Ascites-Derived Organoids to Depict Platinum Resistance in Gynaecological Serous Carcinomas.

Gynaecological serous carcinomas (GSCs) constitute a distinctive entity among female tumours characterised by a very poor prognosis. In addition to late-stage diagnosis and a high rate of recurrent disease associated with massive peritoneal carcinomatosis, the systematic acquisition of resistance to first-line chemotherapy based on platinum determines the unfavourable outcome of GSC patients. To explore the molecular mechanisms associated with platinum resistance, we generated patient-derived organoids (PDOs) from liquid biopsies of GSC patients. PDOs are emerging as a relevant preclinical model system to assist in clinical decision making, mainly from tumoural tissue and particularly for personalised therapeutic options. To approach platinum resistance in a GSC context, proficient PDOs were generated from the ascitic fluid of ovarian, primary peritoneal and uterine serous carcinoma patients in platinum-sensitive and platinum-resistant clinical settings from the uterine aspirate of a uterine serous carcinoma patient, and we also induced platinum resistance in vitro in a representative platinum-sensitive PDO. Histological and immunofluorescent characterisation of these ascites-derived organoids showed resemblance to the corresponding original tumours, and assessment of platinum sensitivity in these preclinical models replicated the clinical setting of the corresponding GSC patients. Differential gene expression profiling of a panel of 770 genes representing major canonical cancer pathways, comparing platinum-sensitive and platinum-resistant PDOs, revealed cellular response to DNA damage stimulus as the principal biological process associated with the acquisition of resistance to the first-line therapy for GSC. Additionally, candidate genes involved in regulation of cell adhesion, cell cycles, and transcription emerged from this proof-of-concept study. In conclusion, we describe the generation of PDOs from liquid biopsies in the context of gynaecological serous carcinomas to explore the molecular determinants of platinum resistance.

Noninvasive detection of microsatellite instability in patients with endometrial cancer.

The analysis of mismatch repair proteins in solid tissue is the standard of care (SoC) for the microsatellite instability (MSI) characterization in endometrial cancer (EC). Uterine aspirates (UAs) or circulating-DNA (cfDNA) samples capture the intratumor heterogeneity and provide a more comprehensive and dynamic molecular diagnosis. Thus, MSI analysis by droplet-digital PCR (ddPCR) in UAs and cfDNA can provide a reliable tool to characterize and follow-up the disease. The UAs, paraffin-embedded tumor tissue (FFPE) and longitudinal plasma samples from a cohort of 90 EC patients were analyzed using ddPCR panel and compared to the SoC. A high concordance (96.67%) was obtained between the analysis of MSI markers in UAs and the SoC. Three discordant cases were validated as unstable by ddPCR on FFPE samples. Besides, a good overall concordance (70.27%) was obtained when comparing the performance of the ddPCR assay on UAs and cfDNA in high-risk tumors. Importantly, our results also evidenced the value of MSI analysis to monitor the disease evolution. MSI evaluation in minimally invasive samples shows great accuracy and sensitivity and provides a valuable tool for the molecular characterization and follow-up of endometrial tumors, opening new opportunities for personalized management of EC.

Red Blood Cells Protein Profile Is Modified in Breast Cancer Patients.

Metastasis is the primary cause of death for most breast cancer (BC) patients who succumb to the disease. During the hematogenous dissemination, circulating tumor cells interact with different blood components. Thus, there are microenvironmental and systemic processes contributing to cancer regulation. We have recently published that red blood cells (RBCs) that accompany circulating tumor cells have prognostic value in metastatic BC patients. RBC alterations are related to several diseases. Although the principal known role is gas transport, it has been recently assigned additional functions as regulatory cells on circulation. Hence, to explore their potential contribution to tumor progression, we characterized the proteomic composition of RBCs from 53 BC patients from stages I to III and IV, compared with 33 cancer-free controls. In this work, we observed that RBCs from BC patients showed a different proteomic profile compared to cancer-free controls and between different tumor stages. The differential proteins were mainly related to extracellular components, proteasome, and metabolism. Embryonic hemoglobins, not expected in adults' RBCs, were detected in BC patients. Besides, lysosome-associated membrane glycoprotein 2 emerge as a new RBCs marker with diagnostic and prognostic potential for metastatic BC patients. Seemingly, RBCs are acquiring modifications in their proteomic composition that probably represents the systemic cancer disease, conditioned by the tumor microenvironment.

