Tathiane Maistro Malta

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.

TCGAbiolinks: an R/Bioconductor package for integrative analysis of TCGA data

The Cancer Genome Atlas (TCGA) research network has made public a large collection of clinical and molecular phenotypes of more than 10 000 tumor patients across 33 different tumor types. Using this cohort, TCGA has published over 20 marker papers detailing the genomic and epigenomic alterations associated with these tumor types. Although many important discoveries have been made by TCGAs research network, opportunities still exist to implement novel methods, thereby elucidating new biological pathways and diagnostic markers. However, mining the TCGA data presents several bioinformatics challenges, such as data retrieval and integration with clinical data and other molecular data types (e.g. RNA and DNA methylation). We developed an R/Bioconductor package called TCGAbiolinks to address these challenges and offer bioinformatics solutions by using a guided workflow to allow users to query, download and perform integrative analyses of TCGA data. We combined methods from computer science and statistics into the pipeline and incorporated methodologies developed in previous TCGA marker studies and in our own group. Using four different TCGA tumor types (Kidney, Brain, Breast and Colon) as examples, we provide case studies to illustrate examples of reproducibility, integrative analysis and utilization of different Bioconductor packages to advance and accelerate novel discoveries.

Forced expression of OCT4 influences the expression of pluripotent genes in human mesenchymal stem cells and fibroblasts.

Genetic reprogramming of adult cells to generate induced pluripotent stem (iPS) cells is a new and important step in sidestepping some of the ethical issues and risks involved in the use of embryonic stem cells. iPS cells can be generated by introduction of transcription factors, such as OCT4, SOX2, KLF4, and CMYC. iPS cells resemble embryonic stem cells in their properties and differentiation potential. The mechanisms that lead to induced pluripotency and the effect of each transcription factor are not completely understood. We performed a critical evaluation of the effect of overexpressing OCT4 in mesenchymal stem cells and fibroblasts and found that OCT4 can activate the expression of other stemness genes, such as SOX2, NANOG, CMYC, FOXD3, KLF4, and βCATENIN, which are not normally or are very weakly expressed in mesenchymal stem cells. Transient expression of OCT4 was also performed to evaluate whether these genes are affected by its overexpression in the first 48 h. Transfected fibroblast cells expressed around 275-fold more OCT4 than non-transfected cells. In transient expression, in which cells were analyzed after 48 h, we detected only the up-regulation of FOXD3, SOX2, and KLF4 genes, suggesting that these genes are the earlier targets of OCT4 in this cellular type. We conclude that forced expression of OCT4 can alter cell status and activate the pluripotent network. Knowledge gained through study of these systems may help us to understand the kinetics and mechanism of cell reprogramming.

The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: Transcriptomic evidence that pericytes are stem cells in human adipose tissue

Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology.

Glioma CpG island methylator phenotype (G-CIMP): biological and clinical implications

Gliomas are a heterogeneous group of brain tumors with distinct biological and clinical properties. Despite advances in surgical techniques and clinical regimens, treatment of high-grade glioma remains challenging and carries dismal rates of therapeutic success and overall survival. Challenges include the molecular complexity of gliomas, as well as inconsistencies in histopathological grading, resulting in an inaccurate prediction of disease progression and failure in the use of standard therapy. The updated 2016 World Health Organization (WHO) classification of tumors of the central nervous system reflects a refinement of tumor diagnostics by integrating the genotypic and phenotypic features, thereby narrowing the defined subgroups. The new classification recommends molecular diagnosis of isocitrate dehydrogenase (IDH) mutational status in gliomas. IDH-mutant gliomas manifest the cytosine-phosphate-guanine (CpG) island methylator phenotype (G-CIMP). Notably, the recent identification of clinically relevant subsets of G-CIMP tumors (G-CIMP-high and G-CIMP-low) provides a further refinement in glioma classification that is independent of grade and histology. This scheme may be useful for predicting patient outcome and may be translated into effective therapeutic strategies tailored to each patient. In this review, we highlight the evolution of our understanding of the G-CIMP subsets and how recent advances in characterizing the genome and epigenome of gliomas may influence future basic and translational research.

