Samantha Mascelli

Evaluation of a methylenetetrahydrofolate-dehydrogenase 1958G>A polymorphism for neural tube defect risk

AbstractGenetic variants of enzymes involved in the folate pathway might be expected to have an impact on neural tube defect (NTD) risk. Given its key role in folate metabolism, the methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene could represent an attractive candidate in NTD aetiology. In this study, the impact of the MTHFD1 1958G>A polymorphism on NTD risk in the Italian population was examined both by hospital-based case-control and family-based studies. The MTHFD1 1958G>A polymorphism was genotyped in 142 NTD cases, 125 mothers, 108 fathers and 523 controls. An increased risk was found for the heterozygous 1958G/A (OR=1.69; P=0.04) and homozygous 1958A/A (OR=1.91; P=0.02) genotypes in the children. Significant association was also found when combined 1958G/A and 1958A/A genotypes of cases were compared with the 1958G/G genotype (OR=1.76; P=0.02). The risk of an NTD-affected pregnancy of the mothers was increased 1.67-fold (P=0.04) only when a dominant effect (1958G/A or 1958A/A vs 1958G/G) of the 1958A allele was analysed. The combined TDT/1-TDT (Z=2.11; P=0.03) and FBAT (Z=2.4; P=0.01) demonstrated a significant excess of transmission of the 1958A allele to affected individuals. In summary, our results indicate that heterozygosity and homozygosity for the MTHFD1 1958G>A polymorphism are genetic determinants of NTD risk in the cases examined.

Current perspectives on the genetic causes of neural tube defects

Neural tube defects (NTDs) are a group of severe congenital abnormalities resulting from the failure of neurulation. The pattern of inheritance of these complex defects is multifactorial, making it difficult to identify the underlying causes. Scientific research has rapidly progressed in experimental embryology and molecular genetics to elucidate the basis of neurulation. Crucial mechanisms of neurulation include the planar cell polarity pathway, which is essential for the initiation of neural tube closure, and the sonic hedgehog signaling pathway, which regulates neural plate bending. Genes influencing neurulation have been investigated for their contribution to human neural tube defects, but only genes with well-established role in convergent extension provide an exciting new set of candidate genes. Biochemical factors such as folic acid appear to be the greatest modifiers of NTDs risk in the human population. Consequently, much research has focused on genes of folate-related metabolic pathways. Variants of several such genes have been found to be significantly associated with the risk of neural tube defects in more studies. In this manuscript, we reviewed the current perspectives on the causes of neural tube defects and highlighted that we are still a long way from understanding the etiology of these complex defects.

Maternal periconceptional factors affect the risk of spina bifida-affected pregnancies: an Italian case–control study

PurposeNeural tube defects, including spina bifida and anencephaly, are the second most common birth defects with an incidence in Italy of 0.4–1/1,000. Information on factors playing a role in the pathogenesis of spina bifida is based on populations with different exposures, lifestyle, social and cultural habits compared to Italian people. Our objective was to fill this gap by using data from a case–control interview study carried out at the G. Gaslini Children’s Hospital, Genoa, from 2000 to 2008.MethodsWe surveyed questionnaires from 133 case mothers and 273 control women providing information on periconceptional risk factors. Univariate and multivariate logistic regression analyses were used to estimate risks by odds ratios (ORs) and 95% confidence intervals (95% CIs).ResultsUnivariate results suggest that birth order, low maternal educational level, age, smoking habits, alcohol consumption, high caffeine intake, lack of folate supplementation, low and high calorie diet, occasional consumption of fruit and vegetables, high emotional stress, and environmental pollution are associated with an increased spina bifida risk. Nevertheless, high caffeine intake (OR = 10.82; 95% CI, 3.78–31), low calorie diet (OR = 5.15; 95%CI, 1.79–14), occasional consumption of fruit and vegetables (OR = 3.38; 95% CI, 1.67–6.82), alcohol consumption (OR = 3.05; 95% CI, 1.24–7.50) and, above all, lack of folate supplementation at any time of pregnancy (OR = 20.54; 95% CI, 5.41–77) mainly determined spina bifida risk in the multivariate analysis.ConclusionOur findings point out that a common underlying mechanism, a disturbed folate/homocysteine metabolism, may be causative for the burden of spina bifida in the Italian population.

Predictability, efficacy and safety of radiosensitization of glioblastoma-initiating cells by the ATM inhibitor KU-60019.

We have previously shown that pharmacological inhibition of ataxia telangiectasia mutated (ATM) protein sensitizes glioblastoma‐initiating cells (GICs) to ionizing radiation (IR). Herein, we report the experimental conditions to overcome GIC radioresistance in vitro using the specific ATM inhibitor KU‐60019, two major determinants of the tumor response to this drug and the absence of toxicity of this treatment in vitro and in vivo. Repeated treatments with KU‐60019 followed by IR substantially delayed GIC proliferation in vitro and even eradicated radioresistant cells, whereas GIC treated with vehicle plus radiation recovered early and expanded. The tumor response to the drug occurred under a cutoff level of expression of TP53 and over a cutoff level of expression of phosphatidylinositol 3‐kinase (PI3K). No increased clastogenicity or point mutagenicity was induced by KU‐60019 plus radiation when compared to vehicle plus radiation. No significant histological changes to the brain or other organs were observed after prolonged infusion into the brain of KU‐60019 at millimolar concentrations. Taken together, these findings suggest that GIC‐driven tumors with low expression of TP53 and high expression of PI3K might be effectively and safely radiosensitized by KU‐60019.

