Carlos Gilberto Carlotti

Molecular Profiling Reveals Biologically Discrete Subsets and Pathways of Progression in Diffuse Glioma

Therapy development for adult diffuse glioma is hindered by incomplete knowledge of somatic glioma driving alterations and suboptimal disease classification. We defined the complete set of genes associated with 1,122 diffuse grade II-III-IV gliomas from The Cancer Genome Atlas and used molecular profiles to improve disease classification, identify molecular correlations, and provide insights into the progression from low- to high-grade disease. Whole-genome sequencing data analysis determined that ATRX but not TERT promoter mutations are associated with increased telomere length. Recent advances in glioma classification based on IDH mutation and 1p/19q co-deletion status were recapitulated through analysis of DNA methylation profiles, which identified clinically relevant molecular subsets. A subtype of IDH mutant glioma was associated with DNA demethylation and poor outcome; a group of IDH-wild-type diffuse glioma showed molecular similarity to pilocytic astrocytoma and relatively favorable survival. Understanding of cohesive disease groups may aid improved clinical outcomes.

Multiple recurrent genetic events converge on control of histone lysine methylation in medulloblastoma

We used high-resolution SNP genotyping to identify regions of genomic gain and loss in the genomes of 212 medulloblastomas, malignant pediatric brain tumors. We found focal amplifications of 15 known oncogenes and focal deletions of 20 known tumor suppressor genes (TSG), most not previously implicated in medulloblastoma. Notably, we identified previously unknown amplifications and homozygous deletions, including recurrent, mutually exclusive, highly focal genetic events in genes targeting histone lysine methylation, particularly that of histone 3, lysine 9 (H3K9). Post-translational modification of histone proteins is critical for regulation of gene expression, can participate in determination of stem cell fates and has been implicated in carcinogenesis. Consistent with our genetic data, restoration of expression of genes controlling H3K9 methylation greatly diminishes proliferation of medulloblastoma in vitro. Copy number aberrations of genes with critical roles in writing, reading, removing and blocking the state of histone lysine methylation, particularly at H3K9, suggest that defective control of the histone code contributes to the pathogenesis of medulloblastoma.

PIK3CA gene mutations in pediatric and adult glioblastoma multiforme

The phosphatidylinositol 3-kinases (PI3K) are a family of enzymes that relay important cellular growth control signals. Recently, a large-scale mutational analysis of eight PI3K and eight PI3K-like genes revealed somatic mutations in PIK3CA, which encodes the p110α catalytic subunit of class IA PI3K, in several types of cancer, including glioblastoma multiforme. In that report, 4 of 15 (27%) glioblastomas contained potentially oncogenic PIK3CA mutations. Subsequent studies, however, showed a significantly lower mutation rate ranging from 0% to 7%. Given this disparity and to address the relation of patient age to mutation frequency, we examined 10 exons of PIK3CA in 73 glioblastoma samples by PCR amplification followed by direct DNA sequencing. Overall, PIK3CA mutations were found in 11 (15%) samples, including several novel mutations. PIK3CA mutations were distributed in all sample types, with 18%, 9%, and 13% of primary tumors, xenografts, and cell lines containing mutations, respectively. Of the primary tumors, PIK3CA mutations were identified in 21% and 17% of pediatric and adult samples, respectively. No evidence of PIK3CA gene amplification was detected by quantitative real-time PCR in any of the samples. This study confirms that PIK3CA mutations occur in a significant number of human glioblastomas, further indicating that therapeutic targeting of this pathway in glioblastomas is of value. Moreover, this is the first study showing PIK3CA mutations in pediatric glioblastomas, thus providing a molecular target in this important pediatric malignancy. (Mol Cancer Res 2006;4(10):709–14)

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

BackgroundConsidering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue.ResultsHere we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold.ConclusionAltogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression.

Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas.

We have performed cDNA microarray analyses to identify gene expression differences between highly invasive glioblastoma multiforme (GBM) and typically benign pilocytic astrocytomas (PA). Despite the significant clinical and pathological differences between the 2 tumor types, only 63 genes were found to exhibit 2‐fold or greater overexpression in GBM as compared to PA. Forty percent of these genes are related to the regulation of the cell cycle and mitosis. QT‐PCR validation of 6 overexpressed genes: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101 confirmed at least a 5‐fold increase in the average expression levels in GBM. Maternal embryonic leucine zipper kinase (MELK) exhibited the most statistically significant difference. A more detailed investigation of MELK expression was undertaken to study its oncogenic relevance. In the examination of more than 100 tumors of the central nervous system, we found progressively higher expression of MELK with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. Similar level of overexpression was also observed in medulloblastoma. We found neither gene promoter hypomethylation nor amplification to be a factor in MELK expression, but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage‐independent growth in in vitro assays. Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK correlated with malignancy grade in astrocytomas and represents a therapeutic target for the management of the most frequent brain tumors in adult and children.

miR-29b and miR-125a regulate podoplanin and suppress invasion in glioblastoma.

