The EPISTOP project consists of eight work packages described below.
WP1. Project and database management
Leader: The Children’s Memorial Health Institute, Warsaw, Poland – professor Sergiusz Jóźwiak
This large and complex project will be conducted by an international multidisciplinary consortium, including nine clinical sites, genetic laboratory, two complementary neuroscience laboratories, and two innovative SMEs. The consortium is comprised of 14 partners from 9 states (8 EC Member States: Poland, Germany, Italy, France, Czech Republic, Netherlands, Belgium, Austria, and USA). Therefore, timely and efficient coordination and daily management of the project is crucial for its development.
The objective of WP1 is to:
- coordinate the work of the consortium with respect to project deadlines, costs and objectives
- to ensure the constant monitoring and reporting of the development of the project
- to assure the flow of communication between the partners and European Commission
- to monitor the gender aspects of the project
- to monitor the ethical issues of the project
- to set up and manage the database for all the clinical and laboratory data obtained during the project by the partners
- to manage of risk and to implement the appropriate contingency plan
WP2. EEG tracking of epilepsy and epileptogenesis in TSC patients
Leader: The University Hospital of the University of Leuven, Belgium - Lieven Lagae, MD, PhD
The main objective of WP2 is to define strict criteria for the interpretation of consecutive EEGs in patients diagnosed with TSC and especially to detect EEG epileptic abnormalities. This will allow all participating centers to enter patients in the randomized prospective trial (WP6). It is well established that interictal epileptic abnormalities (IEA) can be considered as biomarkers for an ongoing epileptogenic process and that these precede the clinical symptoms (seizures) in most cases. More specifically, the presence of spikes and spike-waves patterns on the EEG is highly associated with epileptic changes in the brain. Identifying IEA in the longitudinal EEG follow up in children with TSC will show us the dynamics of the epileptogenic process in TSC and is one of the primary entry criteria into the prospective trial. Therefore, for each child with a proven diagnosis of TSC, video-EEGs will be performed at regular intervals, following a standardized technical protocol (nr electrodes, minimum duration, etc) and scored according to a predetermined accepted interpretation protocol. This will show us to see at what point a possible epileptogenic process in TSC becomes visible on the EEG and to correlate this with the biochemical, molecular and neuroimaging biomarkers (WP 3 and 4). We will define in great detail how to score the IEA, and whenever these become visible on the EEG, the child will enter the study and be randomized to standard clinical follow up or preventative anti-epileptic treatment (vigabatrin). One of the challenges is to critically define IEA so that these can be scored in the same way in every participating center. It is established that when the epileptogenic process becomes more active, repetitive spikes, multifocal spike and spike waves will appear. We can also expect to see continuous epileptic discharges and even subclinical seizures or the development of the typical hypsarrhythmic pattern. Therefore, our EEG scoring system will include both focal criteria as well as temporal criteria. Since we will follow the EEG evolution for 24 months, it will be possible to see the EEG effect of standard care (epileptic treatment) versus anti-epileptogenic treatment. Not only the evolution of the epileptic abnormalities will be followed, but also the maturation of the background activity. Background activity and reactivity is associated with cognitive development and our EEG follow up data will therefore be matched with the neurodevelopmental outcome measurements in the prospective clinical trial. Finally, we hope that these careful prospective EEG assessments will allow us to determine, also at the EEG level, positive or negative predictive factors for epilepsy and cognitive development in TSC.
