Final Report Summary - EPISTOP (Long-term, prospective study evaluating clinical and molecular biomarkers of epileptogenesis in a genetic model of epilepsy – tuberous sclerosis complex)
Executive Summary:
Epilepsy affects 1% of the world’s population. In Europe, 6 million people have epilepsy (World Health Organization, 2010). Despite great progress in the management of epilepsy and increasing numbers of antiepileptic drugs, 30-40% of epilepsy patients are refractory to all available medications and many suffer from epilepsy-related comorbidities (Jensen, 2011).
The EPISTOP project had two major objectives: a clinical trial objective, focused on epilepsy prevention study in infants with Tuberous Sclerosis Complex (TSC); and a laboratory objective, the identification of biomarkers of epilepsy development and its neurodevelopmental comorbidities. EPISTOP was carried out by a multidisciplinary consortium of 10 clinical sites, 5 laboratories, and 1 administrative partner. It was the first prospective study of epileptogenesis in humans and included a unique clinical trial comparing epilepsy and neurodevelopmental outcome in patients receiving either standard or preventive antiepileptic treatment.
The clinical component of EPISTOP has provided first evidence that EEG monitoring of infants at high epilepsy risk and introduction of antiepileptic treatment before the onset of seizures prevents or delays seizures and reduces the risk of drug-resistant epilepsy. It also reduces the risk of infantile spasms and seizure burden measured as the number of days with seizures, indicating that preventive treatment reduces epilepsy severity in TSC children.
One-hundred-one TSC infants participated in the study, and were the primary beneficiaries of the project. TSC affects 1 in 6,000 newborns, 70% of them develop epilepsy within the first year of life, and our project showed that EEG monitoring and early epilepsy management is feasible in the majority of TSC infants (Recommendations: Curatolo et al. 2018). If future studies confirm the benefit of preventive strategies in other types of epilepsy, then early monitoring and treatment might be recommended in as many as 40% of patients with epilepsy, potentially having a major impact in reducing complications and social stigma associated with epilepsy.
The laboratory component of EPISTOP included analysis of a wide range of EEG, neuroimaging, neuropsychological, and molecular data, to identify risk factors and biomarkers of the development of epilepsy and its comorbidities. EPISTOP investigators developed an EEG scoring system, that also may be validated for other types of epilepsy, and used more broadly to enable early preventive treatment or other disease modification strategies.
Project Context and Objectives:
In Europe, 6 million people have epilepsy (World Health Organization, 2010). Despite great progress in the management of epilepsy and increasing numbers of antiepileptic drugs, 30-40% of epilepsy patients are refractory to all available medications and many suffer from epilepsy-related comorbidities (Jensen, 2011). Given these data and the leading position of European epileptology centres, the Consensus Document of European Brain Research in 2011 recommended research focused on preventing epilepsy development to be supported by European funding (Di Luca, 2011).
In more than 65% of patients, epilepsy begins in childhood and the incidence of epilepsy is highest in the first year of life (Hauser, 1993). In children, the problem of epilepsy is far beyond seizures, as about 50% of children with epilepsy suffer from psychiatric and behavioural comorbidities, including developmental delay, learning disabilities, and autism spectrum disorder (Ono, 2012). Furthermore, early onset of seizures is regarded as one of the major risk factors for development of drug-resistant epilepsy. Epilepsy differs from many other neurological conditions due to its extreme heterogeneity in aetiologies and phenotypes. Thus, studies of epilepsy risk factors might be uniquely enabled by analyses in young children where the influence of the external, environmental factors is lower than in adults. Tuberous Sclerosis Complex (TSC) is a genetically determined neurocutaneous syndrome affecting 1 child in 6,000 (Curatolo, 2008). TSC is often considered an excellent clinical model of severe focal epilepsy, as 70 to 90% of patients are affected by epilepsy and in most cases the seizures are drug-resistant. In the majority of patients epilepsy manifests in the first months of life and half of patients develop cognitive impairment, autism spectrum disorder or other neurodevelopmental disturbances (Jozwiak, 1998). Therefore TSC is an excellent model for both focal epilepsy and infantile spasms. Although there is definite clinical heterogeneity, TSC represents a relatively homogenous group of patients for the studies of epileptogenesis, who are at high risk of this disease. Recent studies (Jozwiak, 2011) demonstrated that in infants with paroxysmal discharges on EEG it is possible to modify the outcome of the disease by antiepileptic treatment before the onset of clinical seizures. EPISTOP model gives us a unique opportunity to investigate whether clinically successful epilepsy prevention is possible, and to explore the molecular mechanisms of this effect. EPISTOP is aimed to better understand the complex pathophysiology of epilepsy in order to develop novel preventative strategies in at risk patients, improve diagnostic methods and develop more effective therapeutic strategies. To achieve this aim, the risk factors and biomarkers of epilepsy were identified by a multidisciplinary, systematic approach in three settings:
- a prospective study of epilepsy development in infants with TSC, using a wide range of clinical, neuroimaging, and genetic analyses
- prospective clinical study of TSC infants treated with antiepileptic drugs at the onset of subclinical seizures in comparison to children treated only after clinical seizures appear
- analysis of biomarkers of epileptogenesis and drug-resistant epilepsy in epileptogenic brain specimens obtained from patients with TSC.
