Publications by authors named "Bárbara Meléndez"

51 Publications

APR-246 combined with 3-deazaneplanocin A, panobinostat or temozolomide reduces clonogenicity and induces apoptosis in glioblastoma cells.

Int J Oncol 2021 Mar 26;58(3):312-330. Epub 2021 Jan 26.

Department of Biochemistry and Genetics, University of Navarra School of Sciences, 31008 Pamplona, Spain.

Glioblastoma is the most malignant brain tumor and presents high resistance to chemotherapy and radiotherapy. Surgery, radiotherapy and chemotherapy with temozolomide are the only treatments against this tumor. New targeted therapies, including epigenetic modulators such as 3‑deazaneplanocin A (DZ‑Nep; an EZH2 inhibitor) and panobinostat (a histone deacetylase inhibitor) are being tested in vitro, together with temozolomide. The present study combined APR‑246 with DZ‑Nep, panobinostat and teomozolomide in order to explore the possibility of restoring p53 function in mutated cases of glioblastoma. Following the Chou‑Talalay method it was demonstrated that APR‑246 acts in an additive manner together with the other compounds, reducing clonogenicity and inducing apoptosis in glioblastoma cells independently of p53 status.
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http://dx.doi.org/10.3892/ijo.2021.5177DOI Listing
March 2021

Craniopharyngiomas: A clinicopathological and molecular study of 52 cases - Experience in the Complejo Hospitalario de Toledo and Hospital Universitario 12 de Octubre (Madrid).

Clin Neuropathol 2021 Jan-Feb;40(1):26-35

Craniopharyngiomas (CPs) are histologically benign tumors that are associated with high levels of morbidity. Two clinicopathological variants - adamantinomatous (ACP) and papillary (PCP) - have been described. They differ in their molecular features, whereby activating mutations in (V600E) and genes characterize PCP and ACP, respectively. Recently, both variants have been shown to express elevated PD-L1 protein expression, but ACP also exhibited tumor cell-intrinsic PD-1 expression. In this study we analyze these molecular alterations in 52 cases with a long follow-up and examine their associations with immunohistochemical and clinical characteristics. ACPs comprise 73.1% of cases, while 21.2% are PCPs. Aberrant nuclear immunoreactivity for β-catenin was observed in all ACPs. p.V600E mutations were observed in 90.9% of PCPs. Only one ACP case featured both alterations. Both types of CP exhibited strong nuclear staining for p63 with diffuse and basal distribution. ACP and PCP consistently expressed PD-L1, most in a substantial percentage of tumor cells, with a distinctive spatial distribution of expression in each subtype; only ACP demonstrated PD-1 expression. There was no evidence of differences in clinical prognosis between ACPs and PCPs. The identification of hallmark molecular signatures in the two CP variants is useful for sub-categorization in routine histopathology reporting. It is also pertinent to personalized therapy and for the development of improved non-invasive therapeutic strategies in this disease.
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http://dx.doi.org/10.5414/NP301268DOI Listing
December 2020

Clinical, radiological and molecular characterization of intramedullary astrocytomas.

Acta Neuropathol Commun 2020 08 8;8(1):128. Epub 2020 Aug 8.

Department of Pathology, Erasme University Hospital, Université Libre de Bruxelles (ULB), 808 Route de Lennik, B-1070, Brussels, Belgium.

Intramedullary astrocytomas (IMAs) are rare tumors, and few studies specific to the molecular alterations of IMAs have been performed. Recently, KIAA1549-BRAF fusions and the H3F3A p.K27M mutation have been described in low-grade (LG) and high-grade (HG) IMAs, respectively. In the present study, we collected clinico-radiological data and performed targeted next-generation sequencing for 61 IMAs (26 grade I pilocytic, 17 grade II diffuse, 3 LG, 3 grade III and 12 grade IV) to identify KIAA1549-BRAF fusions and mutations in 33 genes commonly implicated in gliomas and the 1p/19q regions. One hundred seventeen brain astrocytomas were analyzed for comparison. While we did not observe a difference in clinico-radiological features between LG and HG IMAs, we observed significantly different overall survival (OS) and event-free survival (EFS). Multivariate analysis showed that the tumor grade was associated with better OS while EFS was strongly impacted by tumor grade and surgery, with higher rates of disease progression in cases in which only biopsy could be performed. For LG IMAs, EFS was only impacted by surgery and not by grade. The most common mutations found in IMAs involved TP53, H3F3A p.K27M and ATRX. As in the brain, grade I pilocytic IMAs frequently harbored KIAA1549-BRAF fusions but with different fusion types. Non-canonical IDH mutations were observed in only 2 grade II diffuse IMAs. No EGFR or TERT promoter alterations were found in IDH wild-type grade II diffuse IMAs. These latter tumors seem to have a good prognosis, and only 2 cases underwent anaplastic evolution. All of the HG IMAs presented at least one molecular alteration, with the most frequent one being the H3F3A p.K27M mutation. The H3F3A p.K27M mutation showed significant associations with OS and EFS after multivariate analysis. This study emphasizes that IMAs have distinct clinico-radiological, natural evolution and molecular landscapes from brain astrocytomas.
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http://dx.doi.org/10.1186/s40478-020-00962-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414698PMC
August 2020

TP53, ATRX alterations, and low tumor mutation load feature IDH-wildtype giant cell glioblastoma despite exceptional ultra-mutated tumors.

Neurooncol Adv 2020 Jan-Dec;2(1):vdz059. Epub 2020 Jan 24.

Department of Pathology (Neuropathology) and Instituto de Investigación i+12, Hospital Universitario 12 de Octubre, Madrid, Spain.

