Pietro Ciccarino

Detection of IDH1 mutation in the plasma of patients with glioma

Objective: The IDH1R132H mutation is both a strong prognostic predictor and a diagnostic hallmark of gliomas and therefore has major clinical relevance. Here, we developed a new technique to detect the IDH1R132H mutation in the plasma of patients with glioma. Methods: Small-size DNA (150–250 base pairs) was extracted from the plasma of 31 controls and 80 patients with glioma with known IDH1R132H status and correlated with MRI data. The IDH1R132H mutation was detected by a combination of coamplification at lower denaturation temperature and digital PCR. Results: The small size DNA concentration was 1.2 ng/mL (range 0.1–6.6) in controls vs 1.2 ng/mL (range 0.1–50.3) in patients with glioma (p = not significant) and 0.9 ng/mL (0.0–3.0) in low-grade gliomas vs 1.5 ng/mL in high-grade gliomas (p < 0.01). The small size DNA concentration correlated with enhancing tumor volume (1.6 ng/mL [0.4–24.9] when <10 cm3 and 14.0 ng/mL [0.6–50.3] when ≥10 cm3). The IDH1R132H mutation was detected in 15 out of 25 plasma DNA mixtures (60%) from patients with mutated tumors and in none of the 14 patients with a nonmutated tumor. The sensitivity increased with enhancing tumor volume (3/9 in nonenhancing tumors, 6/10 for enhancing volume <10 cm3, and 6/6 for enhancing volume ≥10 cm3). Conclusion: With a specificity of 100% and a sensitivity related to the tumor volume and contrast enhancement, IDH1R132H identification has a valuable diagnostic accuracy in patients not amenable to biopsy.

5-Aminolevulinic Acid Fluorescence in High Grade Glioma Surgery: Surgical Outcome, Intraoperative Findings, and Fluorescence Patterns

Background. 5-Aminolevulinic acid (5-ALA) fluorescence is a validated technique for resection of high grade gliomas (HGG); the aim of this study was to evaluate the surgical outcome and the intraoperative findings in a consecutive series of patients. Methods. Clinical and surgical data from patients affected by HGG who underwent surgery guided by 5-ALA fluorescence at our Department between June 2011 and February 2014 were retrospectively evaluated. Surgical outcome was evaluated by assessing the resection rate as gross total resection (GTR) > 98% and GTR > 90%. We finally stratified data for recurrent surgery, tumor location, tumor size, and tumor grade (IV versus III grade sec. WHO). Results. 94 patients were finally enrolled. Overall GTR > 98% and GTR > 90% was achieved in 93% and 100% of patients. Extent of resection (GTR > 98%) was dependent on tumor location, tumor grade (P < 0.05), and tumor size (P < 0.05). In 43% of patients the boundaries of fluorescent tissue exceeded those of tumoral tissue detected by neuronavigation, more frequently in larger (57%) (P < 0.01) and recurrent (60%) tumors. Conclusions. 5-ALA fluorescence in HGG surgery enables a GTR in 100% of cases even if selection of patients remains a main bias. Recurrent surgery, and location, size, and tumor grade can predict both the surgical outcome and the intraoperative findings.

Low penetrance susceptibility to glioma is caused by the TP53 variant rs78378222

Background:Most of the heritable risk of glioma is presently unaccounted for by mutations in known genes. In addition to rare inactivating germline mutations in TP53 causing glioma in the context of the Li-Fraumeni syndrome, polymorphic variation in TP53 may also contribute to the risk of developing glioma.Methods:To comprehensively evaluate the impact of variation in TP53 on risk, we analysed 23 tagSNPs and imputed 2377 unobserved genotypes in four series totaling 4147 glioma cases and 7435 controls.Results:The strongest validated association signal was shown by the imputed single-nucleotide polymorphism (SNP) rs78378222 (P=6.86 × 10−24, minor allele frequency ∼0.013). Confirmatory genotyping confirmed the high quality of the imputation. The association between rs78378222 and risk was seen for both glioblastoma multiforme (GBM) and non-GBM tumours. We comprehensively examined the relationship between rs78378222 and overall survival in two of the case series totaling 1699 individuals. Despite employing statistical tests sensitive to the detection of differences in early survival, no association was shown.Conclusion:Our data provided strong validation of rs78378222 as a risk factor for glioma but do not support the tenet that the polymorphism being a clinically useful prognostic marker. Acquired TP53 inactivation is a common feature of glioma. As rs78378222 changes the polyadenylation signal of TP53 leading to impaired 3′-end processing of TP53 mRNA, the SNP has strong plausibility for being directly functional contributing to the aetiological basis of glioma.

COLD PCR HRM: a highly sensitive detection method for IDH1 mutations.

