The maspin expression in canine mammary tumors: an immunohistochemical and molecular study

The serpin maspin, a tumor suppressor in breast cancer was described as an inhibitor of cell migration and inducer of cell adhesion between the basement membrane and extracellular matrix resulting in inhibition of tumor metastasis. In contrast, overexpression of maspin is correlated with poor prognosis in other types of cancer. Little is known about expression, regulation and function of maspin in canine mammary tumors. It was demonstrated in this study, a loss of maspin expression in malignant canine mammary cells compared with a pool of normal canine mammary tissue, analyzed by quantitative real-time PCR; weak maspin expression in malignant canine mammary tumors were observed by immunohistochemistry. It was also demonstrated that a correlation with nuclear maspin expression and a good prognosis. It is suggested that maspin could be used as a prognostic marker in canine mammary neoplasia.

Canine papillomatosis: a retrospective study of 24 cases (2001-2011) and immunohistochemical characterization

A retrospective study of 24 cases of papillomas in dogs was performed from January 2001 to March 2011. Additionally, immunohistochemistry (IHC) was used to characterize and evaluate the samples. We found that disease was observed more in mixed breed dogs, ages ranging from 6 months to 10 years (mean 3.1 years), and there was no gender predilection. The main lesion sites were the skin (75%), lips (16.7%), and eyelids (8.3%). Upon histological evaluation, we observed papillary exophytic proliferation of squamous epithelium and papillary endophytic proliferation (inverted) in 87.5% and 12.5% of cases, respectively. The tumors were characterized by spinous layer hyperplasia (87.5%) with koilocytes (70.8%) and intranuclear pale basophilic inclusions bodies (8.3%), prominent granular layer with large amounts of keratohyalin granules (95.8%), and hyperkeratosis in the stratum corneum (100%). Positive immunostaining for Papillomavirus was found in 83.3% of cases, which were distributed between the granular layer and the stratum corneum. These findings indicate the following: that papillomas in dogs are caused by Papillomavirus, the viral cytopathic effect induces epithelial lesions, viral particles are found inside the cell nuclei, and inclusions bodies are rare.

Retrospective canine skin peripheral nerve sheath tumors data with emphasis on histologic, immunohistochemical and prognostic factors

Abstract: In this retrospective study was determined the frequency of canine skin peripheral nerve sheath tumors (PNST) in cases diagnosed by the Setor de Patologia Veterinária of the Universidade Federal do Rio Grande do Sul (SPV-UFRGS), Brazil, between the years 2000 and 2012. The canine profiles, as well as histological, immunohistochemical and prognostic aspects of the tumors were based on 70 samples, comprising 40 females, 29 males and one unspecified sample. Between 2000 and 2012, 2,984 skin tumors of dogs were diagnosed in the SPV-UFRGS, totaling 2.34% of skin neoplasms in dogs. Animals that comprised the largest amount of samples (43%) were those with no breed (SRD), followed by German Shepherds (10%). Females were more affected than males (40/70 - 57% and 29/70 - 41% respectively). Skin PNST of this research showed predominant localization on the limbs (40% in the forelimbs and 29% in the hindlimbs); affecting adult dogs, mostly aged between 8 and 11 years (54%). The samples were routinely processed for hematoxylin and eosin, and were also evaluated by toluidine blue and Masson's trichrome staining, and immunohistochemistry (IHC) anti-vimentin, -S-100, -GFAP, -actin, von Willebrand factor and neurofilament. Anisocytosis and anisokaryosis, mitotic index, intratumoral necrosis, invasion of adjacent tissues, tumor location, local recurrence and metastasis were related to the diagnosis of benign (49/70) or malignant tumor (21/70). The Antoni A histological pattern was observed more frequently in benign tumors. The immunohistochemistry helped to diagnose PNST, and anti-vimentin and anti-protein S-100 showed the highest rates of immunostaining. Throughout statistical analysis of animals with tumor recurrence, it was found that the chance of an animal with a malignant peripheral nerve sheath tumor to develop recurrence is 4.61 times higher than in an animal that had a benign tumor.