Background: Giant cell glioblastoma (gcGBM) is a rare morphological variant of IDH-wildtype (IDHwt) GBM that occurs in young adults and have a slightly better prognosis than "classic" IDHwt GBM.

Methods: We studied 36 GBMs, 14 with a histopathological diagnosis of gcGBM and 22 with a giant cell component. We analyzed the genetic profile of the most frequently mutated genes in gliomas and assessed the tumor mutation load (TML) by gene-targeted next-generation sequencing. We validated our findings using The Cancer Genome Atlas (TCGA) data.

Results: p53 was altered by gene mutation or protein overexpression in all cases, while driver , , , or mutations were infrequent or absent. Compared to IDHwt GBMs, gcGBMs had a significant higher frequency of , , , and mutations, while lower frequency of amplification, deletion, and promoter mutation. Almost all tumors had low TML values. The high TML observed in only 2 tumors was consistent with and mutations. In the histopathological review of TCGA IDHwt, -mutant tumors identified giant cells in 37% of the cases. Considering our series and that of the TCGA, patients with -mutant gcGBMs had better overall survival than those with GBMs (log-rank test,  < .002).

Conclusions: gcGBMs have molecular features that contrast to "classic" IDHwt GBMs: unusually frequent mutations and few amplifications and deletions, especially in tumors with a high number of giant cells. TML is frequently low, although exceptional high TML suggests a potential for immune checkpoint therapy in some cases, which may be relevant for personalized medicine.
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http://dx.doi.org/10.1093/noajnl/vdz059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212869PMC
January 2020

The synergistic effect of DZ‑NEP, panobinostat and temozolomide reduces clonogenicity and induces apoptosis in glioblastoma cells.

Int J Oncol 2020 Jan 30;56(1):283-300. Epub 2019 Oct 30.

Department of Biochemistry and Genetics, University of Navarra School of Sciences, 31008 Pamplona, Spain.

Current treatment against glioblastoma consists of surgical resection followed by temozolomide, with or without combined radiotherapy. Glioblastoma frequently acquires resistance to chemotherapy and/or radiotherapy. Novel therapeutic approaches are thus required. The inhibition of enhancer of zeste homolog 2 (EZH2; a histone methylase) and histone deacetylases (HDACs) are possible epigenetic treatments. Temozolomide, 3‑deazaneplanocin A (DZ‑Nep; an EZH2 inhibitor) and panobinostat (an HDAC inhibitor) were tested in regular and temozolomide‑resistant glioblastoma cells to confirm whether the compounds could behave in a synergistic, additive or antagonistic manner. A total of six commercial cell lines, two temozolomide‑induced resistant cell lines and two primary cultures derived from glioblastoma samples were used. Cell lines were exposed to single treatments of the drugs in addition to all possible two‑ and three‑drug combinations. Colony formation assays, synergistic assays and reverse transcription‑quantitative PCR analysis of apoptosis‑associated genes were performed. The highest synergistic combination was DZ‑Nep + panobinostat. Triple treatment was also synergistic. Reduced clonogenicity and increased apoptosis were both induced. It was concluded that the therapeutic potential of the combination of these three drugs in glioblastoma was evident and should be further explored.
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http://dx.doi.org/10.3892/ijo.2019.4905DOI Listing
January 2020

Blood tumor mutational burden: are we ready for clinical implementation?

J Thorac Dis 2019 Sep;11(Suppl 15):S1906-S1908

Department of Pathology, Erasme University Hospital, Free University of Brussels (ULB), Brussels, Belgium.

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http://dx.doi.org/10.21037/jtd.2019.07.60DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783780PMC
September 2019

A 47-year-old man with a recurrent glioma.

Brain Pathol 2019 07;29(4):581-582

Laboratory of Pathology, Erasme University Hospital, Free University of Brussels (ULB), Brussels, Belgium.

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http://dx.doi.org/10.1111/bpa.12753DOI Listing
July 2019

Epigenetic downregulation of TET3 reduces genome-wide 5hmC levels and promotes glioblastoma tumorigenesis.

Int J Cancer 2020 01 1;146(2):373-387. Epub 2019 Jul 1.

Nanomaterials and Nanotechnology Research Center (CINN-CSIC), Universidad de Oviedo, Oviedo, Spain.

Loss of 5-hydroxymethylcytosine (5hmC) has been associated with mutations of the ten-eleven translocation (TET) enzymes in several types of cancer. However, tumors with wild-type TET genes can also display low 5hmC levels, suggesting that other mechanisms involved in gene regulation might be implicated in the decline of this epigenetic mark. Here we show that DNA hypermethylation and loss of DNA hydroxymethylation, as well as a marked reduction of activating histone marks in the TET3 gene, impair TET3 expression and lead to a genome-wide reduction in 5hmC levels in glioma samples and cancer cell lines. Epigenetic drugs increased expression of TET3 in glioblastoma cells and ectopic overexpression of TET3 impaired in vitro cell growth and markedly reduced tumor formation in immunodeficient mice models. TET3 overexpression partially restored the genome-wide patterns of 5hmC characteristic of control brain samples in glioblastoma cell lines, while elevated TET3 mRNA levels were correlated with better prognosis in glioma samples. Our results suggest that epigenetic repression of TET3 might promote glioblastoma tumorigenesis through the genome-wide alteration of 5hmC.
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http://dx.doi.org/10.1002/ijc.32520DOI Listing
January 2020

Design and Validation of a Gene-Targeted, Next-Generation Sequencing Panel for Routine Diagnosis in Gliomas.

Cancers (Basel) 2019 Jun 4;11(6). Epub 2019 Jun 4.

Department of Pathology, Erasme Hospital, Université Libre de Bruxelles, 1070 Brussels, Belgium.

