Marta Zalacain

Clinical involvement of catechol-O-methyltransferase polymorphisms in schizophrenia spectrum disorders: influence on the severity of psychotic symptoms and on the response to neuroleptic treatment

Genetic variation in the catechol-O-methyltransferase (COMT) gene may influence the susceptibility to schizophrenia and the response to neuroleptic treatment. The authors tested for an association between a COMT haplotype and schizophrenia-spectrum disorders and for an eventual influence of a specific COMT genotype in the clinical outcome and in the response to treatment. The genotypes for single nucleotide polymorphisms rs737865, rs4633, rs6267, rs4680 (Val158Met) and rs165599 were determined in 207 patients with schizophrenia-spectrum disorders and 204 paired controls. Statistical tests for linkage disequilibrium and for case–control differences in haplotype frequencies were performed using log-linear modelling embedded within the expectation-maximization algorithm. P-values based on permutations were calculated using the software UNPHASED, and odds ratios were estimated using the SHEsis platform. The response to neuroleptic treatment was assessed by the Global Assessment of Functioning scale and the severity of psychotic symptoms by the positive and negative syndrome scale (PANSS) scale. The overall disease status was significantly associated with the T-G (Val) diplotype for rs4633–rs4680 (P=0.0049). A significant association was observed between schizophrenia, but not other related disorders, and genotypes GG (Val/Val) for rs4680 and TT for rs4633. Val/Val patients with schizophrenia showed a higher severity of the psychotic symptoms and a worse response to the neuroleptic treatment. COMT genetic variation seems to be involved in the psychotic symptomatology of the schizophrenia-spectrum disorders and specifically in the narrow schizophrenia phenotype. Our results show an influence of the Val158Met polymorphism on the severity of psychotic symptoms and on the response to treatment.

Methotrexate in Pediatric Osteosarcoma: Response and Toxicity in Relation to Genetic Polymorphisms and Dihydrofolate Reductase and Reduced Folate Carrier 1 Expression

OBJECTIVE To determine the influence of the genotype and the level of expression of different enzymes involved in folate metabolism on the response to and toxicity of high-dose methotrexate treatment in pediatric osteosarcomas. STUDY DESIGN DHFR and Reduced folate carrier 1 (RFC1) semiquantitative expression was analyzed in 34 primary and metastatic osteosarcoma tissues by real-time polymerase chain reaction. The following polymorphisms were also analyzed in peripheral blood from 96 children with osteosarcoma and 110 control subjects: C677T, A1298C (MTHFR), G80A (RFC1), A2756G (MTR), C1420T (SHMT), the 28bp-repeat polymorphism, and 1494del6 of the TYMS gene. Treatment toxicity was scored after each cycle according to criteria from the World Health Organization. RESULTS DHFR and RFC1 expression was lower in initial osteosarcoma biopsy specimens than in metastases (P = .024 and P = .041, respectively). RFC1 expression was moderately decreased in samples with poor histologic response to preoperative treatment (P = .053). Patients with osteosarcoma with G3/G4 hematologic toxicity were more frequently TT than CT/CC for C677T/MTHFR (P = .023) and GG for A2756G/MTR (P = .048 and P = .057 for gastrointestinal and hematologic toxicity, respectively). CONCLUSIONS The role of C677T/MTHFR and A2756G/MTR on chemotherapy-induced toxicity should be further investigated in pediatric osteosarcomas receiving high-dose methotrexate. Altered expression of DHFR and RFC1 is a feasible mechanism by which osteosarcoma cells become resistant to methotrexate.

