Characterization of human pancreatic orthotopic tumor xenografts suitable for drug screening

Background Efforts to identify novel therapeutic options for human pancreatic ductal adenocarcinoma (PDAC) have failed to result in a clear improvement in patient survival to date. Pancreatic cancer requires efficient therapies that must be designed and assayed in preclinical models with improved predictor ability. Among the available preclinical models, the orthotopic approach fits with this expectation, but its use is still occasional. Methods An in vivo platform of 11 orthotopic tumor xenografts has been generated by direct implantation of fresh surgical material. In addition, a frozen tumorgraft bank has been created, ensuring future model recovery and tumor tissue availability. Results Tissue microarray studies allow showing a high degree of original histology preservation and maintenance of protein expression patterns through passages. The models display stable growth kinetics and characteristic metastatic behavior. Moreover, the molecular diversity may facilitate the identification of tumor subtypes and comparison of drug responses that complement or confirm information obtained with other preclinical models. Conclusions This panel represents a useful preclinical tool for testing new agents and treatment protocols and for further exploration of the biological basis of drug responses.

Characterization of human pancreatic orthotopic tumor xenografts suitable for drug screening

Background Efforts to identify novel therapeutic options for human pancreatic ductal adenocarcinoma (PDAC) have failed to result in a clear improvement in patient survival to date. Pancreatic cancer requires efficient therapies that must be designed and assayed in preclinical models with improved predictor ability. Among the available preclinical models, the orthotopic approach fits with this expectation, but its use is still occasional. Methods An in vivo platform of 11 orthotopic tumor xenografts has been generated by direct implantation of fresh surgical material. In addition, a frozen tumorgraft bank has been created, ensuring future model recovery and tumor tissue availability. Results Tissue microarray studies allow showing a high degree of original histology preservation and maintenance of protein expression patterns through passages. The models display stable growth kinetics and characteristic metastatic behavior. Moreover, the molecular diversity may facilitate the identification of tumor subtypes and comparison of drug responses that complement or confirm information obtained with other preclinical models. Conclusions This panel represents a useful preclinical tool for testing new agents and treatment protocols and for further exploration of the biological basis of drug responses.

Horizontal transfer of exosomal microRNAs transduce apoptotic signals between pancreatic beta-cells.

BACKGROUND: Diabetes mellitus is a common metabolic disorder characterized by dysfunction of insulin-secreting pancreatic beta-cells. MicroRNAs are important regulators of beta-cell activities. These non-coding RNAs have recently been discovered to exert their effects not only inside the cell producing them but, upon exosome-mediated transfer, also in other recipient cells. This novel communication mode remains unexplored in pancreatic beta-cells. In the present study, the microRNA content of exosomes released by beta-cells in physiological and physiopathological conditions was analyzed and the biological impact of their transfer to recipient cells investigated. RESULTS: Exosomes were isolated from the culture media of MIN6B1 and INS-1 derived 832/13 beta-cell lines and from mice, rat or human islets. Global profiling revealed that the microRNAs released in MIN6B1 exosomes do not simply reflect the content of the cells of origin. Indeed, while a subset of microRNAs was preferentially released in exosomes others were selectively retained in the cells. Moreover, exposure of MIN6B1 cells to inflammatory cytokines changed the release of several microRNAs. The dynamics of microRNA secretion and their potential transfer to recipient cells were next investigated. As a proof-of-concept, we demonstrate that if cel-miR-238, a C. Elegans microRNA not present in mammalian cells, is expressed in MIN6B1 cells a fraction of it is released in exosomes and is transferred to recipient beta-cells. Furthermore, incubation of untreated MIN6B1 or mice islet cells in the presence of microRNA-containing exosomes isolated from the culture media of cytokine-treated MIN6B1 cells triggers apoptosis of recipient cells. In contrast, exosomes originating from cells not exposed to cytokines have no impact on cell survival. Apoptosis induced by exosomes produced by cytokine-treated cells was prevented by down-regulation of the microRNA-mediating silencing protein Ago2 in recipient cells, suggesting that the effect is mediated by the non-coding RNAs. CONCLUSIONS: Taken together, our results suggest that beta-cells secrete microRNAs that can be transferred to neighboring beta-cells. Exposure of donor cells to pathophysiological conditions commonly associated with diabetes modifies the release of microRNAs and affects survival of recipient beta-cells. Our results support the concept that exosomal microRNAs transfer constitutes a novel cell-to-cell communication mechanism regulating the activity of pancreatic beta-cells.