The updated 2016 World Health Organization (WHO) classification system for gliomas integrates molecular alterations and histology to provide a greater diagnostic and prognostic utility than the previous, histology-based classification. The increasing number of markers that are tested in a correct diagnostic procedure makes gene-targeted, next-generation sequencing (NGS) a powerful tool in routine pathology practice. We designed a 14-gene NGS panel specifically aimed at the diagnosis of glioma, which allows simultaneous detection of mutations and copy number variations, including the 1p/19q-codeletion and Epidermal Growth Factor Receptor () amplification. To validate this panel, we used reference mutated DNAs, nontumor and non-glioma samples, and 52 glioma samples that were previously characterized. The panel was then prospectively applied to 91 brain lesions. A specificity of 100% and sensitivity of 99.4% was achieved for mutation detection. Orthogonal methods, such as in situ hybridization and immunohistochemical techniques, were used for validation, which showed high concordance. The molecular alterations that were identified allowed diagnosis according to the updated WHO criteria, and helped in the differential diagnosis of difficult cases. This NGS panel is an accurate and sensitive method, which could replace multiple tests for the same sample. Moreover, it is a rapid and cost-effective approach that can be easily implemented in the routine diagnosis of gliomas.
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http://dx.doi.org/10.3390/cancers11060773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627812PMC
June 2019

Tubastatin A, an inhibitor of HDAC6, enhances temozolomide‑induced apoptosis and reverses the malignant phenotype of glioblastoma cells.

Int J Oncol 2019 May 1;54(5):1797-1808. Epub 2019 Mar 1.

Department of Biochemistry and Genetics, University of Navarra School of Sciences, 31008 Pamplona, Spain.

Glioblastoma or grade IV astrocytoma is the most common and lethal form of glioma. Current glioblastoma treatment strategies use surgery followed by chemotherapy with temozolomide. Despite this, numerous glioblastoma cases develop resistance to temozolomide treatments, resulting in a poor prognosis for the patients. Novel approaches are being investigated, including the inhibition of histone deacetylase 6 (HDAC6), an enzyme that deacetylates α‑tubulin, and whose overexpression in glioblastoma is associated with the loss of primary cilia. The aim of the present study was to treat glioblastoma cells with a selective HDAC6 inhibitor, tubastatin A, to determine if the malignant phenotype may be reverted. The results demonstrated a notable increase in acetylated α‑tubulin levels in treated cells, which associated with downregulation of the sonic hedgehog pathway, and may hypothetically promote ciliogenesis in those cells. Treatment with tubastatin A also reduced glioblastoma clonogenicity and migration capacities, and accelerated temozolomide‑induced apoptosis. Finally, HDAC6 inhibition decreased the expression of mesenchymal markers, contributing to reverse epithelial‑mesenchymal transition in glioblastoma cells.
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http://dx.doi.org/10.3892/ijo.2019.4739DOI Listing
May 2019

Methods of measurement for tumor mutational burden in tumor tissue.

Transl Lung Cancer Res 2018 Dec;7(6):661-667

Department of Pathology, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium.

Immunotherapies based on immune checkpoint inhibitors are emerging as an innovative treatment for different types of advanced cancers. While the utility of immune checkpoint inhibitors has been clearly demonstrated, the response rate is highly variable across individuals. Due to the cost and toxicity of these immunotherapies, a critical challenge in this field is the identification of predictive biomarkers to discriminate which patients may respond to immunotherapy. Recently, a high tumor mutational burden (TMB) has been identified as a genetic signature that is associated with a favorable outcome for immune checkpoint inhibitor therapy. The TMB is defined as the total number of nonsynonymous mutations per coding area of a tumor genome. Initially, it was determined using whole exome sequencing, but due to the high costs and long turnaround time of this method, targeted panel sequencing is currently being explored to measure TMB. In the near future, TMB evaluation may play an important role in immuno-oncology, but its implementation in a routine setting involves robust analytical and clinical validation. Standardization is also needed in order to make informed decisions about patients. This review presents the methodologies employed for determining TMB and discusses the factors that may have an impact on its measurement.
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http://dx.doi.org/10.21037/tlcr.2018.08.02DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249625PMC
December 2018

Molecular Study of Long-Term Survivors of Glioblastoma by Gene-Targeted Next-Generation Sequencing.

J Neuropathol Exp Neurol 2018 08;77(8):710-716

Department of Pathology, Virgen de la Salud Hospital, Toledo, Spain.

Glioblastoma (GBM) is the most common malignant adult primary brain tumor. Despite its high lethality, a small proportion of patients have a relatively long overall survival (OS). Here we report a study of a series of 74 GBM samples from 29 long-term survivors ([LTS] OS ≥36 months) and 45 non-LTS. Using next-generation sequencing, we analyzed genetic alterations in the genes most frequently altered in gliomas. Approximately 20% of LTS had a mutation in the IDH1 or IDH2 (IDH) genes, denoting the relevance of this molecular prognostic factor. A new molecular group of GBMs harbored alterations in ATRX or DAXX genes in the absence of driver IDH or H3F3A mutations. These patients tended to have a slightly better prognosis, to be younger at diagnosis, and to present frontal or temporal tumors, and, morphologically, to present giant tumor cells. A significant fraction of LTS GBM patients had tumors with 1 or more alterations in the relevant GBM signaling pathways (RTK/PI3K, TP53 and RB1). In these patients, the PDGFRA alteration is suggested to be a favorable molecular factor. Our findings here are relevant for developing future targeted therapies and for identifying molecular prognostic factors in GBM patients.
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http://dx.doi.org/10.1093/jnen/nly048DOI Listing
August 2018

Tumor Surface Regularity at MR Imaging Predicts Survival and Response to Surgery in Patients with Glioblastoma.