Survival analysis of female dogs with mammary tumors after mastectomy: epidemiological, clinical and morphological aspects

Abstract: Mammary gland tumors are the most common type of tumors in bitches but research on survival time after diagnosis is scarce. The purpose of this study was to investigate the relationship between survival time after mastectomy and a number of clinical and morphological variables. Data was collected retrospectively on bitches with mammary tumors seen at the Small Animal Surgery Clinic Service at the University of Brasília. All subjects had undergone mastectomy. Survival analysis was conducted using Cox's proportional hazard method. Of the 139 subjects analyzed, 68 died and 71 survived until the end of the study (64 months). Mean age was 11.76 years (SD=2.71), 53.84% were small dogs. 76.92% of the tumors were malignant, and 65.73% had both thoracic and inguinal glands affected. Survival time in months was associated with age (hazard rate ratios [HRR] =1.23, p-value =1.4x10-4), animal size (HRR between giant and small animals =2.61, p-value =0.02), nodule size (HRR =1.09, p-value =0.03), histological type (HRR between solid carcinoma and carcinoma in a mixed tumor =2.40, p-value =0.02), time between diagnosis and surgery (TDS, with HRR =1.21, p-value =2.7x10-15), and the interaction TDS*follow-up time (HRR =0.98, p-value =1.6x10-11). The present study is one of the few on the subject matter. Several important covariates were evaluated and age, animal size, nodule size, histological type, TDS and TDS*follow up time were identified as significantly associated to survival time.

TP53 mutations and loss of heterozygosity of chromosome 17 in colorectal tumors

The incidence of TP53 point mutations and loss of heterozygosity (LOH) of chromosome 17 in colorectal tumors was determined in a group of Brazilian patients. We screened DNA samples from tumors and distal normal mucosa of 39 patients with colorectal cancer, for TP53 mutations by PCR-SSCP (single-strand conformation polymorphism) analysis. Chromosome 17 LOH was investigated using six PCR-based polymorphic markers and one VNTR probe. TP53 mutations were demonstrated in 15/39 of the cases. Mutations were distributed among all exons examined (five to eight), the majority of them being G/C to A/T transitions. LOH of chromosome 17p and 17q was detected in 70 and 46% of the tumors, respectively. There was a significant association between TP53 mutations and LOH in chromosome 17p (P = 0.0035) and 17q (P = 0.03). Although no correlation was observed between TP53 genetic alterations and clinical/ pathological characteristics, the association of TP53 mutations with loss of both chromosome 17 arms may indicate that TP53 inactivation provokes an unstable phenotype in tumor cells in colorectal tumors.

Amplification of 9q34 in childhood adrenocortical tumors: a specific feature unrelated to ethnic origin or living conditions

Adrenocortical tumors (ACT) in children under 15 years of age exhibit some clinical and biological features distinct from ACT in adults. Cell proliferation, hypertrophy and cell death in adrenal cortex during the last months of gestation and the immediate postnatal period seem to be critical for the origin of ACT in children. Studies with large numbers of patients with childhood ACT have indicated a median age at diagnosis of about 4 years. In our institution, the median age was 3 years and 5 months, while the median age for first signs and symptoms was 2 years and 5 months (N = 72). Using the comparative genomic hybridization technique, we have reported a high frequency of 9q34 amplification in adenomas and carcinomas. This finding has been confirmed more recently by investigators in England. The lower socioeconomic status, the distinctive ethnic groups and all the regional differences in Southern Brazil in relation to patients in England indicate that these differences are not important to determine 9q34 amplification. Candidate amplified genes mapped to this locus are currently being investigated and Southern blot results obtained so far have discarded amplification of the abl oncogene. Amplification of 9q34 has not been found to be related to tumor size, staging, or malignant histopathological features, nor does it seem to be responsible for the higher incidence of ACT observed in Southern Brazil, but could be related to an ACT from embryonic origin.

Adrenocortical tumors in children

Childhood adrenocortical tumors (ACT) are rare. In the USA, only about 25 new cases occur each year. In Southern Brazil, however, approximately 10 times that many cases are diagnosed each year. Most cases occur in the contiguous states of São Paulo and Paraná. The cause of this higher rate has not been identified. Familial genetic predisposition to cancer (p53 mutations) and selected genetic syndromes (Beckwith-Wiedemann syndrome) have been associated with childhood ACT in general but not with the Brazilian counterpart. Most of the affected children are young girls with classic endocrine syndromes (virilizing and/or Cushing). Levels of urinary 17-ketosteroids and plasma dehydroepiandrosterone sulfate (DHEA-S), which are abnormal in approximately 90% of the cases, provide the pivotal clue to a diagnosis of ACT. Typical imaging findings of pediatric ACT consist of a large, well-defined suprarenal tumor containing calcifications with a thin capsule and central necrosis or hemorrhage. The pathologic classification of pediatric ACT is troublesome. Even an experienced pathologist can find it difficult to differentiate carcinoma from adenoma. Surgery is the single most important procedure in the successful treatment of ACT. The role of chemotherapy in the management of childhood ACT has not been established although occasional tumors are responsive to mitotane or cisplatin-containing regimens. Because of the heterogeneity and rarity of the disease, prognostic factors have been difficult to establish in pediatric ACT. Patients with incomplete tumor resection or with metastatic disease at diagnosis have a dismal prognosis. In patients with localized and completely resected tumors, the size of the tumor has predictive value. Patients with large tumors have a much higher relapse rate than those with small tumors.