Cytological diagnosis of pancreatic neuroendocrine neoplasms

The main cytological features of neuroendocrine pancreatic neoplasm are described along with a discussion about the difficulties in classification/grading and the new reporting system for reporting pancreatic cytopathology. An overview about the ancillary techniques and the differential diagnosis is also given.

Combinative effects of β-Lapachone and APO866 on pancreatic cancer cell death through reactive oxygen species production and PARP-1 activation.

UNLABELLED: Pancreatic cancer (PC) is one of the most lethal human malignancies and a major health problem. Patients diagnosed with PC and treated with conventional approaches have an overall 5-year survival rate of less than 5%. Novel strategies are needed to treat this disease. Herein, we propose a combinatorial strategy that targets two unrelated metabolic enzymes overexpressed in PC cells: NAD(P)H: quinone oxidoreductase-1 (NQO1) and nicotinamide phosphoribosyl transferase (NAMPT) using β-lapachone (BL) and APO866, respectively. We show that BL tremendously enhances the antitumor activity of APO866 on various PC cell lines without affecting normal cells, in a PARP-1 dependent manner. The chemopotentiation of APO866 with BL was characterized by the following: (i) nicotinamide adenine dinucleotide (NAD) depletion; (ii) catalase (CAT) degradation; (iii) excessive H2O2 production; (iv) dramatic drop of mitochondrial membrane potential (MMP); and finally (v) autophagic-associated cell death. H2O2 production, loss of MMP and cell death (but not NAD depletion) were abrogated by exogenous supplementation with CAT or pharmacological or genetic inhibition of PARP-1. Our data demonstrates that the combination of a non-lethal dose of BL and low dose of APO866 optimizes significantly cell death on various PC lines over both compounds given separately and open new and promising combination in PC therapy.

Hemangioma vertebral sintomático tratado com radioterapia exclusiva: relato de caso e revisão da literatura

Neste trabalho descrevemos a apresentação clínica e dos exames de imagem, bem como a utilização da radioterapia exclusiva, em um caso de hemangioma vertebral sintomático. Após três meses de seguimento, houve melhora parcial da dor. Recomendamos dose total de 40 Gy, em frações diárias de 2 Gy. Em seguida é apresentada revisão da literatura.

Targeting cancer cell metabolism in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is expected to become the second leading cause of cancer death by 2030. Current therapeutic options are limited, warranting an urgent need to explore innovative treatment strategies. Due to specific microenvironment constraints including an extensive desmoplastic stroma reaction, PDAC faces major metabolic challenges, principally hypoxia and nutrient deprivation. Their connection with oncogenic alterations such as KRAS mutations has brought metabolic reprogramming to the forefront of PDAC therapeutic research. The Warburg effect, glutamine addiction, and autophagy stand as the most important adaptive metabolic mechanisms of cancer cells themselves, however metabolic reprogramming is also an important feature of the tumor microenvironment, having a major impact on epigenetic reprogramming and tumor cell interactions with its complex stroma. We present a comprehensive overview of the main metabolic adaptations contributing to PDAC development and progression. A review of current and future therapies targeting this range of metabolic pathways is provided.

High c-Met expression in stage I-II pancreatic adenocarcinoma: proposal for an immunostaining scoring method and correlation with poor prognosis.

AIMS: c-Met is an emerging biomarker in pancreatic ductal adenocarcinoma (PDAC); there is no consensus regarding the immunostaining scoring method for this marker. We aimed to assess the prognostic value of c-Met overexpression in resected PDAC, and to elaborate a robust and reproducible scoring method for c-Met immunostaining in this setting. METHODS AND RESULTS: c-Met immunostaining was graded according to the validated MetMab score, a classic visual scale combining surface and intensity (SI score), or a simplified score (high c-Met: ≥20% of tumour cells with strong membranous staining), in stage I-II PDAC. A computer-assisted classification method (Aperio software) was developed. Clinicopathological parameters were correlated with disease-free survival (DFS) and overall survival(OS). One hundred and forty-nine patients were analysed retrospectively in a two-step process. Thirty-seven samples (whole slides) were analysed as a pre-run test. Reproducibility values were optimal with the simplified score (kappa = 0.773); high c-Met expression (7/37) was associated with shorter DFS [hazard ratio (HR) 3.456, P = 0.0036] and OS (HR 4.257, P = 0.0004). c-Met expression was concordant on whole slides and tissue microarrays in 87.9% of samples, and quantifiable with a specific computer-assisted algorithm. In the whole cohort (n = 131), patients with c-Met(high) tumours (36/131) had significantly shorter DFS (9.3 versus 20.0 months, HR 2.165, P = 0.0005) and OS (18.2 versus 35.0 months, HR 1.832, P = 0.0098) in univariate and multivariate analysis. CONCLUSIONS: Simplified c-Met expression is an independent prognostic marker in stage I-II PDAC that may help to identify patients with a high risk of tumour relapse and poor survival.

