Anna Luisa Di Stefano

Chromosome 7p11.2 (EGFR) variation influences glioma risk

While gliomas are the most common primary brain tumors, their etiology is largely unknown. To identify novel risk loci for glioma, we conducted genome-wide association (GWA) analysis of two case-control series from France and Germany (2269 cases and 2500 controls). Pooling these data with previously reported UK and US GWA studies provided data on 4147 glioma cases and 7435 controls genotyped for 424 460 common tagging single-nucleotide polymorphisms. Using these data, we demonstrate two statistically independent associations between glioma and rs11979158 and rs2252586, at 7p11.2 which encompasses the EGFR gene (population-corrected statistics, P(c) = 7.72 × 10(-8) and 2.09 × 10(-8), respectively). Both associations were independent of tumor subtype, and were independent of EGFR amplification, p16INK4a deletion and IDH1 mutation status in tumors; compatible with driver effects of the variants on glioma development. These findings show that variation in 7p11.2 is a determinant of inherited glioma risk.

Detection, Characterization, and Inhibition of FGFR–TACC Fusions in IDH Wild-type Glioma

Purpose: Oncogenic fusions consisting of fibroblast growth factor receptor (FGFR) and TACC are present in a subgroup of glioblastoma (GBM) and other human cancers and have been proposed as new therapeutic targets. We analyzed frequency and molecular features of FGFR–TACC fusions and explored the therapeutic efficacy of inhibiting FGFR kinase in GBM and grade II and III glioma. Experimental Design: Overall, 795 gliomas (584 GBM, 85 grades II and III with wild-type and 126 with IDH1/2 mutation) were screened for FGFR–TACC breakpoints and associated molecular profile. We also analyzed expression of the FGFR3 and TACC3 components of the fusions. The effects of the specific FGFR inhibitor JNJ-42756493 for FGFR3–TACC3–positive glioma were determined in preclinical experiments. Two patients with advanced FGFR3–TACC3–positive GBM received JNJ-42756493 and were assessed for therapeutic response. Results: Three of 85 IDH1/2 wild-type (3.5%) but none of 126 IDH1/2-mutant grade II and III gliomas harbored FGFR3–TACC3 fusions. FGFR–TACC rearrangements were present in 17 of 584 GBM (2.9%). FGFR3–TACC3 fusions were associated with strong and homogeneous FGFR3 immunostaining. They are mutually exclusive with IDH1/2 mutations and EGFR amplification, whereas they co-occur with CDK4 amplification. JNJ-42756493 inhibited growth of glioma cells harboring FGFR3–TACC3 in vitro and in vivo. The two patients with FGFR3–TACC3 rearrangements who received JNJ-42756493 manifested clinical improvement with stable disease and minor response, respectively. Conclusions: RT-PCR sequencing is a sensitive and specific method to identify FGFR–TACC–positive patients. FGFR3–TACC3 fusions are associated with uniform intratumor expression of the fusion protein. The clinical response observed in the FGFR3–TACC3–positive patients treated with an FGFR inhibitor supports clinical studies of FGFR inhibition in FGFR–TACC–positive patients. Clin Cancer Res; 21(14); 3307–17. ©2015 AACR. See related commentary by Ahluwalia and Rich, p. 3105

Association between glioma susceptibility loci and tumour pathology defines specific molecular etiologies

BACKGROUND Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) at 7 loci influencing glioma risk: rs2736100 (TERT), rs11979158 and rs2252586 (EGFR), rs4295627 (CCDC26), rs4977756 (CDKN2A/CDKN2B), rs498872 (PHLDB1), and rs6010620 (RTEL1). MATERIALS AND METHODS We studied the relationship among these 7 glioma-risk SNPs and characteristics of tumors from 1374 patients, including grade, IDH (ie IDH1 or IDH2) mutation, EGFR amplification, CDKN2A-p16-INK4a homozygous deletion, 9p and 10q loss, and 1p-19q codeletion. RESULTS rs2736100 (TERT) and rs6010620 (RTEL1) risk alleles were associated with high-grade disease, EGFR amplification, CDKN2A-p16-INK4a homozygous deletion, and 9p and 10q deletion; rs4295627 (CCDC26) and rs498872 (PHLDB1) were associated with low-grade disease, IDH mutation, and 1p-19q codeletion. In contrast, rs4977756 (CDKN2A/B), rs11979158 (EGFR), and to a lesser extent, rs2252586 (EGFR) risk alleles were independent of tumor grade and genetic profile. Adjusting for tumor grade showed a significant association between rs2736100 and IDH status (P = .01), 10q loss (P = .02); rs4295627 and 1p-19q codeletion (P = .04), rs498872 and IDH (P = .02), 9p loss (P = .04), and 10q loss (P = .02). Case-control analyses stratified into 4 molecular classes (defined by 1p-19q status, IDH mutation, and EGFR amplification) showed an association of rs4295627 and rs498872 with IDH-mutated gliomas (P < 10(-3)) and rs2736100 and rs6010620 with IDH wild-type gliomas (P < 10(-3) and P = .03). CONCLUSION The frequency of EGFR and CDKN2A/B risk alleles were largely independent of tumor genetic profile, whereas TERT, RTEL1, CCDC26, and PHLDB1 variants were associated with different genetic profiles that annotate distinct molecular pathways. Our findings provide further insight into the biological basis of glioma etiology.