The p.Arg132His mutation of isocitrate dehydrogenase 1 (IDH1R132H) is a frequent alteration and a major prognostic marker in gliomas. However, direct sequencing of highly contaminated tumor samples may fail to detect this mutation. Our objective was to evaluated the sensitivity of a newly described amplification method, coamplification at lower temperature‐PCR (COLD PCR), combined with high‐resolution melting (HRM) for the detection of the IDH1R132H mutation. To this end, we used serial dilutions of mutant DNA with wild‐type DNA. PCR‐HRM assay detects IDH1R132H at an abundance of 25%, similar to the detection limit of direct Sanger sequencing. Introducing a run of COLD PCR allows the detection of 2% mutant DNA. Using two consecutive runs of COLD PCR, we detected 0.25% mutant DNA in a background of wild‐type DNA, that mimics a tumor sample highly contaminated by normal DNA. We then analyzed 10 biopsies of tumor edges, considered free of tumor cells by histological analysis, and showed that immunohistochemistry of IDH1R132H was positive in three cases (30%), whereas double COLD PCR HRM was positive in the 10 cases studied (100%). In summary, COLD PCR HRM analysis is 100‐fold more sensitive than Sanger sequencing, rendering this rapid and powerful strategy particularly useful for samples highly contaminated with normal tissue.Hum Mutat 31:1–6, 2010.

Deciphering the 8q24.21 association for glioma

We have previously identified tagSNPs at 8q24.21 influencing glioma risk. We have sought to fine-map the location of the functional basis of this association using data from four genome-wide association studies, comprising a total of 4147 glioma cases and 7435 controls. To improve marker density across the 700 kb region, we imputed genotypes using 1000 Genomes Project data and high-coverage sequencing data generated on 253 individuals. Analysis revealed an imputed low-frequency SNP rs55705857 (P = 2.24 × 10(-38)) which was sufficient to fully capture the 8q24.21 association. Analysis by glioma subtype showed the association with rs55705857 confined to non-glioblastoma multiforme (non-GBM) tumours (P = 1.07 × 10(-67)). Validation of the non-GBM association was shown in three additional datasets (625 non-GBM cases, 2412 controls; P = 1.41 × 10(-28)). In the pooled analysis, the odds ratio for low-grade glioma associated with rs55705857 was 4.3 (P = 2.31 × 10(-94)). rs55705857 maps to a highly evolutionarily conserved sequence within the long non-coding RNA CCDC26 raising the possibility of direct functionality. These data provide additional insights into the aetiological basis of glioma development.

Hypertension as a biomarker in patients with recurrent glioblastoma treated with antiangiogenic drugs: a single-center experience and a critical review of the literature.

Treatment with angiogenesis inhibitors is becoming a cornerstone of modern anticancer therapy. Hypertension (HTN) is a common adverse event during antiangiogenic treatment and might represent a cancer biomarker in patients with recurrent glioblastoma treated with angiogenesis inhibitors. In a retrospective study, we analyzed 53 patients with recurrent glioblastoma treated with antiangiogenic drugs. Thirty patients were treated with sorafenib and 23 patients were treated with bevacizumab. All patients underwent brain gadolinium-enhanced MRI assessments according to the Radiologic Assessment in Neuro-Oncology criteria every 2 months or when clinically indicated. Blood pressure was measured before and during the treatment. We investigated whether treatment-related HTN may be associated with outcome in patients treated with antiangiogenic drugs. After 2 months of treatment, 24 patients (45%) achieved disease control: stable disease (17 patients) or a partial response (seven patients). The median overall survival from the start of antiangiogenic treatment was 7.3 months [95% confidence interval (CI) 6.02–8.5]; the median progression-free survival (PFS) was 2.7 months (95% CI 1.5–3.5); and the 6-month PFS was 32%. Twenty patients (38%) developed grades 2–3 HTN within 2 months of treatment. A significant association was found between HTN and disease control rate, and HTN and 6-month PFS; no significant association was found between HTN and the median PFS. According to univariate and multivariate analyses, HTN was related to a longer survival from antiangiogenic drug administration: 9.8 versus 4.8 months (P=0.001; hazard ratio=3.5, 95% CI 1.6–7.6). Our data indicate that HTN may be an effective biomarker in patients with recurrent glioblastoma treated with antiangiogenic drugs; in particular, it may be associated with a favorable effect on disease control, 6-month PFS, and the median overall survival.

IDH Mutations: Genotype-Phenotype Correlation and Prognostic Impact

IDH1/2 mutation is the most frequent genomic alteration found in gliomas, affecting 40% of these tumors and is one of the earliest alterations occurring in gliomagenesis. We investigated a series of 1305 gliomas and showed that IDH mutation is almost constant in 1p19q codeleted tumors. We found that the distribution of IDH1R132H, IDH1nonR132H, and IDH2 mutations differed between astrocytic, mixed, and oligodendroglial tumors, with an overrepresentation of IDH2 mutations in oligodendroglial phenotype and an overrepresentation of IDH1nonR132H in astrocytic tumors. We stratified grade II and grade III gliomas according to the codeletion of 1p19q and IDH mutation to define three distinct prognostic subgroups: 1p19q and IDH mutated, IDH mutated—which contains mostly TP53 mutated tumors, and none of these alterations. We confirmed that IDH mutation with a hazard ratio = 0.358 is an independent prognostic factor of good outcome. These data refine current knowledge on IDH mutation prognostic impact and genotype-phenotype associations.