Apresentações incomuns do hemangioma hepático: ensaio iconográfico

O nosso objetivo foi descrever e ilustrar aspectos incomuns do hemangioma hepático na ultra-sonografia (US), tomografia computadorizada (TC) e ressonância magnética (RM). A partir da análise retrospectiva de 300 casos de pacientes com diagnósticos de hemangioma hepático, por meio da análise combinada de exames de imagem, biópsia ou acompanhamento clínico, selecionamos aqueles com apresentação atípica em um ou mais métodos de imagem ou aqueles com evolução não usual, ilustrando os seus principais aspectos de imagem. Entre os casos apresentados, escolhemos pacientes com hemangiomas: hipoecogênicos na US; hipovasculares ou avasculares na TC e RM; com calcificações grosseiras; gigantes e medindo mais de 20 cm de diâmetro; predominantemente exofíticos; hipointensos em T2; promovendo defeito de perfusão; com cicatriz central e simulando hiperplasia nodular focal; com crescimento evolutivo. O hemangioma hepático é o tumor mais comum do fígado e geralmente tem apresentação típica. Porém, os seus diversos aspectos não usuais precisam ser conhecidos para auxiliar na orientação diagnóstica e conduta.

Role of microRNAs in the age-associated decline of pancreatic beta cell function in rat islets.

AIMS/HYPOTHESIS: Ageing can lead to reduced insulin sensitivity and loss of pancreatic beta cell function, predisposing individuals to the development of diabetes. The aim of this study was to assess the contribution of microRNAs (miRNAs) to age-associated beta cell dysfunction. METHODS: The global mRNA and miRNA profiles of 3- and 12-month-old rat islets were collected by microarray. The functional impact of age-associated differences in miRNA expression was investigated by mimicking the observed changes in primary beta cells from young animals. RESULTS: Beta cells from 12-month-old rats retained normal insulin content and secretion, but failed to proliferate in response to mitotic stimuli. The islets of these animals displayed modifications at the level of several miRNAs, including upregulation of miR-34a, miR-124a and miR-383, and downregulation of miR-130b and miR-181a. Computational analysis of the transcriptomic modifications observed in the islets of 12-month-old rats revealed that the differentially expressed genes were enriched for miR-34a and miR-181a targets. Indeed, the induction of miR-34a and reduction of miR-181a in the islets of young animals mimicked the impaired beta cell proliferation observed in old animals. mRNA coding for alpha-type platelet-derived growth factor receptor, which is critical for compensatory beta cell mass expansion, is directly inhibited by miR34a and is likely to be at least partly responsible for the effects of this miRNA. CONCLUSIONS/INTERPRETATION: Changes in the level of specific miRNAs that occur during ageing affect the proliferative capacity of beta cells. This might reduce their ability to expand under conditions of increased insulin demand, favouring the development of type 2 diabetes.

Diferenciação entre cisto simples e hemangioma hepático utilizando seqüência de ressonância magnética ponderada em T2 com técnica gradiente-eco (B-FFE)

