Pedro Miralles Martínez

Identification of novel candidate target genes in amplicons of Glioblastoma multiforme tumors detected by expression and CGH microarray profiling

BackgroundConventional cytogenetic and comparative genomic hybridization (CGH) studies in brain malignancies have shown that glioblastoma multiforme (GBM) is characterized by complex structural and numerical alterations. However, the limited resolution of these techniques has precluded the precise identification of detailed specific gene copy number alterations.ResultsWe performed a genome-wide survey of gene copy number changes in 20 primary GBMs by CGH on cDNA microarrays. A novel amplicon at 4p15, and previously uncharacterized amplicons at 13q32-34 and 1q32 were detected and are analyzed here. These amplicons contained amplified genes not previously reported. Other amplified regions containg well-known oncogenes in GBMs were also detected at 7p12 (EGFR), 7q21 (CDK6), 4q12 (PDGFRA), and 12q13-15 (MDM2 and CDK4). In order to identify the putative target genes of the amplifications, and to determine the changes in gene expression levels associated with copy number change events, we carried out parallel gene expression profiling analyses using the same cDNA microarrays. We detected overexpression of the novel amplified genes SLA/LP and STIM2 (4p15), and TNFSF13B and COL4A2 (13q32-34). Some of the candidate target genes of amplification (EGFR, CDK6, MDM2, CDK4, and TNFSF13B) were tested in an independent set of 111 primary GBMs by using FISH and immunohistological assays. The novel candidate 13q-amplification target TNFSF13B was amplified in 8% of the tumors, and showed protein expression in 20% of the GBMs.ConclusionThis high-resolution analysis allowed us to propose novel candidate target genes such as STIM2 at 4p15, and TNFSF13B or COL4A2 at 13q32-34 that could potentially contribute to the pathogenesis of these tumors and which would require futher investigations. We showed that overexpression of the amplified genes could be attributable to gene dosage and speculate that deregulation of those genes could be important in the development and progression of GBM. Our findings highlight the important influence in GBM of signaling pathways such as the PI3K/AKT, consistent with the invasive features of this tumor.

p16INK4a Gene Alterations Are Frequent in Lesions of Mycosis Fungoides

Mycosis fungoides is usually an indolent disease that, after a variable period of time in a stable phase, evolves into a tumoral form with aggressive behavior. The molecular events that mark this progression remain essentially unknown to date, and this prompted us to investigate the hypothetical role of p16(INK4a) silencing in mycosis fungoides progression. We analyzed the three most frequent mechanisms of inactivation of the p16(INK4a) gene (deletion, promoter hypermethylation, and mutation) in nine cases with patch/plaque and tumoral lesions of mycosis fungoides. The existence of alterations was investigated by microsatellite analysis, methylation-specific polymerase chain reaction, and direct sequencing. Alterations of the p16(INK4a) gene were found in four of nine of the plaque lesions (hypermethylation in three samples and allelic loss in one sample) and seven of nine in the tumor lesions (hypermethylation in five samples and allelic loss in three samples). No case presented point mutations. Although a higher incidence of p16(INK4a) hypermethylation was observed in the cases that failed to achieve a complete remission, the limited size of our series prompted us to evaluate this finding cautiously. The results of this study therefore showed a common genetic alteration that is found more frequently in tumoral lesions than it is in plaque lesions of mycosis fungoides. It also offers data that could suggest a relationship between p16(INK4a) hypermethylation and unfavorable clinical outcome. Broader studies are needed to confirm both relationships.

Identification of survival‐related genes of the phosphatidylinositol 3′‐kinase signaling pathway in glioblastoma multiforme

Knowledge of the molecular mechanisms involved in the biology of glioblastoma multiforme (GBM) is essential for the identification of candidate prognostic markers, new putative therapeutic targets, and early detection strategies predictive of survival.

p16 INK4a Is Selectively Silenced in the Tumoral Progression of Mycosis Fungoides