EPISTOP project is consisted of eight Work Packages (WP). EPISTOP is divided into two parts: clinical study (WP2, WP4, WP6, WP7) and molecular study (WP3 and WP5). Additionally two horizontal WPs (WP1 and WP8) were established. Each WP was devoted for specified tasks which were related to overall study objectives. The consortium is consisted of 16 partners, all partners were involved in the activities of various WPs. Partners participating in the study came from 10 countries from 3 continents (Europe, USA, Australia). IPCZD was a leader of the project as well as leader of WP6. Samples from nine clinical sites (P1-IPCZD, P2-TVG, P3-INS, P4-VUB, P5-UHM, P6-CUB, P7-UMC, P10-MUW, P16-LCCH) have been sent to, and are stored at the Jaworski laboratory at IIMCB (P11-IIMCB). IIMCB has distributed multiple sets of samples to all laboratories participating in this work project, including: the laboratory of Dr. Kwiatkowski at the BWH (P12-BWH), the laboratory of Dr. Aronica at AMC (P8-AMC), Dr. Lehmann at the Proteome Factory (P13-PFA), and Dr. Janssen at GenomeScan (P14-SXS/GS). Computational and statistical analyses were performed by Transition Technologies S.A. Consortium was also supported by Scientific Advisory Board and Ethics Board members. Scientific Advisory Board included experts in clinical and experimental epileptology and molecular biology, as well as the biostatistician (prof. Annamaria Vezzani, prof. Carl Stafstrom, prof. Leszek Kaczmarek, prof. Renzo Guerrini, dr Marcin Balcerzak). Ethics Board included both medical professionals and representatives of patients organizations (prof. Katarzyna Łukasiuk, prof. Steve Roach, prof. Raili Riikonen, Andrzej Chaberka).
The objective of WP2 was to determine electroencephalographic biomarkers of epileptogenesis, and to randomize patients into group A and B (WP6). All the patients underwent videoEEG recording every 4 weeks in the children under the age of 6 months, then every 6 weeks in children under 12 months, and every 8 weeks thereafter till the last follow-up visit at the age of 24 months. In WP2, an EPISTOP scoring system for EEG changes in TSC infants has been elaborated and used to identify patients at high risk of seizure. This system was also used to determine the randomization timepoint.
In the analysis phase, the systematic database was used to analyze the actual EEGs in detail. It was found that EEG monitoring performed before seizures significantly improves the prognosis of psychomotor development, probably influencing the early diagnosis of clinical and electroencephalographic seizures. EEG monitored children are developing significantly better than any previously published group of children with TSC. WP3 was aimed to identify the clinically useful blood biomarkers of epileptogenesis and risk factors for epilepsy. In all EPISTOP participants, blood samples for molecular biomarkers studies were collected: 1/ at entry to the project, 2/at appearance of paroxysmal activity on videoEEG, 3/ at onset of clinical seizures, 4/ at the age of 24 months. The genetic, epigenetic, transcriptome, proteome, metabolomic and immunological parameters were being compared in children with and without epilepsy as well as between the set time points in children with epilepsy or epileptiform discharges on EEG and patients with drug-resistant epilepsy were being analyzed. All data were subject to statistical analysis. The objective of WP4 was to deliver the neuroimaging biomarkers of epileptogenesis. MRI is a standard procedure in epilepsy and TSC patients. In our project, MRI was performed at baseline and at the end of follow-up and whenever indicated clinically. Apart from standard MRI, DTI and DWI were performed in order to identify neuroimaging biomarkers of epilepsy, autism, and neurodevelopmental delay susceptibility. It was found that brain lesions identified in prenatal MRI correlate with epilepsy and neurodevelopmental outcome in TSC children. Neuroimaging findings were included in a set of clinical and molecular epilepsy biomarkers from the blood samples obtained during the study.
