrain and diencephalon, the pons and medulla, the cerebellum, and the trigeminal ganglia. Rabbits similarly infected with the Cooper strain of bovine herpesvirus type 1 showed no neurological signs or meningo-encephalitis, and virus was not recovered from the brain. The brains of BHV-5-infected rabbits showed neuronal degeneration, leptomeningitis, gliosis and perivascular cuffing, predominantly in the olfactory cortex (piriform and entorhinal cortices), amygdala and hippocampus. Mild lymphocytic meningitis was seen in the olfactory bulb and focal lymphocytic infiltration was sometimes present in the medulla and cerebellum. BHV-5, specific antigens and nucleic acids were detected in the olfactory cortex, amygdala and hippocampus by immunohistochemical methods and in-situ hybridization. The results suggested that, after intranasal BHV-5 inoculation, the virus spread to the central nervous system via the olfactory and trigeminal pathways. The olfactory pathway was more susceptible than the trigeminal pathway to neuropathogenic effects.

Study of transneuronal passage of pseudorabies virus in rat central nervous system by use of immunohistochemistry and in situ hybridization.

We studied the uptake and sequential transneuronal passage of pseudorabies virus (PRV) in rat CNS by use of a combination of immunohistochemistry and in situ hybridization. Protocols for rapid detection of PRV by immunohistochemistry and in situ hybridization in rats with PRV infection of the CNS after intranasal instillation of a wild-type strain of PRV were optimized in vitro, using porcine kidney-15 cells. Pseudorabies virus-specific hybridization signals appeared in the cytoplasm and nucleus of PRV-infected porcine kidney-15 cells by postinoculation (PI) hour 6. In tissue sections of PRV-infected rats, PRV nucleic acids were detected in areas of the rat brain in close proximity to the areas in which PRV antigens were evident. The PRV was initially found in the nucleus of trigeminal ganglion neurons at PI hour 24. At PI hour 72, PRV antigens were observed in the mid-brain, and 24 hours later, in the telencephalon. We also found evidence of specific progressive transsynaptic transmission of the virus, and, on the basis of that, we have constructed a map of the synaptic contacts and pathways in the brain. Therefore, combined use of immunohistochemistry and in situ hybridization was useful for characterizing the pathogenesis of PRV in the CNS of rats after intranasal inoculation, following a pattern that mimics PRV infection of the natural host.

[Retrospective analysis of cases with a diagnosis of cerebrocortical necrosis and its relation with type 5 bovine herpesvirus]. / Análisis retrospectivo de casos con diagnóstico de necrosis cerebrocortical y su relación con herpesvirus bovino tipo 5.

In order to demonstrate the association of bovine herpesvirus type 5 (BHV-5) and cerebrocortical necrosis (CCN), 89 such cases were examined in cattle from three regions of Buenos Aires Province, Argentina, registered between 1970-1999. Hematoxylin-eosin staining and BHV-5 in situ hybridization were performed on paraffin-embedded neural tissues. The severity of microscopic lesions was scored according to a 0-3 scale. Morbidity, mortality and lethality rates between groups depending on age and regions were determined. The highest prevalence of CCN was detected between 1979 and 1984, particularly during the spring. Differences in morbidity and mortality rates between groups of age and regions were not detected (P > 0.05). Amaurosis (48%), ptyalism (42%), circling (40%), ataxia (36%) and bruxism (37%) were frequently observed. Lesions were predominantly found in anterior and posterior cortex (90.6%) and diencephalon (36.5%). Meningitis and perivascular cuffing (94.4%) and focal (78%) or diffuse (73%) gliosis were predominant in cerebrum. Focal necrosis was observed in 66.6% of cases. BHV-5 was isolated from 9/19 cases since 1992 and BHV-5 DNA was detected by in situ hybridization in 3/9 cases. No virus was identified in brain tissues with severe lesions. These findings indicate the association of BHV-5 in neurological disease previously reported as CCN.

Evaluation of clinicopathological characteristics and oestrogen receptor gene expression in oestrogen receptor-negative, progesterone receptor-positive canine mammary carcinomas.

The existence of the oestrogen receptor-negative (OR(-))/progesterone receptor-positive (PR(+)) phenotype in canine mammary carcinomas (CMCs) is not well understood, although this phenotype was reported consistently in previous studies. In the present study, immunohistochemistry (IHC) was performed to categorize CMCs with the OR(-)/PR(+) phenotype and compare their clinicopathological features with OR(+)/PR(+) tumours. Of a total of 305 CMCs, 36 (11.8%) were categorized as OR(-)/PR(+) and showed intermediate characteristics between those of OR(+)/PR(+) and OR(-)/PR(-) cases. OR mRNA levels were measured in formalin-fixed, paraffin wax-embedded samples using a novel branched-chain DNA assay method. Similar to the IHC result, one-way analysis of variance showed that the mean normalized OR mRNA level of OR(-)/PR(+) tumours (11.4 ± 16.34) was between that of the OR(-)/PR(-) (mean 4.7 ± 6.35) and OR(+)/PR(+) (mean 15.8 ± 11.95) (P = 0.033) tumours. Only three of the 36 OR(-)/PR(+) tumours completely lacked OR mRNA expression. The OR(-)/PR(+) tumours were not categorized as an independent group nor were they included in the other groups on post-hoc analysis. OR(-)/PR(+) tumours were associated with factors related to poor prognosis compared with OR(+)/PR(+) tumours, but OR(-)/PR(-) tumours were associated with the worst prognostic indicators. Further studies are required in order to determine the clinical significance of the OR(-)/PR(+) phenotype.