Gender-based blood transcriptomes and interactomes in multiple sclerosis: involvement of SP1 dependent gene transcription.

In this study we investigated the contribution of gender to global gene expression in peripheral blood mononuclear cells from multiple sclerosis (MS) patients and healthy controls. We observed that, in contrast to the conventional approach, gender-based case-control comparisons resulted in genelists with significantly reduced heterogeneity in human populations. In addition, MS was characterized by significant changes both in the quantity and in the quality of the sex-specific genes. Application of stringent statistics defined gender-based signatures which classified a second independent MS population with high precision. The global unsupervised cluster analyses for 60 subjects showed that 29/31 female and 27/29 male samples were properly identified. Notably, MS was associated in women and in men with distinct gene signatures which however shared several molecular functions, biological processes and interactors. Issues regarding epigenetic control of gene expression appeared as the main common theme for disease, with a central role for the functional modules related to histone deacetylase, NF-kappaB and androgen receptor signaling. Moreover, in silico analyses predicted that the differential expression in MS women and men were depending on the transcription factor SP1. Specific targeting of this pathway by the bis-anthracycline WP631 impaired T cell responses in vitro and in vivo, and reduced the incidence and the severity of experimental autoimmune encephalomyelitis, indicating that SP1 dependent gene transcription sustains neuroinflammation. Thus, the gender-based approach with its reduced heterogeneity and the systems biology tools with the identification of the molecular and functional networks successfully uncovered the differences but also the commonalities associated to multiple sclerosis in women and men. In conclusion, we propose gender-based systems biology as a novel tool to gain fundamental information on disease-associated functional processes.

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.

Systemic treatments for brain metastases from breast cancer, non-small cell lung cancer, melanoma and renal cell carcinoma: an overview of the literature.

The frequency of metastatic brain tumors has increased over recent years; the primary tumors most involved are breast cancer, lung cancer, melanoma and renal cell carcinoma. While radiation therapy and surgery remain the mainstay treatment in selected patients, new molecular drugs have been developed for brain metastases. Studies so far report interesting results. This review focuses on systemic cytotoxic drugs and, in particular, on new targeted therapies and their clinically relevant activities in brain metastases from solid tumors in adults.

Prognostic Value of CD109+ Circulating Endothelial Cells in Recurrent Glioblastomas Treated with Bevacizumab and Irinotecan

Background Recent data suggest that circulating endothelial and progenitor cells (CECs and CEPs, respectively) may have predictive potential in cancer patients treated with bevacizumab, the antibody recognizing vascular endothelial growth factor (VEGF). Here we report on CECs and CEPs investigated in 68 patients affected by recurrent glioblastoma (rGBM) treated with bevacizumab and irinotecan and two Independent Datasets of rGBM patients respectively treated with bevacizumab alone (n=32, independent dataset A: IDA) and classical antiblastic chemotherapy (n=14, independent dataset B: IDB). Methods rGBM patients with KPS ≥50 were treated until progression, as defined by MRI with RANO criteria. CECs expressing CD109, a marker of tumor endothelial cells, as well as other CEC and CEP subtypes, were investigated by six-color flow cytometry. Results A baseline count of CD109+ CEC higher than 41.1/ml (1st quartile) was associated with increased progression free survival (PFS; 20 versus 9 weeks, P=0.008) and overall survival (OS; 32 versus 23 weeks, P=0.03). Longer PFS (25 versus 8 weeks, P=0.02) and OS (27 versus 17 weeks, P=0.03) were also confirmed in IDA with CD109+ CECs higher than 41.1/ml but not in IDB. Patients treated with bevacizumab with or without irinotecan that were free from MRI progression after two months of treatment had significant decrease of CD109+ CECs: median PFS was 19 weeks; median OS 29 weeks. The presence of two non-contiguous lesions (distant disease) at baseline was an independent predictor of shorter PFS and OS (P<0.001). Conclusions Data encourage further studies on the predictive potential of CD109+ CECs in GBM patients treated with bevacizumab.