Radiology 2018 07;288(1):218-225

From the Mathematical Oncology Laboratory, Instituto de Matemática Aplicada a la Ciencia y la Ingeniería, Universidad de Castilla-La Mancha, Avenida de Camilo Jose Cela 3, 13071 Ciudad Real, Spain (J.P., D.M., J.A.O., A.F., B.L., V.M.P.); Departments of Radiology and Neurosurgery, Hospital General de Ciudad Real, Ciudad Real, Spain (E. Arregui, M.C., J.M.B.); Departments of Pathology, Radiology, and Neurosurgery, Hospital Virgen de la Salud, Toledo, Spain (B.M., Á.R.d.L., R.M.d.l.P.); Department of Neurosurgery, Hospital Clínico San Carlos, Madrid, Spain (L.I.B., J.A.B.); Department of Neurosurgery, Hospital Marqués de Valdecilla, Santander, Spain (J.M., C.V.); Department of Radiology, Complejo Hospitalario Universitario de Granada, Granada, Spain (B.A.); Department of Radiology, Hospital Carlos Haya, Málaga, Spain (M.B., I.H.); Department of Radiology, Hospital de Manises, Valencia, Spain (A.R.); and Department of Radiology, Instituto Valenciano de Oncología, Valencia, Spain (E. Arana).

Purpose To evaluate the prognostic and predictive value of surface-derived imaging biomarkers obtained from contrast material-enhanced volumetric T1-weighted pretreatment magnetic resonance (MR) imaging sequences in patients with glioblastoma multiforme. Materials and Methods A discovery cohort from five local institutions (165 patients; mean age, 62 years ± 12 [standard deviation]; 43% women and 57% men) and an independent validation cohort (51 patients; mean age, 60 years ± 12; 39% women and 61% men) from The Cancer Imaging Archive with volumetric T1-weighted pretreatment contrast-enhanced MR imaging sequences were included in the study. Clinical variables such as age, treatment, and survival were collected. After tumor segmentation and image processing, tumor surface regularity, measuring how much the tumor surface deviates from a sphere of the same volume, was obtained. Kaplan-Meier, Cox proportional hazards, correlations, and concordance indexes were used to compare variables and patient subgroups. Results Surface regularity was a powerful predictor of survival in the discovery (P = .005, hazard ratio [HR] = 1.61) and validation groups (P = .05, HR = 1.84). Multivariate analysis selected age and surface regularity as significant variables in a combined prognostic model (P < .001, HR = 3.05). The model achieved concordance indexes of 0.76 and 0.74 for the discovery and validation cohorts, respectively. Tumor surface regularity was a predictor of survival for patients who underwent complete resection (P = .01, HR = 1.90). Tumors with irregular surfaces did not benefit from total over subtotal resections (P = .57, HR = 1.17), but those with regular surfaces did (P = .004, HR = 2.07). Conclusion The surface regularity obtained from high-resolution contrast-enhanced pretreatment volumetric T1-weighted MR images is a predictor of survival in patients with glioblastoma. It may help in classifying patients for surgery.
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http://dx.doi.org/10.1148/radiol.2018171051DOI Listing
July 2018

Loss of 5hmC identifies a new type of aberrant DNA hypermethylation in glioma.

Hum Mol Genet 2018 09;27(17):3046-3059

Nanomaterials and Nanotechnology Research Center (CINN-CSIC), Universidad de Oviedo, Principado de Asturias, Oviedo, Spain.

Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma.
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http://dx.doi.org/10.1093/hmg/ddy214DOI Listing
September 2018

Clinical application of targeted next-generation sequencing for colorectal cancer patients: a multicentric Belgian experience.

Oncotarget 2018 Apr 17;9(29):20761-20768. Epub 2018 Apr 17.

Department of Pathology, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium.

International guidelines made RAS (KRAS and NRAS) status a prerequisite for the use of anti-EGFR agents for metastatic colorectal cancer (CRC) patients. Daily, new data emerges on the theranostic and prognostic role of molecular biomarkers; this is a strong incentive for a validated, sensitive, and broadly available molecular screening test. Next-generation sequencing (NGS) has begun to supplant other technologies for genomic profiling. We report here our 2 years of clinical practice using NGS results to guide therapeutic decisions. The Ion Torrent AmpliSeq colon/lung cancer panel, which allows mutation detection in 22 cancer-related genes, was prospectively used in clinical practice (BELAC ISO 15189 accredited method). The DNA of 741 formalin-fixed paraffin-embedded CRC tissues, including primary tumors and metastasis, was obtained from 14 different Belgian institutions and subjected to targeted NGS. Of the tumors tested, 98% (727) were successfully sequenced and 89% (650) harbored at least one mutation. KRAS, BRAF and NRAS mutations were found in 335 (46%), 78 (11%) and 32 (4%) samples, respectively. These mutation frequencies were consistent with those reported in public databases. Moreover, mutations and amplifications in potentially actionable genes were identified in 464 samples (64%), including mutations in PIK3CA (14%), ERBB2 (0.4%), AKT1 (0.6%), and MAP2K1 (0.1%), as well as amplifications of ERBB2 (0.3%) and EGFR (0.3%). The median turnaround time between reception of the sample in the laboratory and report release was 8 calendar days. Overall, the AmpliSeq colon/lung cancer panel was successfully applied in daily practice and provided reliable clinically relevant information for CRC patients.
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http://dx.doi.org/10.18632/oncotarget.25099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945518PMC
April 2018

MGMT promoter methylation in Peruvian patients with glioblastoma.