Improving the Management of Endometrial Cancer Patients through the Use of Liquid Biopsy Analyses: A Case Report.

Endometrial cancer (EC) is the 4th most common neoplasm of the female genital tract, with 15-20% of patients being of high risk of recurrence which leads to a significant decrease in patient survival. Current therapeutic options for patients with EC are poor, being the combined therapy of carboplatin and paclitaxel the standard of care, with limited efficacy. Therefore, new therapeutic options and better monitoring tools are needed to improve the management of the disease. In the current case report, we showcase the value of liquid biopsy analyses in a microsatellite instability EC patient with initially good prognosis that however underwent rapid progression disease within 6 months post-surgery; through the study of plasma cfDNA/ctDNA dynamics to assess the tumour evolution during treatment, as well as the study of the uterine aspirate as a valuable sample that captures the intra-tumour heterogeneity that allows a comprehensive genomic profiling of the disease to identify potential therapeutic options. Furthermore, preclinical models were generated at the time of tumour progression to assess the efficacy of the identified targeted therapies.

M-TRAP: Safety and performance of metastatic tumor cell trap device in advanced ovarian cancer patients.

OBJECTIVE: Despite radical surgery and chemotherapy, most patients with ovarian cancer die due to disease progression. M-Trap is an implantable medical device designed to capture peritoneal disseminated tumor cells with the aim to focalize the disease. This trial analyzed the safety and performance of the device. METHODS: This first-in-human prospective, multi-center, non-blinded, single-arm study enrolled 23 women with high-grade serous advanced ovarian cancer. After primary or interval debulking surgery, 3 M-Trap devices were placed in the peritoneum of the abdominal cavity. 18-months post-implantation or at disease progression, devices were initially removed by laparoscopy. The primary safety endpoint was freedom from device and procedure-related major adverse events (MAEs) through 6-months post-implantation compared to an historical control. The primary performance endpoint was histopathologic evidence of tumor cells capture. RESULTS: Only one major adverse event was attributable to the device. 18 women were free of device and procedure related MAEs (78.3%). However, the primary safety endpoint was not achieved (p = 0.131), primarily attributable to the greater surgical complexity of the M-Trap patient population. 62% of recurrent patients demonstrated tumor cell capture in at least one device with a minimal tumor cell infiltration. No other long-term device-related adverse events were reported. The secondary performance endpoint demonstrated a lack of disease focalization. CONCLUSIONS: The M-Trap technology failed to meet its primary safety objective, although when adjusted for surgical complexity, the study approved it. Likewise, the devices did not demonstrate the anticipated benefits in terms of tumor cell capture and disease focalization in recurrent ovarian cancer.

Genomic Profiling of Uterine Aspirates and cfDNA as an Integrative Liquid Biopsy Strategy in Endometrial Cancer.

The incidence and mortality of endometrial cancer (EC) have risen in recent years, hence more precise management is needed. Therefore, we combined different types of liquid biopsies to better characterize the genetic landscape of EC in a non-invasive and dynamic manner. Uterine aspirates (UAs) from 60 patients with EC were obtained during surgery and analyzed by next-generation sequencing (NGS). Blood samples, collected at surgery, were used for cell-free DNA (cfDNA) and circulating tumor cell (CTC) analyses. Finally, personalized therapies were tested in patient-derived xenografts (PDXs) generated from the UAs. NGS analyses revealed the presence of genetic alterations in 93% of the tumors. Circulating tumor DNA (ctDNA) was present in 41.2% of cases, mainly in patients with high-risk tumors, thus indicating a clear association with a more aggressive disease. Accordingly, the results obtained during the post-surgery follow-up indicated the presence of ctDNA in three patients with progressive disease. Moreover, 38.9% of patients were positive for CTCs at surgery. Finally, the efficacy of targeted therapies based on the UA-specific mutational landscape was demonstrated in PDX models. Our study indicates the potential clinical applicability of a personalized strategy based on a combination of different liquid biopsies to characterize and monitor tumor evolution, and to identify targeted therapies.