Intraventricular astroblastoma. Case report.

Astroblastoma is a rare primary brain neoplasm that accounts for 0.45-2.8% of brain gliomas. Intraventricular localization is extremely rare. The authors report a case of well-differentiated completely intraventricular astroblastoma in a 6-year-old girl and review the relevant literature. Their patient presented with a 5-week history of progressive nausea and vomiting. Magnetic resonance (MR) imaging revealed a large, well-demarcated, solid-cystic mass in the left temporooccipital ventricular horn. Macroscopic radical resection of the tumor was performed via the superior temporal sulcus. The postoperative course was uneventful and no adjuvant therapy was administered after surgery. No recurrence was detected at 9-months follow-up. Gross-total resection has the greatest impact on patient survival. In differentiated tumors, recurrence is usually local, and adjuvant therapy is recommended after repeated resection for the treatment of recurrence. In patients harboring anaplastic astroblastoma, gross-total resection and adjuvant therapy after the initial surgery seems to be the best choice. It is important to distinguish astroblastoma from ependymoma in clinical practice because of the differences in therapeutic approaches.

Carmustine Wafer Implantation When Surgical Cavity Is Communicating with Cerebral Ventricles: Technical Considerations on a Clinical Series

BACKGROUND Implantation of carmustine (1,3-bis (2 chloroetyl)-1-nitrosurea [BCNU]) wafers is an approved local treatment after surgical removal of high-grade gliomas. Safety data have been largely reported by phase III studies. The communication between the final surgical cavity and the ventricular cavities is supposed to be a relative contraindication for positioning of the wafers because of the possible development of hydrocephalus. However, at present there are neither data about this topic published with the exception of a few case reports, nor any proposals for selection criteria for wafer implantation in such circumstances. Furthermore, there are no technical suggestions in literature put forward for the surgical repairing of ventricular defects. Our study was particularly focused on addressing these 3 issues. METHODS Forty-three patients affected by a high-grade glioma underwent surgical removal and BCNU wafer implantation between March 2007 and September 2009 at the Department of Neurosurgery of Padua. Among them, we retrospectively reviewed clinical, surgical, and radiological data of 9 patients who had been treated with carmustine wafers after surgical repair of communication between the surgical cavity and the ventricular cavities. We also focused on the technical details concerning wafers positioning in this particular situation. RESULTS Ventricular defects were present in the atrium in 4, frontal horn in 3, and temporal horn in 2 cases. The maximum diameter of the defect was between 6 and 10 mm. In all cases, the defect was intraoperatively repaired in the same way, and up to 8 wafers were implanted in the surgical cavity. In the series reported, no cases of hydrocephalus were detected. CONCLUSIONS In our experience, integrity of wafers, size of ventricular wall defect, and accuracy in repairing the defect were crucial issues. Nevertheless, more experience and prospective studies would be helpful to clarify both in what measure ventricular opening affects safety data and the best reliable way of repairing ventricular defects when BCNU wafers are implanted.

Cervical myelomeningocele in adulthood: case report.

OBJECTIVE Cervical myelomeningocele is an extremely rare condition, accounting for only 1 to 5% of all neural tube defects. These lesions are usually diagnosed in childhood. Here, we report a case of a cervical myelomeningocele diagnosed and treated in adulthood. CLINICAL PRESENTATION A 52-year-old man presented with a 3-year history of progressing weakness and paresthesia in his upper limbs. Physical examination revealed a posterior midline neck mass covered with normal skin. Magnetic resonance imaging showed a soft-tissue mass tethering the cord by a stalk extending from the dorsal spinal cord to the dome of the lesion. Syrinx was evident cranially and caudally to the origin of the posterior stalk. INTERVENTION Surgical resection of the sac and intradural exploration were performed. The subdural space was explored, and the tethered structures were released. Histological examination showed small foci of meningothelial cells with psammoma bodies and rare thin fascicle of glial tissue dispersed in hyaline tissue. Immunohistochemical stains against glial fibrillary acidic protein and S100 confirmed the presence of bands of astrocytic tissue. The patient demonstrated early improvement of neurological deficits. Six months after surgery, he was asymptomatic and magnetic resonance imaging showed resolution of the syrinx. CONCLUSION We believe the syrinx in this patient was caused by a blockade of flow in the central canal and around the spinal cord as a result of the tethered cord. The untethering procedure resulted in the collapse of the syrinx followed by resolution of neurological deficits.