Ecancermedicalscience 2018 14;12:812. Epub 2018 Feb 14.

Department of Neurosurgery, Instituto Nacional de Enfermedades Neoplasicas, Lima 15038, Peru.

Purpose: O-methylguanine-DNA methyltransferase () promoter methylation predicts the outcome and response to alkylating chemotherapy in glioblastoma. The aim of this study is to evaluate the prevalence of methylation in Peruvian glioblastoma cases.

Patients And Methods: We evaluated retrospectively 50 cases of resected glioblastoma during the period 2008-2013 at Instituto Nacional de Enfermedades Neoplasicas in Peru. Samples consisted of paraffin embedded and frozen tumour tissue. -promoter methylation status and the expression level of gene were evaluated by methylation-specific PCR and real-time PCR, respectively.

Results: Unmethylated, methylated and partially methylated statuses were found in 54%, 20% and 26% of paraffin-embedded samples, respectively. Methylation status was confirmed in the Virgen de la Salud Hospital and frozen samples. There was an association between the status of -promoter methylation and the level of gene expression ( = 0.001). Methylation was associated with increased progression-free survival ( = 0.002) and overall survival (OS) ( < 0.001).

Conclusion: -promoter methylation frequency in Peruvian glioblastoma is similar to that reported in other populations and the detection test has been standardised.
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http://dx.doi.org/10.3332/ecancer.2018.812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834313PMC
February 2018

Laminin-adherent versus suspension-non-adherent cell culture conditions for the isolation of cancer stem cells in the DAOY medulloblastoma cell line.

Tumour Biol 2016 Sep 15;37(9):12359-12370. Epub 2016 Jun 15.

Department of Biochemistry and Genetics, University of Navarra School of Sciences, Irunlarrea 1, Pamplona, 31008, Spain.

Medulloblastoma (MB) is a highly malignant tumor of childhood. MB seems to be initiated and maintained by a small group of cells, known as cancer stem cells (CSCs). The CSC hypothesis suggests that a subset of tumor cells is able to proliferate, sustain the tumor, and develop chemoresistance, all of which make of CSC an interesting target for new anticancer therapies. The MB cell line DAOY was cultured in suspension by a medullosphere traditional culturing method and in adherent conditions by laminin-pre-coated flasks and serum-free medium enriched with specific growth factors. An increase in the stem features was shown when cells were successively cultured in hypoxia conditions. By contrast, a reduction in these properties was appreciated when cells were exposed to differentiation conditions. In addition, the CD133+ and CD133- subpopulations were isolated from cells grown in laminin-pre-coated flasks, and in vitro experiments showed that the CD133+ fraction represented the stem population and it could have CSC with a higher probability than the CD133- fraction. We can conclude that the laminin culture method in adherent conditions and the medullosphere traditional culturing method in suspension are similarly good for obtaining stem-like cells in the DAOY cell line.
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http://dx.doi.org/10.1007/s13277-016-5119-6DOI Listing
September 2016

P144, a Transforming Growth Factor beta inhibitor peptide, generates antitumoral effects and modifies SMAD7 and SKI levels in human glioblastoma cell lines.

Cancer Lett 2016 10 26;381(1):67-75. Epub 2016 Jul 26.

Department of Biochemistry and Genetics, University of Navarra, Pamplona, Spain. Electronic address:

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor, accounting for 60-70% of all gliomas. Current median patient survival time is 14-16 months after diagnosis. Numerous efforts in therapy have not significantly altered the nearly uniform lethality of this malignancy. The Transforming Growth Factor beta (TGF-β) signaling pathway plays a key role in GBM and is implicated in proliferation, invasion and therapy resistance. Several inhibitors of the TGF-β pathway have entered clinical trials or are under development. In this work, the therapeutic potential of P144, a TGF-β inhibitor peptide, was analyzed. P144 decreased proliferation, migration, invasiveness, and tumorigenicity in vitro, whereas apoptosis and anoikis were significantly increased for GBM cell lines. SMAD2 phosphorylation was reduced, together with a downregulation of SKI and an upregulation of SMAD7 at both transcriptional and translational levels. Additionally, P144 was able to impair tumor growth and increase survival in an in vivo flank model. Our findings suggest a potential effect of P144 in vitro and in vivo that is mediated by regulation of transcriptional target genes of the TGF-β pathway, suggesting a therapeutic potential of P144 for GBM treatment.
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http://dx.doi.org/10.1016/j.canlet.2016.07.029DOI Listing
October 2016

Secretory meningioma with KLF4 K409Q mutation in collision with glioma.

Clin Neuropathol 2015 Nov-Dec;34(6):322-9

Aims: The simultaneous occurrence of two primary intracranial tumors is a rare event, especially if unrelated to radiotherapy or genetic disorders. We present two patients, both with two primary intracranial tumors simultaneously present at adjacent sites, in order to explore a possible mechanism of synchronous tumor formation.

Methods: We performed a molecular analysis of the K409Q mutation of the KLF4 gene, in addition to conventional immunohistochemistry.

Results: Preoperative gadolinium-enhanced magnetic resonance imaging revealed a necrotic mass with an irregular ring-like enhancement adjacent to a frontal meningioma in patient 1, and an infiltrative non-enhancing glial tumor with no evidence of another tumor in patient 2. Postoperative histological examination revealed the presence of two distinct tumors in both cases: secretory meningioma and glioblastoma in patient 1 and secretory meningioma and anaplastic astrocytoma in patient 2. Secretory meningiomas both showed the KLF4 K409Q mutation, while none of the glial tumors had it.