WP5 aimed at discovery and validation the epileptogenesis and drug-resistance biomarkers in brain samples from TSC patients who underwent epilepsy surgery and TSC autopsy cases. The objective of WP5 was to investigate the epileptogenesis and drug-resistance biomarkers in epileptogenic brain samples, in order to validate the findings from blood analysis and to discover brain-specific biomarkers. In the center of the consortium was WP6 proposing multicenter, prospective clinical study assessing epileptogenesis development in infants with TSC. The number of patients enrolled in the project is 101. All children were followed clinically and with serial videoEEG until 24 month of age. The first phase of the study rested on the observation of patients until subclinical seizures appear on videoEEG. Patients with epileptiform discharges on videoEEG prior to clinical seizures entered the randomized phase of the study. They were randomized into two groups: group A - those who receives vigabatrin for subclinical seizures; and B - those who were treated only after the onset of clinical seizures. Patients without clinical and subclinical seizures were followed up without any medication. Patients with clinical seizures were being given standard treatment with vigabatrin. Preventive treatment was implemented after the onset of epileptiform discharges on videoEEG, but before clinical or subclinical seizures. Conventional treatment included treatment after clinical or subclinical seizures. Thus, EPISTOP included two medical interventions: videoEEG monitoring, which applied to all TSC children in the project, and preventive treatment which was compared to conventional one in the settings of clinical trial.
VideoEEG monitoring enabled implementation of antiepileptic treatment earlier than in any previously reported TSC cohort. It is well established that delayed treatment is associated with significantly worse neuropsychological outcome of epilepsy in TSC infants. The overall neuropsychological outcome in children participating in EPISTOP was better than in any previously reported TSC cohort, likely due to early recognition of seizures and implementation of antiepileptic treatment. Preventive treatment resulted in a significantly longer time to first seizure when compared to conventional therapy. Children who received preventive treatment had also lower risk of clinical seizures, drug-resistant epilepsy and infantile spasms. Conducted analysis also confirmed beneficial effect of preventive treatment on reduction in number of days with epileptic seizures in children with TSC and epilepsy. In children receiving preventive treatment lower number of anti-epileptic drugs necessary to use has been observed. WP7 aimed to identify the clinical (WP2, WP4) and molecular (WP3, WP5) biomarkers of epilepsy co-morbidities: autism and neurodevelopmental delay. Neurodevelopmental assessment was being performed in every child at baseline, and then at 6, 12 and 18 months of age. Follow-up examination was done at the age of 24 months. The changes in biomarkers between the groups with and without autistic features, as well as with and without neurodevelopmental delay have been studied and analyzed. Clinical prognostic factors of neurodevelopmental outcome and early clinical markers of Autism Spectrum Disorders and developmental delay have been identified. With regards to neurodevelopmental outcome, the combined use of BSID (Bayley Scales of Infant Developments) and ADOS (Autism Diagnostic Observation Schedule) can reliably identify TSC infants with a higher risk for ASD at age 6-12 months. Therefore, all children with a diagnosis of TSC should undergo neurodevelopmental evaluations every 6 months of life. In children with BSID developmental quotients at 6 months below the normal range, an early and intensive behavioral treatment should be started. Similarly, children with abnormal ADOS results at 12 months should be warranted a prompt referral to intensive behavioral interventions. Further investigations in this field are warranted, in order to obtain wider sample and a longer follow-up to be able to draw international clinical recommendations. WP8 generated an integrative dissemination strategy, created the dedicated internet platform, prepared educational materials on TSC for various stakeholders, elaborated an exploitation plan and expedited measures to promote teaching in TSC. Tasks within WP8 were focused on EPISTOP project promotion and dissemination.