El ensayo de micronúcleos como medida de inestabilidad genética inducida por agentes genotóxicos

La integridad genetica de la poblacion humana se encuentra comprometida por la gran actividad industrial; por lo que es importante determinar que se conoce como un nivel “aceptable” de dano genetico y realizar ensayos de genotoxicidad de manera rutinaria en poblaciones de riesgo. Los micronucleos son cuerpos citoplasmaticos de naturaleza nuclear, se corresponden con material genetico no incorporado correctamente a las celulas hijas durante la division celular, reflejan aberraciones cromosomicas y se originan por roturas cromosomicas, por errores durante la replicacion y posterior division celular del ADN y/o por la exposicion a agentes genotoxicas. Existen factores capaces de influir o modificar el numero de micronucleos presentes en una celula (edad, genero, vitaminas, tratamientos medicos, exposicion diaria a agentes genotoxicos, etc.). El ensayo citogenetico para la deteccion de micronucleos (CBMN: cytokinesis-block micronucleus) se basa en la utilizacion de un agente quimico, denominado citocalasina-B capaz de impedir la citocinesis permitiendo la division nuclear proporcionando a las celulas un aspecto de celulas binucleadas monodivididas. El recuento de micronucleos se realiza sobre 1.000 celulas binucleadas y la muestra de partida puede variar aunque lo optimo es el uso de linfocitos aislados de sangre periferica. El ensayo de micronucleos esta considerado como un ensayo practico, universalmente validado y accesible tecnologicamente, util para evaluar la inestabilidad genetica inducida por agentes genotoxicos.

Effects of benzopyrene-7,8-diol-9,10-epoxide (BPDE) in vitro and of maternal smoking in vivo on micronuclei frequencies in fetal cord blood

Up to 20% of pregnant women smoke and there is indirect evidence that certain tobacco-specific metabolites can cross the placental barrier and are genotoxic to the fetus. The presence of micronuclei results from chromosome damage and reflects the degree of underlying genetic instability. Fetal blood was obtained from the cord blood of 143 newborns (102 from nonsmoking mothers and 41 from mothers smoking >10 cigarettes/d during pregnancy). The micronucleus assay was performed following the guidelines established by the Human MicroNucleus project with modifications. To test the micronucleus assay, we evaluated the effect of a range of benzopyrene-7,8-diol-9,10-epoxide concentrations (from 3.125 nM to 4 μM) on cord blood from nonsmoking mothers. This validation showed that the number of micronuclei and apoptotic cells increased with benzopyrene-7,8-diol-9,10-epoxide dose (p < 0.0001 and p = 0.001, respectively); the minimal detectable effect was induced by 12.5 nM benzopyrene-7,8-diol-9,10-epoxide. In our sample, the number of MN was significantly higher in the 41 cord blood samples from mothers who smoked during pregnancy [smokers: 4 (1; 10.5); nonsmokers: 3 (0; 8); p = 0.016]. Therefore, the data reported herein support the hypothesis that tobacco compounds are able to induce chromosomal losses and breaks that are detectable as an increased number of micronuclei.

Proteomic analysis of chemonaive pediatric osteosarcomas and corresponding normal bone reveals multiple altered molecular targets.

With a view to identify the proteins involved in transformation, metastasis or chemoresistance in pediatric osteosarcoma, we carried out a new experimental approach based on comparison of the proteomic profile of paired samples of osteosarcoma and normal bone tissues from the same patient. The proteomic profiles of five pairs of cell lines (normal vs tumoral) were obtained by two-dimensional difference gel electrophoresis. We detected 56 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify some of these proteins, 16 of which were chosen on the basis of the change of their relative abundance between osteosarcomas and paired normal bones and also because their involvement was supported by the genomic analysis. Two of the 16 proteins, Alpha-crystallin B chain (CRYAB) and ezrin (EZR1), were selected for further studies: an immunohistochemical analysis of a TMA (tissue microarray) and real-time PCR for a set of 14 osteosarcoma/normal-bone pairs. The results of this second tier of studies confirmed that there were significant increases in the amounts of CRYAB and ezrin, especially in advanced stages of the disease. Our overall conclusion is that proteomic profiling of paired samples of osteosarcoma and normal bone tissues from the same patient is a practicable and potentially powerful way of initiating and proceeding with a search for proteins and genes involved in pediatric osteosarcoma.