Transcriptomic comparisons between cultured human adipose tissue-derived pericytes and mesenchymal stromal cells

Mesenchymal stromal cells (MSCs), sometimes called mesenchymal stem cells, are cultured cells able to give rise to mature mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes, and to secrete a wide range of trophic and immunomodulatory molecules. Evidence indicates that pericytes, cells that surround and maintain physical connections with endothelial cells in blood vessels, can give rise to MSCs (da Silva Meirelles et al., 2008 [1]; Caplan and Correa, 2011 [2]). We have compared the transcriptomes of highly purified, human adipose tissue pericytes subjected to culture-expansion in pericyte medium or MSC medium, with that of human adipose tissue MSCs isolated with traditional methods to test the hypothesis that their transcriptomes are similar (da Silva Meirelles et al., 2015 [3]). Here, we provide further information and analyses of microarray data from three pericyte populations cultured in pericyte medium, three pericyte populations cultured in MSC medium, and three adipose tissue MSC populations deposited in the Gene Expression Omnibus under accession number GSE67747.

T cell receptor signaling pathway is overexpressed in CD4 + T cells from HAM/TSP individuals

Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus related to the chronic neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD4(+) T cells activation appears to play a key role on HTLV-1 infection. Here we investigated the expression of genes associated to T cell activation CD3e molecule, epsilon (CD3ɛ), lymphocyte-specific protein tyrosine kinase (LCK), vav 1 guanine nucleotide exchange factor (VAV1), and zeta-chain (TCR) associated protein kinase 70kDa (ZAP70) on T lymphocytes of HTLV-1-infected individuals and compared to healthy uninfected individuals (CT). We observed that CD3ɛ, LCK, ZAP70, and VAV1 gene expression were increased in CD4(+) T cells from HAM/TSP group compared to HTLV-1 asymptomatic patients (HAC). Moreover, ZAP70 and VAV1 were also upregulated in HAM/TSP compared to CT group. We detected a positive correlation among all these genes. We also observed that CD3ɛ, LCK, and VAV1 genes had a positive correlation with the proviral load (PVL) and Tax expression. These results suggest that PVL and Tax protein could drive CD3ɛ, LCK, and VAV1 gene expression in CD4(+) T cells, and these genes function on a synchronized way on the CD4(+) T cell activation. The elucidation of the mechanisms underlying T cell receptor signaling pathway is of considerable interest and might lead to new insights into the mechanism of HAM/TSP.

Leukotrienes are upregulated and associated with human T-lymphotropic virus type 1 (HTLV-1)-associated neuroinflammatory disease.

Leukotrienes (LTs) are lipid mediators involved in several inflammatory disorders. We investigated the LT pathway in human T-lymphotropic virus type 1 (HTLV-1) infection by evaluating LT levels in HTLV-1-infected patients classified according to the clinical status as asymptomatic carriers (HACs) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Bioactive LTB4 and CysLTs were both increased in the plasma and in the supernatant of peripheral blood mononuclear cell cultures of HTLV-1-infected when compared to non-infected. Interestingly, CysLT concentrations were increased in HAM/TSP patients. Also, the concentration of plasma LTB4 and LTC4 positively correlated with the HTLV-1 proviral load in HTLV-1-infected individuals. The gene expression levels of LT receptors were differentially modulated in CD4+ and CD8+ T cells of HTLV-1-infected patients. Analysis of the overall plasma signature of immune mediators demonstrated that LT and chemokine amounts were elevated during HTLV-1 infection. Importantly, in addition to CysLTs, IP-10 was also identified as a biomarker for HAM/TSP activity. These data suggest that LTs are likely to be associated with HTLV-1 infection and HAM/TSP development, suggesting their putative use for clinical monitoring.