Project Results:
Clinical study results
The clinical part of EPISTOP was composed of two arms: a randomized clinical trial and an open-label observational study. It was carried out from March 2014 to October 2018 in 9 sites in Europe and 1 site in Australia. The inclusion criteria were as follows: age ≤ 4 months, definite TSC according to the Roach criteria or with genetic confirmation of the disease, no seizures reported by caregivers and no clinical or subclinical seizures on baseline video EEG. Exclusion criteria were: any epileptic seizure, antiepileptic treatment, contraindications to magnetic resonance imaging, and any condition considered by the investigator to hinder participation in the study. The study was approved by local Ethics Committees at all study sites, and caregivers of all participants signed informed consent before enrolment. The study adhered to the International Conference on Harmonization Guidelines for Good Clinical Practice and was conducted in accordance with the Declaration of Helsinki.
At enrollment, all patients underwent magnetic resonance imaging (MRI) of the brain to assess for TSC-related brain lesions. From enrollment to study completion at the age of 24 months, patients attended follow-up visits at a frequency recommended by the European guidelines for children with TSC: every 4 weeks in children ≤ 6months; every 6 weeks in children ≤ 12 months; and every 8 weeks in children > 12 months. At enrollment and each follow-up visit, video EEGs was performed and data on the occurrence, frequency, and drug-resistance of epileptic seizures were gathered. Epileptic seizures were captured on video EEGs or reported by patients’ parents in seizure diaries. At enrollment and every six months thereafter, patients were assessed by a neuropsychologist on the following scales: Bayley Scales of Infant Development (BSID), to assess cognitive development, and on the Autistic Diagnostic Observation Schedule (ADOS), to assess for autism.
Video EEGs were recorded for ≥ 1 hour and included periods of both wakefulness and sleep (up to sleep stage 2); intermittent photic stimulation with ≥ 4 frequencies and eyes open and closed was applied to assess background rhythm reactivity. Video EEGs were assessed for pre-seizure epileptiform activity, subclinical epileptic seizures, and clinical epileptic seizures. Pre-seizure epileptiform activity was defined as multifocal (≥ 2 areas involving both brain hemispheres) epileptiform activity that occurred for ≥ 1% of recording time or as generalized epileptiform activity, including hypsarrhythmia. Subclinical seizures were defined as ictal EEG activity with no video correlate. Video EEGs were assessed independently by two investigators: a local and a central one.
Patients received either preventive or conservative antiepileptic treatment. Preventive treatment was defined as treatment started after pre-seizure epileptiform activity and before the first subclinical or clinical seizure. Conservative treatment was defined as treatment started after subclinical seizures or clinical seizures (see Fig. 1).
Both preventive and conservative treatments were started with vigabatrin and then modified according to clinical judgment. Patients were allocated to preventive or conservative treatment in two study arms: a randomized arm and an observational arm.
In the randomized arm, 6 sites randomly allocated patients to either preventive or conservative treatment in a 1:1 ratio. Eligible for randomization were only patients with pre-seizure epileptiform activity observed before the first subclinical or clinical seizure and recognized by both the local and central EEG readers. Patients with clinical or subclinical seizures were excluded, and patients with normal EEGs were followed-up until study completion. The central investigator (LL) randomly allocated eligible patients to either preventive or conservative treatment. Randomization was stratified for study sites. The treatment was always started after subclinical or clinical seizures, but these patients were excluded from the randomized arm and transferred to the observational arm. In the randomized arm, the treating physician was blinded to EEG data and thus did not know whether the treatment was randomly allocated after pre-seizure epileptiform activity or started after subclinical seizures. In the observational arm, 4 sites started antiepileptic treatment according to their routine practice: in two sites all patients received conservative treatment, and in another two sites all patients received preventive treatment. In the observational arm, the criteria for preventive treatment were the same as the criteria for randomization in the randomized arm. In the observational arm, we also included patients whose parents did not agree to random treatment allocation. All these patients received preventive treatment, because all sites that assigned treatment randomly used the preventive treatment as their routine practice. Moreover, patients who were not eligible for randomization because of prior subclinical or clinical seizures were also included in the observational arm as patients who received conservative treatment. The observational arm was open, and the treating physician had access to EEG data. In both study arms, we excluded patients whose EEGs were interpreted as displaying pre-seizure epileptiform activity only by one of the two readers, i.e. the central or local reader. These patients, with discordant readings, were excluded from the study.