WP3. Identification of molecular biomarkers of epilepsy risk and epileptogenesis in TSC patients
Leader: Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, U. S. - David J. Kwiatkowski, MD, PhD
To perform the following studies on subjects enrolled in the EPISTOP trial, all with the aim to identify biomarkers of epileptogenesis and epilepsy in TSC patients:
- Expression (RNA) profiling of serial blood samples by RNA-Seq, with confirmation by qRT-PCR
- Proteomic profiling of serial serum samples by mass spectroscopy, followed by Western blot or immunoassay confirmation
- miRNA profiling of serial serum samples
- metabolomic profiling of serial serum samples
- Whole Genome Sequencing (WGS) of peripheral blood DNA
- Deep (> 1000x read depth) next gen sequencing (NGS) of TSC1 and TSC2 to identify all mutations including large deletions, rearrangements, and mosaicism on peripheral blood DNA
WP4. Neuroimaging findings as biomarkers of epilepsy risk and epileptogenesis in TSC patients
Leader: The University Medical Center Utrecht, The Netherlands - F.E. Jansen, MD, PhD
To deliver the neuroimaging biomarkers of epileptogenesis in patients with TSC. Routine epilepsy protocols allow for morphologic evaluation and detection of cerebral anomalies typical in TSC such as tubers, heterotopia, transmantle signs, blurring of gray and white matter junction. The majority of the brain lesions are not considered to change over time, but tubers have been suggested dynamic lesions. Further, with maturation of the immature brain structural abnormalities in TSC may become more prominent. Serial MRI data is reported seldom in TSC. The present study will thus be unique in analyzing g changes in MRI characteristics over time and relate these findings to the electroclinical course of the disease. It is generally believed that conventional MRI does not differentiate between epileptogenic and non-epileptogenic tubers, although certain tuber characteristics such as size, cystic change, calcifications, associated features of focal cortical dysplasia (FCD) have been suggested as potential epileptogenicity markers. Previously it was found that patients with a greater tuber to brain volume proportion (TBP) were younger when they had their first seizure, had a lower intelligence, and tended to have a lower cognition index. TBP was found to be a better marker than the number of tubers for seizures and cognitive functioning in patients with TSC. However, a large TBP was found neither necessary nor sufficient for early seizure onset or cognitive impairment. The drawbacks of conventional imaging techniques described above warrant a novel approach to attempt to identify MRI biomarkers of epileptogenicity and neurodevelopment in TSC. Promising advanced MR imaging techniques include arterial spin-labeling (ASL) perfusion MRI, and diffusion tensor imaging (DTI). ASL is a technique that allows for quantitative measurements of cerebral blood flow (CBF) in a non-invasive fashion, without the need for intravenously administered contrast agents. Recent studies correlated the perfusion values with electroclinical findings and found that presence of hyperperfused cortical tubers was associated with increased seizure frequency. DTI is a powerful imaging tool to demonstrate the microstructural organization of white matter and allows for the detection of abnormalities of the brain tissue in earlier stages than conventional T1- and T2- weighted images. Studies on DTI characteristics of normal-appearing white matter (NAWM) in patients with TSC, showed an increase in apparent diffusion coefficient (ADC) and radial diffusivity (RD) and a decrease in fractional anisotropy (FA), suggesting white matter abnormalities in TSC patients. With respect to epileptogenicity, diffusivity measures and visualization of tracts may provide complementary information on white matter changes accompanying tubers and may assist to explain ictal spread patterns.
The specific objectives for this work package are:
- to relate quantified MRI lesions to epilepsy characteristics and to neurodevelopment
- to study the effect of recurrent seizures in early immature brain to changes in microstructural organization and cerebral blood flow
- to identify MRI related risk factors of epileptogenesis and mental retardation
WP5. Validation of biomarkers of epilepsy and molecular targets for novel therapies in human TSC specimens
Leader: Academisch Medisch Centrum, University of Amsterdam, The Netherlands – professor Eleonora Aronica
The objective of this work package is to investigate the biomarkers of epilepsy and molecular targets for novel therapies and to validate the peripheral epilepsy biomarkers in human TSC brain tissue. WP5 is designed to validate early diagnostic biomarkers of epileptogenesis and molecular targets for antiepileptogenesis therapy. This will be investigated in human specimens from well-characterized TSC patients during epilepsy development, as well as in patients with established epilepsy. We will use brain tissue obtained at surgery or autopsy from TSC patients. In order to further validate our findings we will study also the cortical specimens from patients operated on for intractable epilepsy related to known dysregulation of mTOR pathway. To study the function of the identified molecular targets, overexpression and loss of function studies will be performed in vitro. Data obtained in WP5 represent a major prerequisite for the accomplishment of the validation of biomarkers of epileptogenesis and may contribute to develop novel therapeutic approaches.
WP6. Prospective clinical study of epileptogenesis and randomized pre-seizure antiepileptic treatment in TSC patients
Leader: The Children’s Memorial Health Institute, Warsaw, Poland – professor Katarzyna Kotulska
WP6 is devoted to the multicentre, prospective clinical investigation of epileptogenesis in TSC infants, including a randomized, double-blind clinical study of the treatment of preclinical vs. clinical seizures.
This is the pivotal work package of EPSITOP. The medical data and biological material from patients to be analyzed in WP2, WP4, WP5,WP6, and WP7 will be collected during this study.
Rational for the sample size: The estimated number of patients enrolled during the first 30 months of the project is 100. Recently, increasing number of TSC infants is diagnosed before birth due to the presence of cardiac tumors on prenatal ultrasound. Given that all the clinical partners have long-term registers of TSC patients and concerning the annual number of such patients diagnosed (for example 12-15 infants at coordinating site) the sample size is rational.