p53 as a prognostic factor in adrenocortical tumors of adults and children

Mutations of the tumor suppressor gene p53 have been considered to be important determinants in several kinds of human cancer. Accumulation of p53 protein has been reported to correlate with more aggressive clinical behavior in some neoplasms. The role of p53 expression in adrenal cortical tumors (ACT) has not been elucidated but some studies have suggested its correlation with malignant behavior. Our objective was to determine if there is a correlation between the expression of immunoreactive p53 and the biological behavior of ACT. Fifty-seven ACT (21 from children and 36 from adults) were evaluated for p53 expression by immunohistochemistry in formalin-fixed paraffin-embedded tissue and analyzed in terms of outcome. The p53 parameter was utilized semiquantitatively. Tumors were classified as p53 negative when no positivity was observed, or when only few cells showed weak positivity (0/1+) and scored as p53 positive when there was a diffuse and strong nuclear positivity (2+/3+). In children, p53 positivity was associated with clinically malignant ACT and p53 negativity was associated with clinically benign ACT (P = 0.026). In adults' ACT, p53 positivity had an effect on disease-free survival (P<0.001) and also correlated with Weiss score, with a cutoff = 4 (P = 0.04). p53 expression was related to the clinical behavior of ACT in both children and adults and these findings seem to support a role for p53 in ACT progression.

c-erbB-2 expression and nuclear pleomorphism in canine mammary tumors

The objective of the present investigation was to study the expression of c-erbB-2 and MIB-1 and try to associate them with morphological features of the cell such as nuclear pleomorphism, mitotic count and histological grade in a series of 70 canine mammary gland tumors, 22 of them benign and 48 malignant. Tumors were collected at the Veterinary Hospital of UFMG (Brazil) and the Veterinary Faculty of Porto University (Portugal). c-erbB-2 expression was determined according to the guidelines provided by the manufacturer of the HercepTest system and nuclear pleomorphism, mitotic count and histological grade according the Elston and Ellis grading system. The HercepTest is the FDA-approved in vitro diagnostic test marketed by Dako. It is a semi-quantitative immunohistochemical assay used to determine overexpression of HER2 protein (human epidermal growth factor receptor) in breast cancer tissue. MIB-1 expression was also evaluated in 28 malignant tumors. Seventeen (35.4%) of the malignant tumors were positive for c-erbB-2 expression, which was positively associated with nuclear pleomorphism (P < 0.0001), histological grade (P = 0.0017) and mitotic count (P < 0.05). Nuclear pleomorphism also showed a positive association with MIB-1 index (P < 0.0001). These results suggest that some of the biological and morphological characteristics of the tumor are associated in canine mammary gland tumors, as also reported for human breast cancer. It was also possible to show that the immunoexpression of c-erbB-2 can be a factor in mammary carcinogenesis. This fact opens the possibility of using anti-c-erbB-2 antibodies in the treatment of canine mammary tumors.

Manual and semi-automatic quantification of in vivo ¹H-MRS data for the classification of human primary brain tumors

In vivo proton magnetic resonance spectroscopy (¹H-MRS) is a technique capable of assessing biochemical content and pathways in normal and pathological tissue. In the brain, ¹H-MRS complements the information given by magnetic resonance images. The main goal of the present study was to assess the accuracy of ¹H-MRS for the classification of brain tumors in a pilot study comparing results obtained by manual and semi-automatic quantification of metabolites. In vivo single-voxel ¹H-MRS was performed in 24 control subjects and 26 patients with brain neoplasms that included meningiomas, high-grade neuroglial tumors and pilocytic astrocytomas. Seven metabolite groups (lactate, lipids, N-acetyl-aspartate, glutamate and glutamine group, total creatine, total choline, myo-inositol) were evaluated in all spectra by two methods: a manual one consisting of integration of manually defined peak areas, and the advanced method for accurate, robust and efficient spectral fitting (AMARES), a semi-automatic quantification method implemented in the jMRUI software. Statistical methods included discriminant analysis and the leave-one-out cross-validation method. Both manual and semi-automatic analyses detected differences in metabolite content between tumor groups and controls (P < 0.005). The classification accuracy obtained with the manual method was 75% for high-grade neuroglial tumors, 55% for meningiomas and 56% for pilocytic astrocytomas, while for the semi-automatic method it was 78, 70, and 98%, respectively. Both methods classified all control subjects correctly. The study demonstrated that ¹H-MRS accurately differentiated normal from tumoral brain tissue and confirmed the superiority of the semi-automatic quantification method.

