Study of related senescence pathways in soft tissue tumors development /

Introducción: En esta tesis doctoral hemos estudiado y analizado los marcadores de senescencia celular en un subgrupo de tumores de partes blandas, los tumores de la vaina nerviosa periférica (PNSTs), neoplasias que pueden ocurrir de forma esporádica o estar asociadas a síndromes clínicos como las neurofibromatosis (NFs). La senescencia inducida por oncogenes (OIS) es un tipo específico de senescencia y es un mecanismo de detención del crecimiento celular demostrado en líneas celulares y en algunos tipos de cánceres humanos. Por esta razón se considera un proceso de supresión tumoral contra la transformación maligna de las células. Diferentes vías moleculares están involucradas en este proceso, como RAS/RAF/MAPK o ARF/p53 y p16INK4a/ pRb. Entre los PNSTs, los schwannomas representan un subgrupo de tumores benignos que sólo muy raramente se transforman a neoplasias malignas. Estos tumores, al igual que el nevus, se componen de células derivadas de la cresta neural. Mientras en el nevus está claro que el mecanismo principal que evita la transformación maligna es la entrada en senescencia, en respuesta a la activación de la vía de RAS/MAPK/RAF (mutación de BRAF V600E), poco se conoce todavía de los mecanismos involucrados en el desarrollo de los PNSTs. Los PNSTs que ocurren en las NFs están relacionados con alteraciones de dos genes específicos de estos síndromes, NF1 y NF2, capaces de activar la vía de RAS/MAPK/RAF. Sin embargo, muy poco se conoce sobre la patogénesis de los PNSTs esporádicos y sobre la transformación maligna de los schwannomas. Este estudio analiza los marcadores de senescencia en los PNSTs, con la finalidad de demostrar el posible papel de la senescencia en sus tumorigénesis, junto con la presencia o ausencia de mutaciones BRAF V600E en estos tumores, especialmente en los esporádicos. Métodos: Se ha realizado un estudio retrospectivo de inmunohistoquímica en 39 schwannomas y 18 tumores malignos de la vaina nerviosa periférica tumores (MPNSTs), utilizando tinción para marcadores de senescencia p16INK4a, Ki67, p53 y ciclina D1 en todos los casos. Además, se analizó la actividad de la SA-β-Gal, utilizando una tinción histoquímica específica en los casos en que se disponía de tejido congelado (n = 8). Por otra parte se analizaron las mutaciones del exón 15 de BRAF y de los exones 2 y 3 del gen KRAS, a través de la reacción en cadena de la polimerasa (PCR), y la secuenciación se realizó en muestras fijadas en formalina e incluidas en parafina de 59 schwannomas, 16 neurofibromas y 24 MPNSTs, de pacientes relacionados y no relacionados con las NFs. Resultados: En los schwannomas encontramos una elevada expresión inmunohistoquímica de p16INK4a, baja o ausentes niveles de Ki67 y observamos positividad de la actividad β-SA-Gal. Un patrón opuesto se encontró en los MPNSTs. Se observaron mutaciones de BRAF V600E en 4/40 y 1/13 schwannomas MPNSTs, no asociados con NFs. En un caso de schwannoma esporádico encontramos la mutación de gen KRAS G12S. Conclusión: Nuestros resultados apoyan la hipótesis que los schwannomas son lesiones senescentes y que en los MPNSTs este fenotipo de senescencia se pierde durante la trasformación maligna. Además, el hallazgo de mutaciones de BRAF y KRAS en un subgrupo de PNSTs no relacionadas con NFs representa un evento importante, especialmente para el desarrollo de un tratamiento específico novedoso para estos tumores.Introduction: In this thesis we have studied and analyzed markers of cellular senescence in a subset of soft tissue tumors, peripheral nerve sheath tumors (PNSTs), neoplasms that may occur sporadically or be associated with clinical syndromes such as neurofibromatosis (NFs). Oncogenic-induced senescence (OIS) is a growth-arrest mechanism demonstrated in cells and in some type of human cancers. For this reason it is considered a tumor suppressor process against malignant transformation. Different molecular pathways are involved in this process, such as RAS/RAF/MAPK or ARF/p53 and p16INK4a/pRb. Among the PNSTs, schwannomas represent a subset of benign tumors that only very rarely undergo malignant changes. These tumors, like nevus, are composed of cells derived from the neural crest. While in the nevus is clear that the main mechanism preventing malignant transformation is the entry into senescence, in response to activation of the RAS/MAPK/RAF pathway (BRAF mutation V600E), the mechanisms involved in the development of PNSTs remain still unknown. The PNSTs occurring in NFs are related to alterations in two specific genes of these syndromes, NF1 and NF2, able to activate the RAS/MAPK/RAF pathway. However, very little is known about the pathogenesis of sporadic PNSTs and malignant transformation of schwannomas. This study analyzes the markers of senescence in PNSTs, in order to demonstrate the potential role of senescence in their tumorigenesis, along with the presence or absence of BRAF V600E mutations in these tumors, especially in sporadic ones. Methods: A retrospective immunohistochemical study was done in 39 schwannomas and 18 malignant peripheral nerve sheath tumors (MPNSTs). Staining for p16INK4a, Ki67, p53 and Cyclin D1 was performed in all the cases. Additionally, SA-β-Gal staining was done in those cases in which frozen tissue was available (n=8). Moreover BRAF exon 15 and KRAS exons 2 and 3 polymerase chain reaction (PCR) sequencing was performed in formalin-fixed/paraffin-embedded samples of 59 schwannomas, 16 neurofibromas and 24 MPNSTs, related and non-related to NF types 1 and 2. Results: In schwannomas we found high expression of p16INK4a, low or absent levels of Ki67 and positivity of SA-β-Gal activity. An opposite pattern was found in MPNSTs. Oncogenic BRAF V600E mutations were observed in 4/40 schwannomas and 1/13 MPNST, not associated with NF. A KRAS G12S mutation was also evident in one sporadic schwannoma. Conclusions: Our results support the senescence nature of schwannomas and the absence of senescence phenotype in MPNSTs. Moreover the finding of BRAF V600E and KRAS mutations in a subset of PNSTs not related to NF represents an important event especially for the development of novel specific treatment for these tumors