Genes related to antiviral activity are differentially expressed in CD4+ T cell in HAM/TSP patients

CD4+ T cells play a central role in HTLV-1 infection. We investigated the global gene expression profile of circulating CD4+ T cells in HTLV-1-infected individuals. The microarray platform was performed using 12 individual RNA samples: healthy control (CT, n=4), asymptomatic HTLV-1 carrier (HAC, n=4) and HAM/TSP group (n=4). Proviral load (PVL), Tax expression and the percentage of CD4+Foxp3+ cells were analyzed. Hierarchical clustering analysis showed that CT and HTLV-infected groups clustered separately. We identified 25 differentially expressed genes in common between CT vs. HAM/TSP and HAM/TSP vs. HAC analyses and we observed their participation in granzyme A (GZMA) signaling pathways. GZMA, GZMB and PRF1 were validated by qRT-PCR. GZMA and PRF1 gene expression was significantly increased in HAM/TSP group compared to CT and HAC groups. No difference was observed in gene expression level of GZMB. Regulatory T cells (Treg) cells have cytolytic capacity and perforin/granzyme pathways are required for this activity. Foxp3 gene expression was evaluated and it was significantly increased in HAM/TSP group compared to CT and HAC groups. GZMA, GZMB, and PRF1 genes were positively correlated to Foxp3 gene expression. PRF1 and Foxp3 genes were positively correlated with Tax expression and PVL. The percentage of CD4+Foxp3+ cells revealed a significant increase in HAC and HAM/TSP groups compared to CT. Our results suggest that Treg cells may use the perforin/granzyme pathway as a system to suppress the immune cells and may contribute to immunodeficiency, which is observed in HTLV-1 infection.

Abstract 780: Multi-omic profiling of gliomas reveals distinct DNA methylation changes at tumor recurrence

Varying possibilities of tumor relapse for lower grade glioma (LGG, WHO grade II and III) and glioblastoma (GBM, WHO grade IV) have led to heterogenous clinical outcomes for patients. Our current study aims to establish a molecular profile of recurrence of matched primary and recurrent LGG (n = 28) and recurrent GBM (n = 24) tumor samples. The Cancer Genome Atlas (TCGA) has comprehensively profiled these matched primary/recurrent tumor sets; whole genomes, coding exomes, methylomes, and transcriptomes have been sequenced, and the samples have undergone targeted profiling of the proteome. While unsupervised analysis techniques have not led to a clear recurrence signature, supervised analysis methods have revealed interesting patterns. Protein profiling has shown that recurrent gliomas retain the overall molecular signature of their primary counterpart, but the DNA damage response, apoptosis and RTK pathways are downregulated in the recurrent gliomas, in contrast to RAS/MAPK, PI3K/AKT, and EMT pathways, which are upregulated. Whole genome sequencing and rearrangement analysis have revealed increased genomic complexity among most recurrent gliomas as well as new fusions of interest in recurrent LGG samples (PTPRZ1-MET and involving ATRX). Using genome-wide Illumina HumanMethylation 450K data we observed that 78.6% of LGGs showed depletion of DNA methylation at recurrence and 50% of GBM tumors showed an enrichment of DNA methylation at recurrence. Patient centric enrichment analysis allowed us to discover a candidate biological subgroup characterized by a subset of LGG recurrences (50%) exhibiting an aberrant CpG methylation loss at inintergenic opensea regions when compared with canonical CpG islands and shores (fold > 1.3 and confidence intervals of 99%). Importantly, inspection of CpG sites significantly hypomethylated at openseas showed that this pronounced epigenetic signature maps to candidate TSS distal and hypomethylated enhancers. The gene-targets of these hypomethylated CpG sites show a corresponding up-regulation of expression. Pathway analysis has demonstrated that these upregulated genes are involved in cellular growth and proliferation, cellular function and maintenance, and cell cycle regulation. Our results provide evidence that DNA methylation may represent a stable signature of glioma recurrence and that the crosstalk between DNA hypomethylation at openseas and chromosomal instability may be involved in glioma recurrence. We plan to further integrate our findings between data types and correlate with treatment and patient clinical outcome. Citation Format: Lindsay C. Stetson, Camila Ferreira de Souza, Tathiane Maistro Malta, Thais Sarraf Sabedot, Quinn Ostrom, Peter Liao, Daniela Pretti da Cunha Tirapelli, Luciano Neder, Carlos Gilberto Carlotti, Rehan Akbani, Sofie Salama, Laila Poisson, Daniel Brat, The Cancer Genome Atlas Network, Houtan Noushmehr, Jill Barnholtz-Sloan. Multi-omic profiling of gliomas reveals distinct DNA methylation changes at tumor recurrence. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 780.