Profiling of Chemonaive Osteosarcoma and Paired-Normal Cells Identifies EBF2 as a Mediator of Osteoprotegerin Inhibition to Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis

Purpose: Osteosarcoma is the most prevalent bone tumor in children and adolescents. At present, the mechanisms of initiation, maintenance, and metastasis are poorly understood. The purpose of this study was to identify relevant molecular targets in the pathogenesis of osteosarcoma. Experimental Design: Tumor chemonaive osteoblastic populations and paired control normal osteoblasts were isolated and characterized phenotypically from seven osteosarcoma patients. Global transcriptomic profiling was analyzed by robust microarray analysis. Candidate genes were confirmed by real-time PCR and organized in molecular pathways. EBF2 and osteoprotegerin (OPG) levels were determined by real-time PCR and OPG protein levels were assessed by ELISA. Immunohistochemical analysis was done in a panel of 46 osteosarcoma samples. Silencing of EBF2 was achieved by lentiviral transduction of short hairpin RNA. Apoptosis was determined by caspase-3/7 activity. Results: A robust clustered transcriptomic signature was obtained in osteosarcoma. Transcription factor EBF2, a known functional bone regulator, was among the most significantly overexpressed genes. Immunohistochemical analysis showed that osteosarcoma is expressed in ∼70% of tumors analyzed. Because EBF2 was shown previously to act as a transcriptional activator of OPG, elevated levels of EBF2 were associated with high OPG protein levels in osteosarcoma samples compared with normal osteoblastic cells. Knockdown of EBF2 led to stunted abrogation of OPG levels and increased sensitivity to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis. Conclusions: These findings suggest that EBF2 represents a novel marker of osteosarcoma. EBF2 up-regulation may be one of the mechanisms involved in the high levels of OPG in osteosarcoma, contributing to decrease TRAIL-induced apoptosis and leading to TRAIL resistance. (Clin Cancer Res 2009;15(16):5082–91)

The oncolytic adenovirus Δ24-RGD in combination with cisplatin exerts a potent anti-osteosarcoma activity

Osteosarcoma is the most common malignant bone tumor in children and adolescents. The presence of metastases and the lack of response to conventional treatment are the major adverse prognostic factors. Therefore, there is an urgent need for new treatment strategies that overcome both of these problems. Our purpose was to elucidate whether the use of the oncolytic adenovirus Δ24‐RGD alone or in combination with standard chemotherapy would be effective, in vitro and in vivo, against osteosarcoma. Our results showed that Δ24‐RGD exerted a potent antitumor effect against osteosarcoma cell lines that was increased by the addition of cisplatin. Δ24‐RGD osteosarcoma treatment resulted in autophagy in vitro that was further enhanced when combined with cisplatin. Of importance, administration of Δ24‐RGD and/or cisplatin, in novel orthotopic and two lung metastatic models in vivo resulted in a significant reduction of tumor burden meanwhile maintaining a safe toxicity profile. Together, our data underscore the potential of Δ24‐RGD to become a realistic therapeutic option for primary and metastatic pediatric osteosarcoma. Moreover, this study warrants a future clinical trial to evaluate the safety and efficacy of Δ24‐RGD for this devastating disease.

Cortactin (CTTN) overexpression in osteosarcoma correlates with advanced stage and reduced survival

BACKGROUND The cortactin (CTTN) gene has been found, by transcriptomic profiling, to be overexpressed in pediatric osteosarcoma. The location of CTTN at 11q13 and the role of cortactin in cytoskeleton restructuring make CTTN of interest as a potential biomarker for osteosarcoma. MATERIALS AND METHODS Osteoblasts were isolated from 20 high-grade osteosarcomas before chemotherapy, and paired with cell samples from normal tissue, prior to RNA expression analysis on HG-U133A chips (Affymetrix). Semiquantitative CTTN mRNA expression was analyzed by real-time PCR. An osteosarcoma tissue microarray (TMA) containing 233 tissue spots from 48 patients was used for an immunohistochemical (IHC) study of cortactin. RESULTS Transcriptomic profiling and real-time PCR analysis indicated increased CTTN expression in osteosarcomas (p = 0.001, Students T test). TMA IHC showed cortactin to be present more frequently and in greater abundance in osteosarcomas than non-tumoral osteoblastic samples (p< 0.006, Mann-Withney test). Analysis of clinical outcomes indicated that overall survival for patients with primary tumors positive for cortactin was significantly lower than that for patients with cortactin negative (or only weakly staining) tumors (p = 0.0278, Log-rank test). CONCLUSIONS Our preliminary data support the hypothesis that over-expression of cortactin, contained in the 11q13 amplicon, is involved in osteosarcoma carcinogenesis. The potential of cortactin overexpression as a biomarker for osteosarcoma is consolidated.