Extracellular Vesicles-Based Biomarkers Represent a Promising Liquid Biopsy in Endometrial Cancer.

Tumor-derived extracellular vesicles (EVs) are secreted in large amounts into biological fluids of cancer patients. The analysis of EVs cargoes has been associated with patient´s outcome and response to therapy. However, current technologies for EVs isolation are tedious and low cost-efficient for routine clinical implementation. To explore the clinical value of circulating EVs analysis we attempted a proof-of-concept in endometrial cancer (EC) with ExoGAG, an easy to use and highly efficient new technology to enrich EVs. Technical performance was first evaluated using EVs secreted by Hec1A cells. Then, the clinical value of this strategy was questioned by analyzing the levels of two well-known tissue biomarkers in EC, L1 cell adhesion molecule (L1CAM) and Annexin A2 (ANXA2), in EVs purified from plasma in a cohort of 41 EC patients and 20 healthy controls. The results demonstrated the specific content of ANXA2 in the purified EVs fraction, with an accurate sensitivity and specificity for EC diagnosis. Importantly, high ANXA2 levels in circulating EVs were associated with high risk of recurrence and non-endometrioid histology suggesting a potential value as a prognostic biomarker in EC. These results also confirmed ExoGAG technology as a robust technique for the clinical implementation of circulating EVs analyses.

Spatial cluster analysis for bovine paratuberculosis in Paraiba State, Northeastern Brazil / Análise de aglomerados espaciais para paratuberculose bovina no Estado da Paraíba, Nordeste do Brasil

The aim of this survey was to identify spatial clustering of bovine paratuberculosis positive herds in the State of Paraíba, Northeastern Brazil. The state was divided into three sampling groups: sampling stratum 1 (mesoregion of Sertão), sampling stratum 2 (mesoregion of Borborema), and sampling stratum 3 (mesoregions of Zona da Mata and Agreste). Ten animals were sampled in herds with up to 99 cows aged over 24 months; 15 animals were sampled in herds with 100 or more cows aged over 24 months; and all animals were sampled in those with up to 10 cows aged over 24 months. In total, 2504 cows aged ≥ 24 months were sampled from 480 herds. Indirect enzyme-linked immunosorbant assay (ELISA) test kits were used for Mycobacterium avium subsp. paratuberculosis (MAP) antibody detection. A herd was deemed positive for paratuberculosis if it included at least one positive animal in herds of up to 24 females, and two positive animals in herds with more than 24 females. Spatial clustering was assessed using the Cuzick-Edwards' k-nearest neighbor method and spatial scan statistics. Two significant clustering of positive herds were detected in Northern part of Borborema mesoregion, a border region with the State of Rio Grande do Norte, in which there is a large animal movement from different locations without knowing the sanitary condition of animals. As serological tests for MAP diagnosis are not widely available and are very expensive, as well as replacement or maintenance of livestock by animal purchasing is common in the region, it is concluded that prevention measures should be applied at herd level.(AU)