Glioblastoma is the most frequent and malignant brain tumor, characterized by an elevated capacity for cellular proliferation and invasion. Recently, it was demonstrated that podoplanin membrane sialo‐glycoprotein encoded by PDPN gene is over‐expressed and related to cellular invasion in astrocytic tumors; however the mechanisms of regulation are still unknown. MicroRNAs are noncoding RNAs that regulate gene expression and several biological processes and diseases, including cancer. Nevertheless, their roles in invasion, proliferation, and apoptosis of glioblastoma are not completely understood. In this study, we focused on miR‐29b and miR‐125a, which were predicted to regulate PDPN, and demonstrated that these microRNAs directly target the 3′ untranslated region of PDPN and inhibit invasion, apoptosis, and proliferation of glioblastomas. Furthermore, we report that miR‐29b and miR‐125a are downregulated in glioblastomas and also in CD133‐positive cells. Taken together, these results suggest that miR‐29b and miR‐125a represent potential therapeutic targets in glioblastoma.

Intertumoral Heterogeneity within Medulloblastoma Subgroups

While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.

Plasticity‚ synaptic strength‚ and epilepsy: what can we learn from ultrastructural data?

Summary:  Central nervous system synapses have an intrinsic plastic capacity to adapt to new conditions with rapid changes in their structure. Such activity‐dependent refinement occurs during development and learning, and shares features with diseases such as epilepsy. Quantitative ultrastructural studies based on serial sectioning and reconstructions have shown various structural changes associated with synaptic strength involving both dendritic spines and postsynaptic densities (PSDs) during long‐term potentiation (LTP). In this review, we focus on experimental studies that have analyzed at the ultrastructural level the consequences of LTP in rodents, and plastic changes in the hippocampus of experimental models of epilepsy and human tissue obtained during surgeries for intractable temporal lobe epilepsy (TLE). Modifications in spine morphology, increases in the proportion of synapses with perforated PSDs, and formation of multiple spine boutons arising from the same dendrite are the possible sequence of events that accompany hippocampal LTP. Structural remodeling of mossy fiber synapses and formation of aberrant synaptic contacts in the dentate gyrus are common features in experimental models of epilepsy and in human TLE. Combined electrophysiological and ultrastructural studies in kindled rats and chronic epileptic animals have indicated the occurrence of seizure‐ and neuron loss‐induced changes in the hippocampal network. In these experiments, the synaptic contacts on granule cells are similar to those described for LTP. Such changes could be associated with enhancement of synaptic efficiency and may be important in epileptogenesis.

Modulation of HJURP (Holliday Junction-Recognizing Protein) Levels Is Correlated with Glioblastoma Cells Survival

Background Diffuse astrocytomas are the most common type of primary brain cancer in adults. They present a wide variation in differentiation and aggressiveness, being classified into three grades: low-grade diffuse astrocytoma (grade II), anaplastic astrocytoma (grade III) and glioblastoma multiforme (grade IV), the most frequent and the major lethal type. Recent studies have highlighted the molecular heterogeneity of astrocytomas and demonstrated that large-scale analysis of gene expression could help in their classification and treatment. In this context, we previously demonstrated that HJURP, a novel protein involved in the repair of DNA double-strand breaks, is highly overexpressed in glioblastoma. Methodology/Principal Findings Here we show that HJURP is remarkably overexpressed in a cohort composed of 40 patients with different grade astrocytomas. We also observed that tumors presenting the higher expression levels of HJURP are associated with poor survival prognosis, indicating HJURP overexpression as an independent prognostic factor of death risk for astrocytoma patients. More importantly, we found that HJURP knockdown strongly affects the maintenance of glioblastoma cells in a selective manner. Glioblastoma cells showed remarkable cell cycle arrest and premature senescence that culminated in elevated levels of cell death, differently from non-tumoral cells that were minimally affected. Conclusions These data suggest that HJURP has an important role in the maintenance of extremely proliferative cells of high-grade gliomas and point to HJURP as a potential therapeutic target for the development of novel treatments for glioma patients.

Seizure outcome after surgery for epilepsy due to focal cortical dysplastic lesions

Neocortical development is a highly complex process encompassing cellular proliferation, neuronal migration and cortical organization. At any time this process can be interrupted or modified by genetic or acquired factors causing malformations of cortical development (MCD). Epileptic seizures are the most common type of clinical manifestation, besides developmental delay and focal neurological deficits. Seizures due to MCD are frequently pharmacoresistant, especially those associated to focal cortical dysplasia (FCD). Surgical therapy results have been reported since 1971, however, currently available data from surgical series are still limited, mainly due to small number of patients, distinct selection of candidates and surgical strategies, variable pathological diagnosis and inadequate follow-up. This study addresses the possibilities of seizure relief following resection of focal cortical dysplasia, and the impact of presurgical evaluation, extent of resection and pathological findings on surgical outcome. We included 41 patients, 22 adults and 19 children and adolescents, with medically intractable seizures operated on from 1996 to 2002. All were submitted to standardized presurgical evaluation including high-resolution MRI, Video-EEG monitoring and ictal SPECT. Post-surgical seizure outcome was classified according to Engels schema. Univariate and multivariate analysis were performed. Fifteen patients had temporal and 26 extratemporal epilepsies. Of the total 26 patients (63.4%) reached seizure-free status post-operatively. There was no correlation between outcome and age at surgery, duration of epilepsy, frequency of seizures, and pathological findings. There was, however, a clear correlation with topography of FCD (temporal versus extratemporal) and regional ictal EEG onset, on univariate as well as multivariate analysis.