Knowledge about the molecular mechanisms involved in the pathogenesis of tumoral progression in mycosis fungoides (MF) is still scarce. Because the 9p21 locus seems to be a good target for a detailed study in MF, this prompted us to compare the mechanisms of inactivation of the p16INK4a, p15INK4b, and p14ARF genes in aggressive and stable forms of MF, performing microsatellite analysis, methylation-specific polymerase chain reaction, direct sequencing, and p16INK4a protein expression by immunohistochemistry. Additionally, the p53 gene was also sequenced in tumoral lesions. Thirty-nine patients with stable MF were studied. Alterations in p16INK4a and p15INK4b genes were detected in 18% and 5% of the cases, respectively. None of the cases analyzed showed alterations of the p14ARF gene. In contrast with these findings, in the 11 patients with aggressive MF, alterations of the p16INK4a, p15INK4b, or p14ARF genes were found in 8 (73%), 3 (27%), and 2 (18%) cases, respectively. A significant proportion (4/11) of these alterations were already present in the p16INK4a gene in the initial plaque lesions in these aggressive forms of MF. Alterations in the p16INK4a gene, either methylation or loss of heterozygosity, were clearly more frequent than those in the p15INK4b and p14ARF genes. These p16INK4A alterations were confirmed using immunohistochemistry. None of the nine tumoral lesions analyzed showed mutations in exons 1-2 of the p16INK4a gene or in exons 5-8 of the p53 gene. These results seem to suggest that 9p21 alterations, and selectively p16INK4a silencing, could be a characteristic phenomenon in MF progression.

Analysis of the frequency of microsatellite instability and p53 gene mutation in splenic marginal zone and MALT lymphomas.

AIMS: Studies of the genetic characteristics of splenic marginal zone lymphoma (SMZL) have failed to identify genetic changes specific to this tumour. Microsatellite instability is a type of genomic instability associated with different types of human cancer. Although microsatellite instability is rare in B cell non-Hodgkins lymphomas, it has been found in some specific subsets, such as marginal zone lymphomas arising in mucosa associated lymphoid tissue (MALT), where an association with p53 mutation has been described. Because it has been proposed that SMZL and MALT are close in histogenetic terms, this study investigated the comparative frequency of microsatellite instability and p53 mutation in patients with SMZL and MALT lymphomas. METHODS: Microsatellite instability was investigated using seven microsatellite marker loci in 14 patients with SMZL and 20 patients with MALT lymphomas. In an attempt to clarify the role of p53 gene mutation in the pathogenesis of SMZL, exons 5-8 were also investigated by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and direct sequencing in a total of 20 patients with SMZL and 22 patients with MALT lymphomas. RESULTS: Microsatellite instability was not detected in patients with SMZL, although five of 20 patients with MALT lymphomas had microsatellite instability. The frequency of p53 mutation was low in both series (two of 20 patients with SMZL and one of 22 patients with MALT lymphomas). No significant association was found between p53 mutation and microsatellite instability. CONCLUSIONS: These results indicate that microsatellite instability is not associated with the molecular pathogenesis of SMZL, confirming the relatively increased frequency of microsatellite instability in MALT lymphomas, and perhaps suggesting that MALT and SMZL have different mechanisms of tumorigenesis.

BCR gene disruption in a pilomyxoid astrocytoma.

We report here a 4‐month‐old child with a large, solid enhancing mass involving predominantly the suprasellar and diencephalic regions, with extension of both hemispheres. The patient underwent partial resection of the mass by right temporal craniotomy. Histological diagnosis was of a low‐grade glioma consistent with pilomyxoid astrocytoma. Cytogenetic analyses revealed an insertion on chromosome 17 that involved disruption of the BCR gene. This finding suggests a possible rearrangement of this gene that could act in a similar way to chronic myeloid leukemia with formation of a chimeric tyrosine kinase protein. This study may suggest the use of inhibitors of tyrosine kinase proteins as an alternative treatment approach in cases of refractory or disseminated pilocytic astrocytomas.

Microarray-Based Comparative Genomic Hybridization (Array-CGH) as a Useful Tool for Identifying Genes Involved in Glioblastoma (GB)

Alterations in the genome that lead to changes in DNA sequence copy number are a characteristic of Glioblastomas (GBs). Microarray-based comparative genomic hybridization (array-CGH) is a high-throughput technology that allows the hybridization of genomic DNA onto conventional cDNA microarrays, normally used in expression profiling, to analyze genomic copy number imbalances. In this way, thousands of genes can be reviewed in a high resolution analysis to define amplicons and to identify? candidate genes showing recurrent genomic copy number changes in GB tumors.