Conclusions: To our knowledge, these are the first two cases reported of the simultaneous occurrence of secretory meningiomas with mutation of KLF4 in collision with a glioblastoma and an anaplastic astrocytoma, respectively. These collision tumors presumably have different molecular origins.
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http://dx.doi.org/10.5414/NP300860DOI Listing
September 2016

Whole exome sequencing in a case of sporadic multiple meningioma reveals shared NF2, FAM109B, and TPRXL mutations, together with unique SMARCB1 alterations in a subset of tumor nodules.

Cancer Genet 2015 Jun 11;208(6):327-32. Epub 2015 Apr 11.

Research Unit, La Paz University Hospital, IdiPAZ, Madrid, Spain.

Meningiomas are common intracranial tumors derived from arachnoid cells. Multiple meningiomas are occasionally present even in patients with no history of neurofibromatosis type 2, a condition that can cause the formation of this neoplasm. Previous studies have shown that most multiple meningiomas are monoclonal in origin. In this study, exome sequencing was performed on four meningiomas and the corresponding peripheral blood DNA from a 61-year-old woman with sporadic multiple meningioma. At least three common mutational events (at the NF2, FAM109B, and TPRXL genes) were detected in the tumors' DNA when they were compared with the lymphocyte DNA from the patient as control. Additionally, an array of unique mutations was detected in each tumor, including in SMARCB1 in two of the samples, a gene whose alteration leads to the development of meningioma. Mutations in other genes, such as IRS4, GULP1, NHSL1, and C10orf53, accounted for one alteration in each meningioma nodule. Our data suggest a monoclonal origin of the meningiomas in this patient, although the numerous alterations contained in each sample indicated multiple secondary variable changes in each tumor nodule. Whether the alterations described in this work are drivers of tumorigenesis or are simply passengers requires further study.
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http://dx.doi.org/10.1016/j.cancergen.2015.03.012DOI Listing
June 2015

Impact on prognosis of the regional distribution of MGMT methylation with respect to the CpG island methylator phenotype and age in glioma patients.

J Neurooncol 2015 May 15;122(3):441-50. Epub 2015 Feb 15.

Molecular Pathology Research Unit, Department of Pathology, Virgen de la Salud Hospital, Avda Barber 30, 45004, Toledo, Spain.

Clinical and molecular prognostic factors in gliomas include age, IDH mutation, the glioma CpG island methylator phenotype (G-CIMP+) and promoter methylation of the O(6)-methylguanine DNA-methyltransferase (MGMT) gene. Among these markers, a predictive value was reported in glioblastomas (GBM) for MGMT promoter methylation, in particular in elderly GBM patients. In this study, methylation data from 46 glioma samples with the Illumina 450K platform were obtained and extended using external data to include a total of 247 glioma samples. Methylation analysis of the whole MGMT gene with this platform revealed two strongly survival-associated CpG regions within the promoter and the gene body, which were confirmed in a reported dataset of high grade-gliomas. Methylation at the promoter (CpG 25, cg12981137 and the prognostic model MGMT-STP27) and at the gene body CpG 165 (cg07933035), were significantly associated with better overall survival, and strongly correlated with G-CIMP+ status. In this series, the prognostic value of MGMT methylation at the promoter was not observed in G-CIMP- cases, although around 50 % of them were MGMT-methylated. These results were also obtained in an homogeneously-treated series of chemoradiated G-CIMP- GBMs analyzed by MSP and qMSP, and confirmed in a reported pyrosequencing-analyzed series of gliomas. Interestingly, in contrast to the MGMT promoter, gene body methylation was of prognostic value in G-CIMP-patients older than 65 years. Our study highlights the relevance of the prognostic value of the different regions of methylation throughout the MGMT gene that could be affected by specific G-CIMP profiles and age groups.
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http://dx.doi.org/10.1007/s11060-015-1738-9DOI Listing
May 2015

Molecular classification defines 4 prognostically distinct glioma groups irrespective of diagnosis and grade.

J Neuropathol Exp Neurol 2015 Mar;74(3):241-9

From the Molecular Pathology Research Unit (PM, MM, THI, BM) and Departments of Pathology (MM) and Neurosurgery (ARDL), Virgen de la Salud Hospital, Toledo; Department of Biochemistry and Genetics, University of Navarra School of Sciences, Pamplona (JSC); Department of Pathology, MD Anderson International, Madrid (JFG); Department of Pathology, University Hospital Complex of Vigo, Vigo (CF); Department of Pathology, University Clinic Hospital, Barcelona (TR); and IdiPaz Research Unit, La Paz University Hospital, Madrid (JAR), Spain.

According to World Health Organization criteria, diffuse gliomas are divided into several histological subtypes, including astrocytomas, oligodendrogliomas, and oligoastrocytomas, and 4 malignancy grades (I-IV). Molecular alterations, such as the isocitrate dehydrogenase gene (IDH) mutation or 1p/19q loss, are found in these tumors but are not included in the current classification system. Recently, mutation of α thalassemia/mental retardation syndrome X-linked (ATRX) gene and its loss of expression have been reported in infiltrating gliomas. We evaluated ATRX protein expression in 272 gliomas and its association with molecular and clinical features. Loss of ATRX expression was more common in tumors with an astrocytic component (astrocytomas II/III, 46.4%; oligoastrocytomas, 47.5%) but was uncommon in oligodendrogliomas (7.3%) and glioblastomas (0.9%). In astrocytic tumors, loss of ATRX expression was significantly associated with longer overall survival. Remarkably, on the basis of IDH mutation, 1p/19q codeletion, and ATRX expression, our study defined 4 molecularly and prognostically different groups of gliomas, showing the relevance of ATRX expression as a new marker for refining the molecular classification of gliomas and for distinguishing clinically distinct prognostic subgroups of patients.
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http://dx.doi.org/10.1097/NEN.0000000000000167DOI Listing
March 2015

Genome-wide methylation analysis in vestibular schwannomas shows putative mechanisms of gene expression modulation and global hypomethylation at the HOX gene cluster.