O objetivo deste estudo foi identificar agrupamentos espaciais de rebanhos positivos para paratuberculose bovina no Estado da Paraíba, Nordeste do Brasil. O estado foi dividido em três grupos amostrais: estrato amostral 1 (mesorregião do Sertão), estrato amostral 2 (mesorregião da Borborema), e estrato amostral 3 (mesorregiões da Zona da Mata e Agreste). Dez animais foram amostrados em rebanhos com até 99 vacas com idade maior ou igual a 24 meses; 15 animais foram amostrados em rebanhos com 100 ou mais vacas com idade maior ou igual a 24 meses; e todos os animais foram amostrados naqueles rebanhos com até 10 vacas. No total, foram amostradas 2504 vacas com idade ≥24 meses de 480 rebanhos. Para a detecção de anticorpos anti-Mycobacterium avium subsp. paratuberculosis (MAP) foram utilizados kits do teste imunoenzimático indireto (ELISA). Um rebanho foi considerado positivo para paratuberculose se apresentasse pelo menos um animal positivo em rebanhos de até 24 fêmeas, e dois animais positivos em rebanhos com mais de 24 fêmeas. Os agrupamentos espaciais foram avaliados com o uso da metodologia k-vizinhos mais próximos de Cuzick-Edwards e estatística espacial de varredura. Dois agrupamentos significativos de rebanhos positivos foram detectados na parte norte da mesorregião da Borborema, uma região de fronteira com o Estado do Rio Grande do Norte onde há intenso movimento de animais de diferentes locais sem o conhecimento do estado sanitário desses animais. Tendo em vista que os testes sorológicos para diagnóstico de MAP não são amplamente disponíveis e muito caros, bem como é comum na região a reposição e manutenção dos rebanhos por compra de animais, conclui-se que medidas de prevenção devem ser aplicadas em nível de rebanho.(AU)

Estudo retrospectivo da raiva no Estado da Paraíba, Brasil, 2004 a 2011 / Retrospective survey of rabies in Paraíba State, Brazil, from 2004 to 2011

O objetivo deste trabalho foi realizar um estudo retrospectivo da raiva em herbívoros no estado da Paraíba, Brasil, avaliando aspectos epidemiológicos da doença no período de 2004 a 2011. Foram utilizados os registros da notificação dos casos de raiva contidos no Sistema Continental de Vigilância Epidemiológica (SIVCONT). Durante o período avaliado, foram submetidas para diagnóstico amostras de 17.454 animais de todo o Brasil, das quais 43 (0,25%) foram procedentes do estado da Paraíba, e todas elas foram positivas para raiva. A frequência de focos de raiva por município variou de 2,3% a 11,6%. A distribuição dos casos variou conforme a espécie afetada, havendo predominância nos bovinos (93%), seguidos de equinos (4,7%) e um morcego (2,3%) (p < 0,001). Foi observada predominância de casos positivos nos quais a notificação foi efetuada por proprietários (53,5%), seguido de terceiros (39,5%) e vigilância (7%) (p = 0,001). Com relação aos meses de ocorrência dos focos em todos os anos do período avaliado, as frequências variaram de 4,7% no mês de agosto a 14% no mês de abril. Houve predominância de focos no ano de 2010 (41,9%), com diferença significativa entre os anos (p < 0,001). Em conclusão, sugere-se a adoção de medidas estratégicas de controle da raiva e vigilância contínua das doenças nervosas no estado da Paraíba.

The aim of this work was to perform a retrospective study of rabies in herbivores in the state of Paraíba, Brazil, in order to evaluate epidemiological aspects of the disease from 2004 to 2011. Data on notification of rabies cases included in the Continental Epidemiological Surveillance System (SIVCONT) were used. During the study period samples from 17,454 animals from Brazil were sent to rabies diagnosis, from which 43 (0.25%) samples from the state of Paraiba, and all of them were positive for rabies. The frequency of rabies outbreaks by county ranged from 2.3% to 11.6%. The distribution of cases ranged according to species affected, with predominance in cattle (93%), followed by horses (4.7%) and one bat (2.3%) (p < 0.001). It was observed a predominance of positive cases when the notification was made by owners (53.5%), followed by others (39.5%) and surveillance (7%) (p = 0.001). Related to occurrence of outbreaks by month in all years of the study period, the frequencies ranged from 4.7% in August to 14% in April. It was found predominance of outbreaks in 2010 (41.9%), with significant differences among years (p < 0.001). In conclusion, it is suggested the adoption of strategic measures for the control of rabies and continuous surveillance of nervous diseases in herbivores in the state of Paraíba.

