Melanin bleaching with potassium permanganate for melanocytic lesions diagnosis in veterinary medicine / Despigmentação de melanócitos com permanganato de potássio para diagnóstico de lesões melanocíticas na medicina veterinária

The aim of this study was to microscopically re-evaluate the melanocytic lesions diagnoses established by the Animal Pathology Laboratory of the Federal University of Uberlândia, in the state of Minas Gerais, Brazil, over a period of eleven years; in addition, to perform a comparative analysis between the conventional histopathological (CH) method and the use of the melanin bleaching (MB) technique with potassium permanganate, sulfuric acid, and oxalic acid solutions. The results of the MB method presented a disagreement in 24.32% of the diagnosis previously by CH, with low agreement (61.0%) and low Kappa coefficient (0.2267). Melanoma was the most frequent lesion, more frequent in elderly and non-breed female dogs. The most frequent melanoma location was in the cutaneous tissue. The presence or absence of a pagetoid spread in cutaneous samples, distribution of melanin, pattern of cell layout, cell morphology, degree of cellular atypia, and the number of mitoses verified after MB were the most important criteria to confirm the diagnosis of malignancy or benignity of the lesions. Evaluating pathologists considered MB to be essential for the majority of diagnoses and an efficient complementary method for the diagnosis of melanocytic lesions, even in cases with a moderate degree of pigmentation.(AU)

Este estudo objetivou reavaliar microscopicamente os diagnósticos de lesões melanocíticas estabelecidos pelo setor de Patologia Animal da Universidade Federal de Uberlândia, em um período de 11 anos, e, com base nesse levantamento, realizar uma análise comparativa entre o método histopatológico convencional (HC) e o método de despigmentação de melanócitos (DM) com permanganato de potássio, ácido sulfúrico e ácido oxálico. A DM revelou discordância em 24,32% dos diagnósticos previamente estabelecidos por HC, apresentando baixa concordância (61,0%) e baixo valor de coeficiente Kappa (0,2267). A alteração mais frequente foi o melanoma, com maior ocorrência em cadelas idosas sem raça definida (SRD). A localização mais frequente dos melanomas foi cutânea. A presença ou ausência de disseminação pagetoide nos casos cutâneos, a forma de distribuição da melanina, o padrão de disposição das células, a morfologia celular, o grau de atipia celular e a quantidade de mitoses verificada após a despigmentação foram critérios de elevada importância para firmar o diagnóstico quanto à malignidade ou benignidade da lesão. A despigmentação foi considerada pelos patologistas avaliadores como essencial para o diagnóstico na maioria dos casos, o que leva a concluir que ela constitui um método complementar eficiente no diagnóstico das lesões melanocíticas, mesmo em casos com grau moderado de pigmentação.(AU)

Prognostic Value of Occult Isolated Tumour Cells within Regional Lymph Nodes of Dogs with Malignant Mammary Tumours.

Canine mammary tumours (CMTs) are the most common type of neoplasm in bitches. As in women, the presence of metastasis in regional lymph nodes is an important prognostic factor in bitches with mammary carcinomas, but the clinical significance of occult isolated tumour cells (ITCs) within lymph nodes is still undefined in this species. The effectiveness of immunohistochemistry (IHC) in identifying occult ITCs and micrometastasis (MIC) was compared with that of the conventional haematoxylin and eosin staining technique. The relationship between tumour size, histological type, histological grade and the presence of metastasis was evaluated. The overall survival (OS) of female dogs with occult mammary carcinomas and ITCs within lymph nodes was analysed. Fragments of mammary carcinoma and regional lymph nodes of 59 female dogs were also evaluated. Histological sections of mammary carcinoma and lymph node samples were studied for tumour diagnosis and lymph node samples were tested by IHC using a pan-cytokeratin antibody. It was found that 35.2% of occult ITCs and 2.8% of hidden MIC were detected when IHC was used. There was a good correlation between the size of the tumour and metastasis to the lymph nodes (P = 0.77). ITCs were observed more frequently in the medullary region (60.7%) and metastases in the cortical region (44.4%). There was no significant difference in the OS between female dogs with occult ITCs and lymph nodes without ITCs. IHC can detect occult tumour cells in lymph nodes that are negative by histopathological examination. Female dogs with nodal ITCs do not have lower survival.

Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants.

Autophagy is a critical regulator of organellar homeostasis, particularly of mitochondria. Upon the loss of membrane potential, dysfunctional mitochondria are selectively removed by autophagy through recruitment of the E3 ligase Parkin by the PTEN-induced kinase 1 (PINK1) and subsequent ubiquitination of mitochondrial membrane proteins. Mammalian sequestrome-1 (p62/SQSTM1) is an autophagy adaptor, which has been proposed to shuttle ubiquitinated cargo for autophagic degradation downstream of Parkin. Here, we show that loss of ref(2)P, the Drosophila orthologue of mammalian P62, results in abnormalities, including mitochondrial defects and an accumulation of mitochondrial DNA with heteroplasmic mutations, correlated with locomotor defects. Furthermore, we show that expression of Ref(2)P is able to ameliorate the defects caused by loss of Pink1 and that this depends on the presence of functional Parkin. Finally, we show that both the PB1 and UBA domains of Ref(2)P are crucial for mitochondrial clustering. We conclude that Ref(2)P is a crucial downstream effector of a pathway involving Pink1 and Parkin and is responsible for the maintenance of a viable pool of cellular mitochondria by promoting their aggregation and autophagic clearance.

Drosophila happyhour modulates JNK-dependent apoptosis.

Mitogen-activated protein kinase kinase kinase kinase-3 (MAP4K3) is a Ste20 kinase family member that modulates multiple signal transduction pathways. We recently identified MAP4K3 as proapoptotic kinase using an RNA interference screening approach. In mammalian cells, MAP4K3 enhances the mitochondrial apoptosis pathway through the post-transcriptional modulation of selected proapoptotic Bcl-2 homology domain 3-only proteins. Recent data suggest that MAP4K3 mutations contribute to pancreatic cancer, which highlights the importance of studying the in vivo function of this kinase. To determine whether the cell death function is conserved in vivo and which downstream signalling pathways are involved, we generated transgenic flies expressing happyhour (hppy), the Drosophila MAP4K3 orthologue. Here, we show that the overexpression of hppy promotes caspase-dependent apoptosis and that the hypothetical kinase domain is essential for inducing cell death. In addition, we show that hppy expression triggers the activation of both the c-Jun N-terminal kinase (JNK) and target of rapamycin (TOR) signalling pathways; however, only JNK signalling is required for apoptosis. Together, our results show that hppy has a JNK-dependent proapoptotic function in Drosophila, which reinforces the hypothesis that MAP4K3 might act as tumour suppressor by regulating apoptosis in higher eukaryotes.

Cooperative alterations of Rb pathway regulators in mouse primary T cell lymphomas.

Alterations in the Rb pathway have been described in many different tumors. In order to study this cell cycle regulatory mechanism in murine T cell lymphomas, we have analyzed the RNA and protein expression of the cyclin D1, cdk4 and retinoblastoma genes in primary tumor samples. We have detected overexpression of the cyclin D1 gene and deficient expression of the retinoblastoma gene in 42 and 28% of these tumors, respectively. The immunohistochemical analysis showed that these RT-PCR results are correlated with a significant increase in the number of positive cells for cyclin D1 and a moderate decrease in the expression of Rb protein, respectively. The analysis of cyclin D1, Rb, p15(INK4b) and p16(INK4a) showed that 75% of lymphomas had alterations in these genes and indicates that the Rb pathway is frequently altered in mouse primary T cell lymphomas. Moreover, 31% of lymphomas presented simultaneous alterations in at least two of these genes, suggesting the importance of concurrent alteration of different Rb pathway regulators. In addition, we have characterized these samples for mutational status of the N-ras and K-ras genes. We have only detected mutations in codon 12 of K-ras in six of 49 lymphomas (12%). Interestingly, five of these lymphomas also showed alterations in at least one of the Rb pathway regulators analyzed here. Taken together, these data suggest that deregulation of the Rb pathway regulators and/or oncogenic activation of K-ras may represent a common important clue in progression of murine T cell lymphomas.