Social sciences teaching and information processing. An experience using WebQuests in primary education teacher training

The main aim of this study is to show the utility of information and communication technologies (ICTs) in primary education teacher training. A series of experiences using these resources was produced and then undertaken by students to facilitate their learning of the curricular components of social sciences teaching. This practical exercise was implemented on the Didactic Methodology subject for social sciences teaching, offered on the Primary Education bachelor’s degree course. The experience shows how the students produced a WebQuest and developed the learning process for both the subject’s curricular content and competencies.ResumenEl objetivo principal de este trabajo es mostrar la utilidad de las tecnologías de la información y de la comunicación en la formación del profesorado de educación primaria. Con el uso de estos recursos, se han elaborado una serie de experiencias, llevadas a cabo con los estudiantes, para facilitar el aprendizaje de los elementos curriculares necesarios en la enseñanza de las ciencias sociales. Esta práctica se ha desarrollado en la asignatura Metodología didáctica para la enseñanza de las ciencias sociales, ofertada en el grado de Educación Primaria. La experiencia muestra cómo los alumnos elaboraron una webquest y el proceso de aprendizaje tanto de los contenidos curriculares de la asignatura como de las competencias desarrolladas.The main aim of this study is to show the utility of information and communication technologies (ICTs) in primary education teacher training. A series of experiences using these resources was produced and then undertaken by students to facilitate their learning of the curricular components of social sciences teaching. This practical exercise was implemented on the Didactic Methodology subject for social sciences teaching, offered on the Primary Education bachelor’s degree course. The experience shows how the students produced a WebQuest and developed the learning process for both the subject’s curricular content and competencies. El objetivo principal de este trabajo es mostrar la utilidad de las tecnologías de la información y de la comunicación en la formación del profesorado de educación primaria. Con el uso de estos recursos, se han elaborado una serie de experiencias, llevadas a cabo con los estudiantes, para facilitar el aprendizaje de los elementos curriculares necesarios en la enseñanza de las ciencias sociales. Esta práctica se ha desarrollado en la asignatura Metodología didáctica para la enseñanza de las ciencias sociales, ofertada en el grado de Educación Primaria. La experiencia muestra cómo los alumnos elaboraron una webquest y el proceso de aprendizaje tanto de los contenidos curriculares de la asignatura como de las competencias desarrolladas.

Intelligent and adaptive middleware to improve user-perceived QoS in multimedia applications

We investigate the use of adaptive multimedia applications to improve the user-perceived QoS in next-generation wireless networks. These networks will be characterized by the heterogeneity of the access technologies including ad hoc wireless network extensions. In these network scenarios in which the links are error-prone, their capacity is extremely variable and even the topology can change due to handovers and mobility of the nodes, it is unrealistic to think of traditional network-layer QoS mechanisms alone, as a means to offer strict end-to-end QoS guarantees. As a complement to the network-layer QoS protocols, this paper presents an intelligent adaptation middleware allowing multimedia applications to dynamically adapt their internal settings. This intelligence enables the applications to minimize the impact of the adverse and changing network conditions in the QoS level perceived by the user. We use a machine learning technique to model the users perception of QoS from a large set of QoS scores given by real users. We present some results demonstrating that our proposal clearly outperforms traditional multimedia internetworking.

Extending seamless IP multicast edge-coverage through mobile ad hoc access networks

The provision of multicast communications in wireless and wired networks has followed different paths which have led to different solutions. Little has been accomplished to-date in bringing together the traditional IP multicast model used in fixed networks and multicast routing protocols for wireless ad hoc networks. We analyse the provision of an integrated IP multicast service in which mobile hosts can seamlessly participate in IP multicast sessions regardless of the currently underlying network type. We propose a multicast architecture in combination with a new ad hoc multicast routing protocol called MMARP. MMARP nodes are challenged with special IGMP-handling capabilities allowing our solution to combine the efficiency of multicast ad hoc routing protocols with the support of standard-IP nodes without an impairment in the performance of the protocol. Our empirical results demonstrate that such kind of multicast ad hoc access networks offer a good performance when compared with the traditional single-hop wireless multicast access.