Atitudes dos enfermeiros face à família: validação da escala Families' Importance in Nursing Care - Nurses Attitudes / Attitudes of nurses towards families: validation of the scale Families' Importance in Nursing Care - Nurses Attitudes / Actitudes de los enfermeros frente a la familia: validación de la escala Families Importance in Nursing Care - Nurses Attitudes

As atitudes que os enfermeiros adotam em relação à família condicionam o processo de cuidar. Nosso propósito com este estudo foi nesse sentido: dispor de um instrumento que nos permitisse conhecer esta variável. Assim, nossa proposta foi efetuar a adaptação transcultural e avaliar as propriedades psicométricas da versão portuguesa do instrumento Families' Importance in Nursing Care - Nurses Attitudes (FINC-NA), que visa avaliar as atitudes dos enfermeiros acerca da importância de envolver a família nos cuidados de enfermagem. Foi seguido o método preconizado pela literatura. A amostra foi constituída por 136 enfermeiros dos Cuidados de Saúde Primários. Os resultados obtidos nos testes de confiabilidade revelam uma boa consistência interna para o total dos itens (Alpha de Cronbach = 0,87). O estudo psicométrico permite-nos afirmar que a versão em português da FINC-NA, que denominamos A importância das famílias nos cuidados de enfermagem - atitudes dos enfermeiros (IFCE-AE), é um instrumento fidedigno e válido.

The attitudes of nurses towards families determine the care process. With this study, we aimed to obtain an instrument that would allow us to learn about this variable. Hence, our purpose was to perform the cross-cultural adaptation and evaluate the psychometric features of the Portuguese version of the instrument Families' Importance in Nursing Care - Nurses Attitudes (FINC-NA), which aims to evaluate the attitudes of nurses towards the importance of involving the patient's family in the nursing care. The method recommended by the literature was followed. The sample consisted of 136 nurses working in primary health care. The results obtained in the reliability tests showed good internal consistency (Cronbach's Alpha=0.87). The psychometric study permits us to state that the Portuguese version of the FINC-NA, which in Portuguese is referred to as A importância das famílias nos cuidados de enfermagem - atitudes dos enfermeiros (IFCE-AE), is a reliable and valid tool.

Las actitudes adoptadas por enfermeros en relación a la familia condicionan el proceso de cuidar. En tal sentido, nos propusimos con este estudio, disponer de un instrumento que nos permita conocer esta variable. Así, nos propusimos efectuar la adaptación transcultural y evaluar las propiedades psicométricas de la versión portuguesa del instrumento Families Importance in Nursing Care - Nurses Attitudes (FINC-NA), que apunta a evaluar actitudes de enfermeros acerca de involucrar a la familia en los cuidados de enfermería. Se siguió el método sugerido por la literatura. Muestra constituida por 136 Enfermeros de Atención Primaria de Salud. Los resultados obtenidos en pruebas de fiabilidad revelan buena consistencia interna para el total de ítems (Alpha de Cronbach=0,87). El estudio psicométrico nos permite afirmar que la versión en Portugués del FINC-NA, que denominamos La importancia de las familias en el cuidado de enfermería - actitudes de los enfermeros (IFCE-AE) es un instrumento fidedigno y válido.

Imunidade passiva, morbidade neonatal e desempenho de cabritos em diferentes manejos de colostro / Passive immunity, neonatal morbidity and performance of kids in different colostrum management

Objetivando determinar o manejo de colostro que permitisse a melhor aquisição de imunidade passiva em cabritos e avaliar possíveis relações entre imunidade, morbidade e desempenho, amostras de sangue foram obtidas de 58 cabritos da raça Saanen antes e 30 horas após a primeira ingestão de colostro. Os cabritos foram distribuídos em cinco grupos experimentais. No Tratamento 1 (T1) o colostro foi ingerido ad libitum durante 24 horas. Nos demais tratamentos o colostro foi fornecido em mamadeira; os cabritos do T2 ingeriram 200mL de colostro após o parto; do T3, ingeriram 400mL de colostro sendo 200mL após o parto e 200mL após 8 horas; do T4, ingeriram também 400mL de colostro, sendo 200mL após o parto, 200mL após 14 horas; e os do T5 ingeriram 600mL de colostro, 200mL após o parto, 200mL as 12 e 200mL as 24 horas. Os valores séricos de gamaglobulinas foram avaliados por eletroforese. O ganho de peso diário foi utilizado para avaliação do desempenho no período de aleitamento. A ocorrência de doenças foi registrada do nascimento até 28 dias. A menor concentração de gamaglobulinas foi encontrada nos animais do Grupo 2 (1,65g/dL) e a maior concentração foi observada no Grupo 3 (2,60g/dL). Foi observado no Grupo 3 mais animais com diarréia, porém não foram encontradas diferenças estatísticas significativas ao nível de 5 por cento. Os diferentes manejos de colostro não estiveram associados com o desempenho dos cabritos até o final do período neonatal.