The Oncolytic Adenovirus VCN-01 as Therapeutic Approach Against Pediatric Osteosarcoma

Purpose: Osteosarcoma is the most common malignant bone tumor in children and adolescents. Despite aggressive chemotherapy, more than 30% of patients do not respond and develop bone or lung metastasis. Oncolytic adenoviruses engineered to specifically destroy cancer cells are a feasible option for osteosarcoma treatment. VCN-01 is a replication-competent adenovirus specifically engineered to replicate in tumors with a defective RB pathway, presents an enhanced infectivity through a modified fiber and an improved distribution through the expression of a soluble hyaluronidase. The aim of this study is to elucidate whether the use of VCN-01 would be an effective therapeutic strategy for pediatric osteosarcoma. Experimental Design: We used osteosarcoma cell lines established from patients with metastatic disease (531MII, 678R, 588M, and 595M) and a commercial cell line (143B). MTT assays were carried out to evaluate the cytotoxicity of VCN-01. Hexon assays were used to evaluate the replication of the virus. Western blot analysis was performed to assess the expression levels of viral proteins and autophagic markers. The antitumor effect of VCN-01 was evaluated in orthotopic and metastatic osteosarcoma murine animal models. Results: This study found that VCN-01, a new generation genetically modified oncolytic adenovirus, administered locally or systemically, had a potent antisarcoma effect in vitro and in vivo in mouse models of intratibial and lung metastatic osteosarcoma. Moreover, VCN-01 administration showed a safe toxicity profile. Conclusions: These results uncover VCN-01 as a promising strategy for osteosarcoma, setting the bases to propel a phase I/II trial for kids with this disease. Clin Cancer Res; 22(9); 2217–25. ©2015 AACR.

Matrix‐Gla protein promotes osteosarcoma lung metastasis and associates with poor prognosis

Osteosarcoma (OS) is the most prevalent osseous tumour in children and adolescents and, within this, lung metastases remain one of the factors associated with a dismal prognosis. At present, the genetic determinants driving pulmonary metastasis are poorly understood. We adopted a novel strategy using robust filtering analysis of transcriptomic profiling in tumour osteoblastic cell populations derived from human chemo‐naive primary tumours displaying extreme phenotypes (indolent versus metastatic) to uncover predictors associated with metastasis and poor survival. We identified MGP, encoding matrix‐Gla protein (MGP), a non‐collagenous matrix protein previously associated with the inhibition of arterial calcification. Using different orthotopic models, we found that ectopic expression of Mgp in murine and human OS cells led to a marked increase in lung metastasis. This effect was independent of the carboxylation of glutamic acid residues required for its physiological role. Abrogation of Mgp prevented lung metastatic activity, an effect that was rescued by forced expression. Mgp levels dramatically altered endothelial adhesion, trans‐endothelial migration in vitro and tumour cell extravasation ability in vivo. Furthermore, Mgp modulated metalloproteinase activities and TGFβ‐induced Smad2/3 phosphorylation. In the clinical setting, OS patients who developed lung metastases had high serum levels of MGP at diagnosis. Thus, MGP represents a novel adverse prognostic factor and a potential therapeutic target in OS. Microarray datasets may be found at: http://bioinfow.dep.usal.es/osteosarcoma/ Copyright