Genes Chromosomes Cancer 2015 Apr 23;54(4):197-209. Epub 2014 Dec 23.

Molecular Neuro-oncogenetics Laboratory, Research Unit, Hospital Universitario La Paz, IdiPAZ, Madrid, Spain.

Schwannomas are tumors that develop from Schwann cells in the peripheral nerves and commonly arise from the vestibular nerve. Vestibular schwannomas can present unilaterally and sporadically or bilaterally when the tumor is associated with neurofibromatosis Type 2 (NF2) syndrome. The molecular hallmark of the disease is biallelic inactivation of the NF2 gene. The epigenetic signature of schwannomas remains poorly understood and is mostly limited to DNA methylation of the NF2 gene, whose altered expression due to epigenetic factors in this tumor is controversial. In this study, we tested the genomewide DNA methylation pattern of schwannomas to shed light on this epigenetic alteration in these particular tumors. The methodology used includes Infinium Human Methylation 450K BeadChip microarrays in a series of 36 vestibular schwannomas, 4 nonvestibular schwannomas, and 5 healthy nerves. Our results show a trend toward hypomethylation in schwannomas. Furthermore, homeobox (HOX) genes, located at four clusters in the genome, displayed hypomethylation in several CpG sites in the vestibular schwannomas but not in the nonvestibular schwannomas. Several microRNA (miRNA) and protein-coding genes were also found to be hypomethylated at promoter regions and were confirmed as upregulated by expression analysis; including miRNA-21, Met Proto-Oncogene (MET), and PMEPA1. We also detected methylation patterns that might be involved in alternative transcripts of several genes such as NRXN1 or MBP, which would increase the complexity of the methylation and expression patterns. Overall, our results show specific epigenetic signatures in several coding genes and miRNAs that could potentially be used as therapeutic targets.
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http://dx.doi.org/10.1002/gcc.22232DOI Listing
April 2015

Epigenetic regulation of human hedgehog interacting protein in glioma cell lines and primary tumor samples.

Tumour Biol 2015 Apr 22;36(4):2383-91. Epub 2014 Nov 22.

Brain Tumor Biology Unit, University of Navarra School of Sciences, Pamplona, Spain.

Glioma constitutes one of the most common groups of brain tumors, and its prognosis is influenced by different genetic and epigenetic modulations. In this study, we demonstrated low or no expression of hedgehog interacting protein (HHIP) in most of the cell lines and primary glioma tumor samples. We further proceeded to promoter methylation study of this gene in the same cell lines and primary tumor samples and found 87 % (7/8) HHIP methylation in glioblastoma cell lines and 75 % (33/44) in primary tumor samples. These methylation pattern correlates with low or unexpressed HHIP in both cell lines and primary tumor samples. Our results suggest the possibility of epigenetic regulation of this gene in glioma, similarly to medulloblastoma, gastric, hepatic, and pancreatic cancers. Also, HHIP might be a diagnostic or prognostic marker in glioma and help to the detection of these tumors in early stages of disease.
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http://dx.doi.org/10.1007/s13277-014-2846-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012430PMC
April 2015

Genome-wide microarray expression and genomic alterations by array-CGH analysis in neuroblastoma stem-like cells.

PLoS One 2014 13;9(11):e113105. Epub 2014 Nov 13.

Department of Biochemistry and Genetics, University of Navarra School of Sciences, Pamplona, Spain.

Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0113105PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231109PMC
December 2015

Global expression profile in low grade meningiomas and schwannomas shows upregulation of PDGFD, CDH1 and SLIT2 compared to their healthy tissue.

Oncol Rep 2014 Dec 3;32(6):2327-34. Epub 2014 Oct 3.

Molecular Neuro-Oncogenetics Laboratory, Research Unit, Hospital Universitario La Paz, IdiPAZ, Madrid, Spain.

Schwannomas and grade I meningiomas are non‑metastatic neoplasms that share the common mutation of gene NF2. They usually appear in neurofibromatosis type 2 patients. Currently, there is no drug treatment available for both tumors, thus the use of wide expression technologies is crucial to identify therapeutic targets. Affymetrix Human Gene 1.0 ST was used to test global gene expression in 22 meningiomas, 31 schwannomas and, as non-tumoral controls, 3 healthy meningeal tissues, 8 non-tumoral nerves and 1 primary Schwann cell culture. A non-stringent P-value cut-off and fold change were used to establish deregulated genes. We identified a subset of genes that were upregulated in meningiomas and schwannomas when compared to their respectively healthy tissues, including PDGFD, CDH1 and SLIT2. Thus, these genes should be thoroughly studied as targets in a possible combined treatment.
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http://dx.doi.org/10.3892/or.2014.3526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240498PMC
December 2014

Mycotic popliteal aneurysm rupture secondary to Campylobacter fetus.

Ann Vasc Surg 2015 Jan 12;29(1):122.e9-11. Epub 2014 Jun 12.

Department of Vascular Therapy, Colorado Permanente Medical Group, Denver, CO.

Background: Mycotic aneurysms of the popliteal artery are uncommon. Popliteal aneurysms rarely rupture. The authors present the second reported case of popliteal artery rupture as a result of Campylobacter fetus infection. This report confirms the arterial destructive potential of C. fetus infection in a peripheral artery.