The G-power analysis, based on available data and previous statistical analysis revealed that the planned number of patients should be sufficient to obtain the scientific meaningful results. The G-Power analysis was done using GPower 3.1 software with two assumptions: alpha level 0.05, and G-Power=80%.
Key inclusion criteria: Infants with the diagnosis of TSC made in the first 4 months of life and before the onset of clinical seizures.
Key exclusion criteria: Clinical seizures at baseline
Study synopsis: The detailed medical history of the child and family will be collected at baseline.
Each subject will be followed with video-EEG (vEEG) starting from baseline until 24 month of age (WP2).
The local electroencephalographer will be blinded to the clinical data. The EEG reports will be sent to the central EEG coordinator (WP2). Children with no epileptiform discharges on vEEG and no seizures will not receive any antiepileptic treatment. Children with clinical seizures, either noticed by caregivers or visible on vEEG will be given standard, recommended treatment for TSC patients with epilepsy which is vigabatrin in standard doses. Children with epileptiform discharges on vEEG prior to clinical seizures will enter the randomized phase of the study. They will be randomized (1:1) by central EEG coordinator (WP2) into two groups: group A- those who will receive vigabatrin and group B- those who will not receive treatment unless having clinical seizures. Neither the treating neurologist, nor the patient’s caregivers will not be aware of the result of EEG and if the patient is given preventative or standard therapy (double-blind trial).
The neuropsychological examination will be performed every six months (WP7). MRI will be performed at baseline, at the end of follow-up, and whenever clinically indicated (WP4).
After 24 months of life, in infants who did not present seizures on follow-up, the treatment will be gradually discontinued. Children with seizures will continue standard epilepsy management.
Molecular biomarkers of epileptogenesis will be determined at baseline, at the onset of epileptiform discharges on EEG or at the age of 6 months in children who do not develop EEG abnormalities, at the onset of clinical seizures, and at the end of follow-up (24th month of life). Forty age-matched children, who will undergo routine diagnostic blood sampling and MRI due to non-epilepsy reasons, will be enrolled to the control group (WP3). The only project related procedure in the control group is single blood sampling.
WP7. Identification of the prognostic factors of neurodevelopmental outcome in TSC patients and possible treatment strategies
Leader: Tor Vergata University Hospital, Rome, Italy – professor Paolo Curatolo
The objective of WP7 is to identify the biomarkers of epilepsy comorbidities: autism and neurodevelopmental delay. Intellectual disabilities and autism spectrum disorders are frequent comorbidities in TSC children with epilepsy. However, there are no clinical or molecular biomarkers to discriminate the patient who will or will not develop autism and cognitive impairment. Nowadays, more and more TSC patients are diagnosed prenatally or soon after birth, enabling serial observation before the onset of epilepsy. Early antiepileptic treatment immediately after seizure onset or at the first appearance of EEG abnormalities could modify the outcome of the disease. Such approaches resulted in significant reduction of clinical seizures, of the risk of drug-resistant epilepsy and relevant improvement in neurodevelopmental outcome. Identification of the risk factors and the biomarkers will allow the implementation of preventive therapeutic strategies to lower the incidence of neuropsychiatric comorbidities of childhood epilepsy, including autism and neurodevelopmental delay in infants and young children, in order to give clinical management recommendations for patients at high risk of developing cognitive impairment and autism. Neuropsychological examinations results will be integrated with the relevant clinical data (WP1, WP2), neuroimaging findings (WP4), to help define pathogenetic mechanisms of cognitive impairment and autism and provide insights for early diagnosis and early intervention (WP6).
WP8. Exploitation and Dissemination
WP leader: Vrije Universiteit Brussel, Belgium - Anna Jansen, MD, PhD
This WP is dedicated to the dissemination and exploitation of the project’s results, including IPR management. It will be carried out during the whole lifecycle of the project. The specific objectives of these WP are:
- Dissemination of information about the project, its objectives and results at the:
- Consortium level: effective exchange of information between the project partners to guarantee project progression
- Scientific/Technical level: Dissemination of research results to healthcare professionals, researchers, and industry for validation (international peer reviewed journals, conferences.
- End User Community: Dissemination to the TSC and epilepsy communities
- Public level: Dissemination to public authorities, the wider community and general society
- Exploitation of the results to develop new diagnostic and therapeutic strategies for individuals with epilepsy and to develop further research on epilepsy and epileptogenesis
- Management of intellectual property