O direito internacional e a biopirataria na Amazônia: Uma análise das ações das empresas privadas multinacionais à luz dos deveres fundamentais

This paper aims to analyze the actions of private international companies that commit biopiracy in the Brazilian Amazon Forest. And it intends to investigate whether, even without specific legislation that criminalizes this action, there is some fence to it in the country, and what are the legal consequences of the recognition of a fundamental duty in the Brazilian legal system. To achieve this goal, the article addresses the concept of biopiracy, existing cases, global social relations, international and national legislation and finally the Theory of Fundamental Duties to understand the application of art caput. 225 of CF/88. This research used a theoretical basis based on bibliographic and documentary research, of the current legislation. The methodology used in the research was the dialectical method. It was obtained as a result of the present work that biopiracy, despite not having any fencing in the treaties in which Brazil is a signatory nor in the national environmental legislation, finds a fence in the Constitution, especially in the fundamental duty of preserving the environment. Although little addressed, the application of the fundamental duty imposes legal consequences on the whole community and may restrict the action of multinationals and impose on the State the obligation to carry out the control of constitutionality.Este trabalho tem como objetivo analisar a atuação de empresas privadas internacionais que cometem biopirataria na Floresta Amazônica Brasileira. E pretende averiguar se mesmo sem legislação especifica que criminalize essa ação, há alguma vedação a ela no país, e quais as consequências jurídicas do reconhecimento de um dever fundamental no ordenamento jurídico brasileiro. Para atingir esse objetivo, o artigo aborda o conceito da biopirataria, os casos existentes, as relações sociais globais, a legislação internacional e nacional e finalmente a Teoria dos Deveres Fundamentais para compreender a aplicação do caput do art. 225 da CF/88. A presente pesquisa utilizou um embasamento teórico fundamentado em pesquisas bibliográficas e documentais, das legislações vigentes. A metodologia utilizada na pesquisa foi o método dialético. Obteve-se como resultado do presente trabalho que a biopirataria, apesar de não possuir nenhuma vedação nos tratados em que o Brasil é signatário nem na legislação ambiental nacional, encontra vedação na Constituição, especialmente no dever fundamental de preservação do meio ambiente. Apesar de pouco abordados, a aplicação do dever fundamental, impõe consequências jurídicas a toda a coletividade e pode restringir a ação das multinacionais e impor ao Estado a obrigação de realizar o controle de constitucionalidade

Two cases of sternal osteomyelitis due to Mycobacterium africanum: a casual or causal association

Abstract The sternal localization of bone and joint tuberculosis (TB) is rare, has an insidious clinical presentation, and usually affects young adults living in endemic areas. Mycobacterium africanum causes a relevant proportion of human TB in West Africa and in migrants from endemic countries . Here, we report two cases of sternal osteomyelitis due to M. africanum in migrants

A systematic analysis of orphan cyclins reveals CNTD2 as a new oncogenic driver in lung cancer

As lung cancer has increased to the most common cause of cancer death worldwide, prognostic biomarkers and effective targeted treatments remain lacking despite advances based on patients' stratification. Multiple core cyclins, best known as drivers of cell proliferation, are commonly deregulated in lung cancer where they may serve as oncogenes. The recent expansion of the cyclin family raises the question whether new members might play oncogenic roles as well. Here, we investigated the protein levels of eight atypical cyclins in lung cancer cell lines and formalin-fixed and paraffin-embedded (FFPE) human tumors, as well as their functional role in lung cancer cells. Of the new cyclins evaluated, CNTD2 was significantly overexpressed in lung cancer compared to adjacent normal tissue, and exhibited a predominant nuclear location. CNTD2 overexpression increased lung cancer cell viability, Ki-67 intensity and clonogenicity and promoted lung cancer cell migration. Accordingly, CNTD2 enhanced tumor growth in vivo on A549 xenograft models. Finally, the analysis of gene expression data revealed a high correlation between elevated levels of CNTD2 and decreased overall survival in lung cancer patients. Our results reveal CNTD2 as a new oncogenic driver in lung cancer, suggesting value as a prognostic biomarker and therapeutic target in this disease