Pharmacokinetics, pharmacodynamics and efficacy on pediatric tumors of the glioma radiosensitizer KU60019

We have recently reported that glioblastoma (GB)‐initiating cells (GIC) with low expression and/or mutation of TP53 and high expression of PI3K (“responder” genetic profile) can be effectively and safely radiosensitized by the ATM inhibitor KU60019. We report here on drugs diffusion and elimination from the animal body and brain, its effects on orthotopic GB and efficacy toward pediatric GIC. Healthy mice were infused by convection enhanced delivery (CED) with KU60019 and the drug kinetics followed by high performance liquid chromatography–mass spectrometry. Already at the end of CED, KU60019 had diffused from the injection site to the ipsilateral and, to a lower extent, controlateral hemisphere. After 24 hr, no drug could be detected all over the brain or in other organs, indicating rapid draining and excretion. After intraperitoneal injection, traces only of KU60019 could be detected in the brain, indicating inability to cross the brain–blood barrier. Consistent with the induction of cell cycle progression previously observed in vitro, KU60019 stimulated proliferation of orthotopic GB cells with the highest effect observed 96 hr after drug delivery. Adult GIC with high expression of TP53 and low expression of PI3K could be radiosensitized by KU60019, although less promptly than GIC bearing the “responder” profile. Consistent with the kinetics of proliferation induction, the highest radiosensitizing effect was observed 96 hr after delivery of KU60019 to GIC. Pediatric GIC could be similarly radiosensitized after exposure to KU60019. The results indicate that ATM inhibition may allow to radiosensitize a wide range of adult and pediatric high‐grade gliomas.

High levels of PROM1 (CD133) transcript are a potential predictor of poor prognosis in medulloblastoma

The surface marker PROM1 is considered one of the most important markers of tumor-initiating cells, and its expression is believed to be an adverse prognostic factor in gliomas and in other malignancies. To date, to our knowledge, no specific studies of its expression in medulloblastoma series have been performed. The aims of our study were to evaluate the expression profile of the PROM1 gene in medulloblastoma and to assess its possible role as a prognostic factor. The PROM1 gene expression was evaluated by quantitative- polymerase chain reaction on 45 medulloblastoma samples by using specific dye-labeled probe systems. A significantly higher expression of PROM1 was found both in patients with poorer prognosis (P= .007) and in those with metastasis (P= .03). Kaplan-Meier analysis showed that both overall survival (OS) and progression-free survival (PFS) were shorter in patients with higher PROM1 mRNA levels than in patients with lower expression, even when the desmoplastic cases were excluded (P= .0004 and P= .002, for OS and PFS for all cases, respectively; P= .002 and P= .008 for OS and PFS for nondesmoplastic cases, respectively). Cox regression model demonstrated that PROM1 expression is an independent prognostic factor (hazard ratio, 4.56; P= .008). The result was validated on an independent cohort of 42 cases by microarray-based analysis (P= .019). This work suggests that high mRNA levels of PROM1 are associated with poor outcome in pediatric medulloblastoma. Furthermore, high PROM1 expression levels seem to increase the likelihood of metastases. Such results need to be confirmed in larger prospective series to possibly incorporate PROM1 gene expression into risk classification systems to be used in the clinical setting.

Therapeutic and prognostic implications of BRAF V600E in pediatric low-grade gliomas

Purpose BRAF V600E is a potentially highly targetable mutation detected in a subset of pediatric low-grade gliomas (PLGGs). Its biologic and clinical effect within this diverse group of tumors remains unknown. Patients and Methods A combined clinical and genetic institutional study of patients with PLGGs with long-term follow-up was performed (N = 510). Clinical and treatment data of patients with BRAF V600E mutated PLGG (n = 99) were compared with a large international independent cohort of patients with BRAF V600E mutated-PLGG (n = 180). Results BRAF V600E mutation was detected in 69 of 405 patients (17%) with PLGG across a broad spectrum of histologies and sites, including midline locations, which are not often routinely biopsied in clinical practice. Patients with BRAF V600E PLGG exhibited poor outcomes after chemotherapy and radiation therapies that resulted in a 10-year progression-free survival of 27% (95% CI, 12.1% to 41.9%) and 60.2% (95% CI, 53.3% to 67.1%) for BRAF V600E and wild-type PLGG, respectively ( P < .001). Additional multivariable clinical and molecular stratification revealed that the extent of resection and CDKN2A deletion contributed independently to poor outcome in BRAF V600E PLGG. A similar independent role for CDKN2A and resection on outcome were observed in the independent cohort. Quantitative imaging analysis revealed progressive disease and a lack of response to conventional chemotherapy in most patients with BRAF V600E PLGG. Conclusion BRAF V600E PLGG constitutes a distinct entity with poor prognosis when treated with current adjuvant therapy.