hMOF histone acetyltransferase is required for histone H4 lysine 16 acetylation in mammalian cells.

Reversible histone acetylation plays an important role in regulation of chromatin structure and function. Here, we report that the human orthologue of Drosophila melanogaster MOF, hMOF, is a histone H4 lysine K16-specific acetyltransferase. hMOF is also required for this modification in mammalian cells. Knockdown of hMOF in HeLa and HepG2 cells causes a dramatic reduction of histone H4K16 acetylation as detected by Western blot analysis and mass spectrometric analysis of endogenous histones. We also provide evidence that, similar to the Drosophila dosage compensation system, hMOF and hMSL3 form a complex in mammalian cells. hMOF and hMSL3 small interfering RNA-treated cells also show dramatic nuclear morphological deformations, depicted by a polylobulated nuclear phenotype. Reduction of hMOF protein levels by RNA interference in HeLa cells also leads to accumulation of cells in the G(2) and M phases of the cell cycle. Treatment with specific inhibitors of the DNA damage response pathway reverts the cell cycle arrest caused by a reduction in hMOF protein levels. Furthermore, hMOF-depleted cells show an increased number of phospho-ATM and gammaH2AX foci and have an impaired repair response to ionizing radiation. Taken together, our data show that hMOF is required for histone H4 lysine 16 acetylation in mammalian cells and suggest that hMOF has a role in DNA damage response during cell cycle progression.

Estudo comparativo das capacidades aeróbica e anaeróbica de adolescentes com obesidade severa da cidade de Säo Paulo / Comparative study of the aerobic and anaerobic capacities of adolescents with severe obesity in the Säo Paulo city

A obesidade, antes um problema de saúde nos países desenvolvidos, tem-se tornado, ultimamente, um problema de saúde pública também nos países em desenvolvimento, sendo mais preocupante em crianças e adolescentes,uma vez que, estabelecida nesta faixa etária, determina uma potencial piora das condiçöes de saúde associada à obesidade na vida adulta. O estudo comparou a capacidade aeróbica e anaeróbica de meninos e meninas, com obesidade severa da cidade de Säo Paulo-Brasil.Sessenta meninos e sessenta meninas com obesidade severa (índice de massa corpórea - IMC 30.0) com idade entre 14 e 19 anos realizaram densidometria óssea (DEXA), avaliaçåo da capacidade anaeróbia em bicicleta.Os meninos apresentam valores aumentados para consumo máximo de oxigênio (VO2 MÄX), limiar anaeróbio ventilatório (VO2 LV-I), frequência cardíaca do limiar aeróbio ventilatório (FC LV-I), potência máxima (PT máx) e potência média (PT med), embora apresentassem mesma gordura corporal total (MG total) e uma massa magra total (MM total) maior se comparados com as meninas: O dados apresentam valores semelhantes aos apresentados em outros trabalhos de normalidade para capacidade aeróbia e anaeróbia em adolescentes obesos e näo obesos, confirmando a existência de diferenças entre os sexos na performance física.Quando analisamos as diferenças de composiçåo corporal, embora haja uma igualdade se comparando o IMC e a quantidade de gordura corporal total de ambos os grupos, podemos constatar uma diferença significativa quanto a quantidade de massa muscular total, o que poderia explicar tal diferença na performance física entre os sexos