Parametric Response Maps of Perfusion MRI May Identify Recurrent Glioblastomas Responsive to Bevacizumab and Irinotecan

Background Perfusion weighted imaging (PWI) can be used to measure key aspects of tumor vascularity in vivo and recent studies suggest that perfusion imaging may be useful in the early assessment of response to angiogenesis inhibitors. Aim of this work is to compare Parametric Response Maps (PRMs) with the Region Of Interest (ROI) approach in the analysis of tumor changes induced by bevacizumab and irinotecan in recurrent glioblastomas (rGBM), and to evaluate if changes in tumor blood volume measured by perfusion MRI may predict clinical outcome. Methods 42 rGBM patients with KPS ≥50 were treated until progression, as defined by MRI with RANO criteria. Relative cerebral blood volume (rCBV) variation after 8 weeks of treatment was calculated through semi-automatic ROI placement in the same anatomic region as in baseline. Alternatively, rCBV variations with respect to baseline were calculated into the evolving tumor region using a voxel-by-voxel difference. PRMs were created showing where rCBV significantly increased, decreased or remained unchanged. Results An increased blood volume in PRM (PRMCBV+) higher than 18% (first quartile) after 8 weeks of treatment was associated with increased progression free survival (PFS; 24 versus 13 weeks, p = 0.045) and overall survival (OS; 38 versus 25 weeks, p = 0.016). After 8 weeks of treatment ROI analysis showed that mean rCBV remained elevated in non responsive patients (4.8±0.9 versus 5.1±1.2, p = 0.38), whereas decreased in responsive patients (4.2±1.3 versus 3.8±1.6 p = 0.04), and re-increased progressively when patients approached tumor progression. Conclusions Our data suggest that PRMs can provide an early marker of response to antiangiogenic treatment and warrant further confirmation in a larger cohort of GBM patients.

Acute late-onset encephalopathy after radiotherapy: an unusual life-threatening complication.

Unusual late-onset complications of brain irradiation, characterized by reversible neurologic focal signs, seizures, and MRI alterations, have recently been reported and classified as stroke-like migraine attacks after radiation therapy (SMART)1 and peri-ictal pseudoprogression (PIPG).2

Highly specific determination of IDH status using edited in vivo magnetic resonance spectroscopy

Background Mutations in the isocitrate dehydrogenase (IDH) enzyme affect 40% of gliomas and represent a major diagnostic and prognostic marker. The goals of this study were to evaluate the performance of noninvasive magnetic resonance spectroscopy (MRS) methods to determine the IDH status of patients with brain gliomas through detection of the oncometabolite 2-hydroxyglutarate (2HG) and to compare performance of these methods with DNA sequencing and tissue 2HG analysis. Methods Twenty-four subjects with suspected diagnosis of low-grade glioma were included prospectively in the study. For all subjects, MRS data were acquired at 3T using 2 MRS methods, edited MRS using Mescher-Garwood point-resolved spectroscopy (MEGA-PRESS) sequence and a PRESS sequence optimized for 2HG detection, using a volume of interest larger than 6 mL. IDH mutational status was determined by a combination of automated immunohistochemical analysis and Sanger sequencing. Levels of 2HG in tissue samples measured by gas chromatography-mass spectrometry were compared with those estimated by MRS. Results Edited MRS provided 100% specificity and 100% sensitivity in the detection of 2HG. The 2HG levels estimated by this technique were in line with those derived from tissue samples. Optimized PRESS provided lower performance, in agreement with previous findings. Conclusions Our results suggest that edited MRS is one of the most reliable tools to predict IDH mutation noninvasively, showing high sensitivity and specificity for 2HG detection. Integrating edited MRS in clinical practice may be highly beneficial for noninvasive diagnosis of glioma, prognostic assessment, and treatment planning.