Successful isolation and long-term establishment of a cell line with stem cell-like features from an anaplastic medulloblastoma

Aims: Herein we report on the successful isolation and establishment of a novel, long‐term, primary, neurosphere‐like cell line called 1603‐MED from a 5‐year‐old boy affected by a highly aggressive anaplastic medulloblastoma. Methods: Elaboration of the new protocol for neurosphere assay is extensively discussed, together with a complete immuno‐histochemical and cytogenetic characterization of 1603‐MED. Results: Clinical course and histopathology are briefly discussed. The 1603‐MED possesses a high capacity for proliferation, CD133 expression, self‐renewal and differentiation, thus indicating that anaplastic medulloblastoma contains a subpopulation of cancer stem cells as observed in classic medulloblastoma. Conclusions: 1603‐MED provides us with the first in vitro model of anaplastic medulloblastoma that may be suitable for studying both tumour progression and the genetic mechanisms related to therapy resistance, and may lead to the development and testing of chemosensitivity and new therapeutic targets.

Characterization of Glioma Stem Cells Through Multiple Stem Cell Markers and Their Specific Sensitization to Double-Strand Break-Inducing Agents by Pharmacological Inhibition of Ataxia Telangiectasia Mutated Protein

Previous studies have shown that tumor‐driving glioma stem cells (GSC) may promote radio‐resistance by constitutive activation of the DNA damage response started by the ataxia telangiectasia mutated (ATM) protein. We have investigated whether GSC may be specifically sensitized to ionizing radiation by inhibiting the DNA damage response. Two grade IV glioma cell lines (BORRU and DR177) were characterized for a number of immunocytochemical, karyotypic, proliferative and differentiative parameters. In particular, the expression of a panel of nine stem cell markers was quantified by reverse transcription‐polymerase chain reaction (RT‐PCR) and flow cytometry. Overall, BORRU and DR177 displayed pronounced and poor stem phenotypes, respectively. In order to improve the therapeutic efficacy of radiation on GSC, the cells were preincubated with a nontoxic concentration of the ATM inhibitors KU‐55933 and KU‐60019 and then irradiated. BORRU cells were sensitized to radiation and radio‐mimetic chemicals by ATM inhibitors whereas DR177 were protected under the same conditions. No sensitization was observed after cell differentiation or to drugs unable to induce double‐strand breaks (DSB), indicating that ATM inhibitors specifically sensitize glioma cells possessing stem phenotype to DSB‐inducing agents. In conclusion, pharmacological inhibition of ATM may specifically sensitize GSC to DSB‐inducing agents while sparing nonstem cells.

Diagnostic and prognostic value of 18F-DOPA PET and 1H-MR spectroscopy in pediatric supratentorial infiltrative gliomas: a comparative study.

BACKGROUND (1)H-MR spectroscopy (MRS) and (18)F-dihydroxyphenylalanine (DOPA) PET are noninvasive imaging techniques able to assess metabolic features of brain tumors. The aim of this study was to compare diagnostic and prognostic information gathered by (18)F-DOPA PET and (1)H-MRS in children with supratentorial infiltrative gliomas or nonneoplastic brain lesions suspected to be gliomas. METHODS We retrospectively analyzed 27 pediatric patients with supratentorial infiltrative brain lesions on conventional MRI (21 gliomas and 6 nonneoplastic lesions) who underwent (18)F-DOPA PET and (1)H-MRS within 2 weeks of each other. (1)H-MRS data (choline/N-acetylaspartate, choline-to-creatine ratios, and presence of lactate) and (18)F-DOPA uptake parameters (lesion-to-normal tissue and lesion-to-striatum ratios) were compared and correlated with histology, WHO tumor grade, and patient outcome. RESULTS (1)H-MRS and (18)F-DOPA PET data were positively correlated. Sensitivity, specificity, and accuracy in distinguishing gliomas from nonneoplastic lesions were 95%, 83%, and 93% for (1)H-MRS and 76%, 83%, and 78% for (18)F-DOPA PET, respectively. No statistically significant differences were found between the 2 techniques (P > .05). Significant differences regarding (18)F-DOPA uptake and (1)H-MRS ratios were found between low-grade and high-grade gliomas (P≤.001 and P≤.04, respectively). On multivariate analysis, (18)F-DOPA uptake independently correlated with progression-free survival (P≤.05) and overall survival (P = .04), whereas (1)H-MRS did not show significant association with outcome. CONCLUSIONS (1)H-MRS and (18)F-DOPA PET provide useful complementary information for evaluating the metabolism of pediatric brain lesions. (1)H-MRS represents the method of first choice for differentiating brain gliomas from nonneoplastic lesions.(18)F-DOPA uptake better discriminates low-grade from high-grade gliomas and is an independent predictor of outcome.