Relationship between deep venous thrombosis and inflammatory cytokines in postoperative patients with malignant abdominal tumors

Deep venous thrombosis (DVT) is a common surgical complication in cancer patients and evidence that inflammation plays a role in the occurrence of DVT is increasing. We studied a population of cancer patients with abdominal malignancies with the aim of investigating whether the levels of circulating inflammatory cytokines were associated with postoperative DVT, and to determine the levels in DVT diagnoses. The serum levels of C-reactive protein (CRP), interleukins (IL)-6 and IL-10, nuclear transcription factor-κB (NF-κB) and E-selectin (E-Sel) were determined in 120 individuals, who were divided into 3 groups: healthy controls, patients with and patients without DVT after surgery for an abdominal malignancy. Data were analyzed by ANOVA, Dunnet's T3 test, chi-square test, and univariate and multivariate logistic regression as needed. The CRP, IL-6, NF-κB, and E-Sel levels in patients with DVT were significantly higher than those in the other groups (P<0.05). The IL-10 level was higher in patients with DVT than in controls but lower than in patients without DVT. Univariate analysis revealed that CRP, IL-6, NF-κB, and E-Sel were statistically associated with the risk of DVT (OR=1.98, P=0.002; OR=1.17, P=0.000; OR=1.03, P=0.042; and OR=1.38, P=0.003; respectively), whereas IL-10 had a protective effect (OR=0.94, P=0.011). Multivariate analysis showed that E-Sel was an independent risk factor (OR=1.41, P=0.000). Thus, this study indicated that an increased serum level of E-Sel was associated with increased DVT risk in postoperative patients with abdominal malignancy, indicating that E-Sel may be a useful predictor of diagnosis of DVT.

Intensive chemotherapy as salvage treatment for solid tumors: focus on germ cell cancer

Germ cell tumors present contrasting biological and molecular features compared to many solid tumors, which may partially explain their unusual sensitivity to chemotherapy. Reduced DNA repair capacity and enhanced induction of apoptosis appear to be key factors in the sensitivity of germ cell tumors to cisplatin. Despite substantial cure rates, some patients relapse and subsequently die of their disease. Intensive doses of chemotherapy are used to counter mechanisms of drug resistance. So far, high-dose chemotherapy with hematopoietic stem cell support for solid tumors is used only in the setting of testicular germ cell tumors. In that indication, high-dose chemotherapy is given as the first or late salvage treatment for patients with either relapsed or progressive tumors after initial conventional salvage chemotherapy. High-dose chemotherapy is usually given as two or three sequential cycles using carboplatin and etoposide with or without ifosfamide. The administration of intensive therapy carries significant side effects and can only be efficiently and safely conducted in specialized referral centers to assure optimum patient care outcomes. In breast and ovarian cancer, most studies have demonstrated improvement in progression-free survival (PFS), but overall survival remained unchanged. Therefore, most of these approaches have been dropped. In germ cell tumors, clinical trials are currently investigating novel therapeutic combinations and active treatments. In particular, the integration of targeted therapies constitutes an important area of research for patients with a poor prognosis.

Expression of NR1I3 in mouse lung tumors induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanone

Nuclear receptor subfamily 1, group I, member 3 (NR1I3) is reported to be a possible novel therapeutic target for some cancers, including lung, brain and hematopoietic tumors. Here, we characterized expression of NR1I3 in a mouse model of lung carcinogenesis induced by 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanone (NNK), the most potent tobacco carcinogen. Lung tumors were collected from mice treated with NNK (400 mg/kg) and euthanized after 52 weeks. Benign and malignant lesions were formalin-fixed and paraffin-embedded for histology and immunohistochemistry, with samples snap-frozen for mRNA analysis. Immunohistochemically, we found that most macrophages and type I and II pneumocytes expressed NR1I3, whereas fibroblasts and endothelial cells were NR1I3−. Compared with benign lesions, malignant lesions had less NR1I3+ tumor cells. Gene expression analysis also showed an inverse correlation between NR1I3 mRNA expression and tumor size (P=0.0061), suggesting that bigger tumors expressed less NR1I3 transcripts, in accordance with our immunohistochemical NR1I3 tests. Our results indicate that NR1I3 expression decreased during progression of malignant lung tumors induced by NNK in mice.