Methods: An 85-year-old male who had previously undergone endovascular abdominal aortic aneurysm repair in 2007 presented with positive blood cultures for C. fetus. No endocarditis was detected. No periprosthetic fluid to suggest aortic endograft infection was present. During hospitalization for sepsis, he developed acute right knee pain and swelling. A 5.2-cm popliteal aneurysm, with contained rupture, was found on ultrasound and confirmed by computed tomography and angiography. Recommendations for treatment and a literature review are provided.

Results: This patient was successfully managed with total excision of the aneurysm via a posterior approach with reconstruction through a medial approach using autologous saphenous vein bypass. Culture-directed antibiotic therapy (6 weeks of intravenous ertapenem) to eradicate the pathogen completed the therapy. The patient is doing well at 18- month follow-up.

Conclusions: Mycotic popliteal aneurysm associated with C. fetus is a rare but potentially fatal condition. Isolating C. fetus should alert the surgeon to the peripheral arterial destructive potential of this pathogen, as manifested by acute rupture in this patient. Traditional resection through a posterior approach and revascularization through noncontaminated tissue with culture-directed therapy are the treatments of choice.
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http://dx.doi.org/10.1016/j.avsg.2014.05.021DOI Listing
January 2015

Homozygous deletion of TNFRSF4, TP73, PPAP2B and DPYD at 1p and PDCD5 at 19q identified by multiplex ligation-dependent probe amplification (MLPA) analysis in pediatric anaplastic glioma with questionable oligodendroglial component.

Mol Cytogenet 2014 Jan 6;7(1). Epub 2014 Jan 6.

Molecular Neuro-oncogenetics Laboratory, Research Unit-Unidad de Investigación, Hospital Universitario La Paz, Paseo de la Castellana 261, 28046 Madrid, Spain.

Background: Pediatric oligodendrogliomas are rare and appear to show a different molecular profile from adult tumors. Some gliomas display allelic losses at 1p/19q in pediatric patients, although less frequently than in adult patients, but this is rare in tumors with an oligodendroglial component. The molecular basis of this genomic abnormality is unknown in pediatric gliomas, but it represents a relatively common finding in pediatric oligodendroglioma-like neoplasms with leptomeningeal dissemination.

Results: Multiplex ligation-dependent probe amplification (MLPA) analysis using SALSA P088-B1 for the analysis of the 1p/19q allelic constitution in a pediatric anaplastic (oligodendro)-glioma showed homozygous co-deletion for markers: TNFRSF4 (located at 1p36.33), TP73 (1p36.32), PPAP2B (1pter-p22.1), DPYD (1p21.3), and PDCD5 (19q13.12), and hemizygous deletion of BAX (19q13.3-q13.4). No sequence changes for R132 and R172 of the IDH1/2 genes were identified.

Conclusions: The molecular findings in this pediatric anaplastic glioma do not allow for a clearly definitive pathological diagnosis. However, the findings provide data on a number of 1p/19q genomic regions that, because of homozygotic deletion, might be the location of genes that are important for the development and clinical evolution of some malignant gliomas in children.
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http://dx.doi.org/10.1186/1755-8166-7-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3905963PMC
January 2014

Gene expression analysis of aberrant signaling pathways in meningiomas.

Oncol Lett 2013 Jul 23;6(1):275-279. Epub 2013 May 23.

Neuro-Oncogenetics Laboratory, Research Unit, Madrid, Spain ;

Examining aberrant pathway alterations is one method for understanding the abnormal signals that are involved in tumorigenesis and tumor progression. In the present study, expression arrays were performed on tumor-related genes in meningiomas. The GE Array Q Series HS-006 was used to determine the expression levels of 96 genes that corresponded to six primary biological regulatory pathways in a series of 42 meningiomas, including 32 grade I, four recurrent grade I and six grade II tumors, in addition to three normal tissue controls. Results showed that 25 genes that were primarily associated with apoptosis and angiogenesis functions were downregulated and 13 genes frequently involving DNA damage repair functions were upregulated. In addition to the inactivation of the neurofibromin gene, , which is considered to be an early step in tumorigenesis, variations of other biological regulatory pathways may play a significant role in the development of meningioma.
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http://dx.doi.org/10.3892/ol.2013.1363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3742750PMC
July 2013

Global profiling in vestibular schwannomas shows critical deregulation of microRNAs and upregulation in those included in chromosomal region 14q32.

PLoS One 2013 11;8(6):e65868. Epub 2013 Jun 11.

Neuro-Oncology Laboratory, Research Unit, La Paz University Hospital, IdiPAZ, Madrid, Spain.

Background: Vestibular schwannomas are benign tumors that arise from Schwann cells in the VIII cranial pair and usually present NF2 gene mutations and/or loss of heterozygosity on chromosome 22q. Deregulation has also been found in several genes, such as ERBB2 and NRG1. MicroRNAs are non-coding RNAs approximately 21 to 23 nucleotides in length that regulate mRNAs, usually by degradation at the post-transcriptional level.

Methods: We used microarray technology to test the deregulation of miRNAs and other non-coding RNAs present in GeneChip miRNA 1.0 (Affymetrix) over 16 vestibular schwannomas and 3 control-nerves, validating 10 of them by qRT-PCR.

Findings: Our results showed the deregulation of 174 miRNAs, including miR-10b, miR-206, miR-183 and miR-204, and the upregulation of miR-431, miR-221, miR-21 and miR-720, among others. The results also showed an aberrant expression of other non-coding RNAs. We also found a general upregulation of the miRNA cluster located at chromosome 14q32.

Conclusion: Our results suggest that several miRNAs are involved in tumor formation and/or maintenance and that global upregulation of the 14q32 chromosomal site contains miRNAs that may represent a therapeutic target for this neoplasm.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065868PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3679163PMC
January 2014