OBJETIVO: Estabelecer o valor das seqüências ponderadas em T2 para diferenciar cistos simples de hemangiomas hepáticos. MATERIAIS E MÉTODOS: Estudo prospectivo, observacional, transversal e duplo-cego em 52 pacientes com 91 lesões hepáticas (34 cistos simples e 57 hemangiomas) submetidos a ressonância magnética de abdome. A análise conjunta de todas as seqüências realizadas foi considerada o padrão-ouro. Dois observadores independentes avaliaram, subjetivamente, as seqüências TSE com TE longo e B-FFE, procurando diferenciar cistos de hemangiomas. Foram calculadas a eficácia das seqüências e a concordância interobservador e intra-observador por meio do teste kappa (κ) (p < 0,05*). RESULTADOS: As dimensões dos cistos variaram entre 0,5 e 6,5 cm (média de 1,89 cm) e as dos hemangiomas, entre 0,8 e 11 cm (média de 2,62 cm). A concordância entre a avaliação da seqüência com TE longo e o padrão-ouro foi insignificante (κ: 0,00-0,10). A concordância entre a avaliação da seqüência B-FFE e o padrão-ouro variou de substancial (κ: 0,62-0,71) a quase perfeita (κ: 0,86) para ambos os examinadores. A concordância interobservador e intra-observador para a seqüência B-FFE variou entre substancial (κ: 0,62-0,70) e quase perfeita (κ: 0,85-0,91). CONCLUSÃO: A técnica B-FFE apresenta eficácia e reprodutibilidade elevadas na diferenciação de cistos e hemangiomas.

Normal values for pancreatic stone protein in different age groups.

BACKGROUND: Pancreatic stone protein (PSP) has been identified as a promising sepsis marker in adults, children and neonates. However, data on population-based reference values are lacking. This study aimed to establish age-specific reference values for PSP. METHODS: PSP was determined using a specific ELISA. PSP serum concentrations were determined in 372 healthy subjects including 217 neonates, 94 infants and children up to 16 years, and 61 adults. The adjacent categories method was used to determine which age categories had significantly different PSP concentrations. RESULTS: PSP circulating levels were not gender-dependent and ranged from 1.0 to 99.4 ng/ml with a median of 9.2 ng/ml. PSP increased significantly between the age categories, from a median of 2.6 ng/ml in very preterm newborns, to 6.3 ng/ml in term newborns, to 16.1 ng/ml in older children (p < 0.001). PSP levels were higher on postnatal day three compared to levels measured immediately post delivery (p < 0.001). Paired umbilical artery and umbilical vein samples were strongly correlated (p < 0.001). Simultaneously obtained capillary heel-prick versus venous samples showed a good level of agreement for PSP (Rho 0.89, bias 19 %). CONCLUSIONS: This study provides age-specific normal values that may be used to define cut-offs for future trials on PSP. We demonstrate an age-dependent increase of PSP from birth to childhood.

Hemangioma cavernoso hepático roto: relato de caso e revisão da literatura

O hemangioma hepático é frequentemente assintomático e, mesmo quando de grandes dimensões, não mostra tendência a sangramento. Os autores relatam o caso de uma paciente de 31 anos de idade com dor abdominal aguda, cujos exames de imagem evidenciaram hematoma no lobo hepático esquerdo, associado a hemoperitônio. A paciente foi submetida a hepatectomia esquerda, com o diagnóstico histopatológico de hemangioma cavernoso.

Hemangioma hepático subcapsular com realce perilesional: achados de RM

OBJETIVO: Descrever os achados de imagem de RM no hemangioma hepático com realce perilesional. MATERIAIS E MÉTODOS: Realizou-se uma pesquisa no banco de dados da unidade de RM para identificar todos os casos de hemangioma hepático com realce perilesional entre março de 2008 e julho de 2009. Todos os pacientes foram submetidos a RM pré-contraste em imagens ponderadas em T1 e T2 e em imagens dinâmicas após injeção de gadolínio. Características do hemangioma e do realce perilesional foram avaliadas nas imagens de RM. RESULTADOS: Sete hemangiomas em sete pacientes (cinco homens, duas mulheres; faixa etária entre 41-69 anos; média de 57 anos) foram incluídos no presente estudo. O tamanho das lesões variou de 7 a 20 mm (média de 12,4 mm). Na fase dominante arterial hepática, todos os sete hemangiomas mostraram realce perilesional cuneiforme que se atenuou nas imagens dois minutos após injeção de gadolínio. Quatro dessas lesões demonstraram realce capsular adjacente. CONCLUSÃO: Todos os hemangiomas hepáticos com realce perilesional eram lesões capsulares medindo menos que 2 cm. Tal localização sugere que esses hemangiomas podem recrutar vasos capsulares responsáveis pelo realce perilesional