Carfilzomib, lenalidomide, and dexamethasone in relapsed refractory multiple myeloma: a prospective real-life experience of the Regional Tuscan Myeloma Network

IntroductionCarfilzomib, a potent, irreversible, selective proteasome inhibitor has demonstrated consistent results in relapsed/refractory multiple myeloma (RRMM) combined with lenalidomide and dexamethasone (KRd). No prospective studies are yet available that analyzed the efficacy of the KRd combination.MethodsHerein, we report a multicenter prospective observational study on 85 patients who were treated with KRd combination as the second or third line of treatment, according to standard practice.ResultsThe median age was 61 years; high-risk cytogenetic was found in 26% and renal impairment (estimated glomerular filtration rate (eGFR) <60 ml/min) in 17%. After a median follow-up of 40 months, patients received a median number of 16 cycles of KRd, with a median duration of treatment (DoT) of 18 months (range, 16.1–19.2 months). The overall response rate was 95%, with a high-quality response (≥very good partial remission [VGPR]) in 57% of the patients. The median progression-free survival (PFS) was 36 months (range, 29.1–43.2 months). Achievement of at least VGPR and a previous autologous stem cell transplantation (ASCT) were associated with longer PFS. The median overall survival (OS) was not reached (NR); the 5-year OS rate was 73%. Nineteen patients underwent KRd treatment as a bridge to autologous transplantation, obtaining a post-transplant minimal residual disease (MRD) negativity in 65% of cases. The most common adverse events were hematological, followed by infection and cardiovascular events, rarely G3 or higher, with a discontinuation rate for toxicities of 6%. Our data confirmed the feasibility and safety of the KRd regimen in real life

An 1H NMR study of the cytarabine degradation in clinical conditions to avoid drug waste, decrease therapy costs and improve patient compliance in acute leukemia

Cytarabine, the 4-amino-1-(β-D-arabinofuranosyl)-2(1H)-pyrimidinone, (ARA-C) is an antimetabolite cytidine analogue used worldwide as key drug in the management of leukaemia. As specified in the manufacturers' instructions, once the components-sterile water and cytarabine powder-are unpackaged and mixed, the solution begins to degrade after 6 hours at room temperature and 12 hours at 4°C. To evaluate how to avoid wasting the drug in short-term, low-dose treatment regimens, the reconstituted samples, stored at 25°C and 4°C, were analyzed every day of the test week by reversed-phase HPLC and high-field NMR spectroscopy. All the samples remained unchanged for the entire week, which corresponds to the time required to administer the entire commercial drug package during low-dose therapeutic regimens. The drug solution was stored in a glass container at 4°C in an ordinary freezer and drawn with sterile plastic syringes; during this period, no bacterial or fungal contamination was observed. Our findings show that an cytarabine solution prepared and stored in the original vials retains its efficacy and safety and can, therefore, be divided into small doses to be administered over more days, thus avoiding unnecessary expensive and harmful waste of the drug preparation. Moreover, patients who require daily administration of the drug could undergo the infusion at home without need to go to hospital. The stability of the aliquots would help decrease hospitalization costs

Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer

<p>Abstract</p> <p>Background</p> <p>Immunohistochemistry (IHC) with mutation-specific antibodies may be an ancillary method of detecting EGFR mutations in lung cancer patients.</p> <p>Methods</p> <p>EGFR mutation status was analyzed by DNA assays, and compared with IHC results in five non-small-cell lung cancer (NSCLC) cell lines and tumor samples from 78 stage IV NSCLC patients.</p> <p>Results</p> <p>IHC correctly identified del 19 in the H1650 and PC9 cell lines, L858R in H1975, and wild-type EGFR in H460 and A549, as well as wild-type EGFR in tumor samples from 22 patients. IHC with the mAb against EGFR with del 19 was highly positive for the protein in all 17 patients with a 15-bp (ELREA) deletion in exon 19, whereas in patients with other deletions, IHC was weakly positive in 3 cases and negative in 9 cases. IHC with the mAb against the L858R mutation showed high positivity for the protein in 25/27 (93%) patients with exon 21 EGFR mutations (all with L858R) but did not identify the L861Q mutation in the remaining two patients.</p> <p>Conclusions</p> <p>IHC with mutation-specific mAbs against EGFR is a promising method for detecting EGFR mutations in NSCLC patients. However these mAbs should be validated with additional studies to clarify their possible role in routine clinical practice for screening EGFR mutations in NSCLC patients.</p