The primary end point was the time from birth to the first clinical seizure. The secondary end points were assess at the 24th month of life and included:
• proportion of patients with clinical seizures
• proportion of patients with drug resistant epilepsy. Resistance to antiepileptic drugs was defined as failure of adequate trials of two tolerated, appropriately chosen and used antiepileptic drug schedules (whether as monotherapies or in combination) to achieve sustained seizure freedom.
• proportion of patients with infantile spasms
• proportion of patients with EEG abnormalities, defined as EEG without pre-seizure epileptiform activity and subclinical and clinical seizures.
• proportion of patients with hypsarrhythmia
• proportion of patients with autism (low autism risk on ADOS)
• proportions of patients with impaired cognitive function (BSID cognitive score < 70).
The safety of patients in the study was monitored by an independent ethics board supported by a biostatistician. Adverse events were recorded during follow-up visits.
Survival analysis with Kaplan-Meier curves and proportional hazards regression were used to study the primary end point among all patients who received at least one dose of preventive or conservative treatment. Survival curves were compared with the log-rank test. In both study arms, differences in the frequency of secondary end points between patients who completed the study on either preventive or conservative treatment were reported as odds rations, with the Fisher’s test of significance. In addition to separate analyses for the randomized and observational arms, we performed a pooled analysis that included both arms. Statistical significance was set at p < 0.05; two-tailed for the primary end points and one-tailed for the secondary end points. All calculations were completed in the R software (version 3.5.2) with the packages tidyverse, survival, and survminer.
Of 101 patients assessed for eligibility, 4 were excluded due to misdiagnosis (Fig. 2).
Of 97 patients enrolled, 54 were included in the randomized arm and 43 in the observational arm. In the randomized arm, 21 patients were not eligible for randomization: 9 had clinical and 6 had subclinical seizures before pre-seizure epileptiform changes, 3 had normal EEG throughout the study, and 3 dropped out before randomization. In 6 patients the central and local analyses of EEG were discordant. Of 27 patients eligible for randomization, 13 were randomly allocated to preventive treatment and 14 to conservative treatment. Of 14 patients randomly allocated to conservative treatment, 1 patient dropped out.
Of 43 patients in the observational arm, 22 patients were recruited in sites with routine preventive treatment and 21 patients in sites with routine conservative treatment. Of the 43 patients, 4 did not need treatment and 7 dropped out. Twelve patients received preventive treatment and 23 patients received conservative treatment; additional 12 patients were transferred to the observational arm from the randomized arm. These 12 patients were not eligible for randomization due to prior subclinical or clinical seizures and thus received conservative treatment (Fig. 2). Baseline clinical and anthropometric characteristics did not differ significantly in both study arms between the patients who received preventive or conservative treatment.
In both study arms, patients who received preventive treatment were about 3 times more likely to remain seizure-free over the study period than the patients who received conservative treatment (randomized arm: hazard ratio, 3.19; 95% CI, 1.22 to 8.32; P=0.013; observational arm: hazard ratio, 2.89; 95% CI, 1.19 to 7.00; P=0.014; Fig. 3). In the randomized arm, the median time to the first seizure was more than twice as long with preventive than with conservative treatment (297 vs. 124 days). In the observational arm, the median time to the first seizure was more than 5 times longer with preventive than with conservative treatment (397 vs. 73 days). At 24 months, in both study arms, patients who received preventive treatment were less likely to have experienced a clinical seizure than the patients who received conservative treatment (odds ratio [OR, 95% CI]; randomized arm: 0.23 [0.02 1.74]; observational arm: 0.35 [0.07 1.7)]) but this difference did not reach statistical significance (P≥0.158,). However, in the pooled analysis, the reduction of the risk of clinical seizures was significant with preventive treatment (OR, 0.33 95% CI, 0.1 1.03 P=0.036).
At 24 months, the risk of drug-resistant epilepsy was lower with preventive than conservative treatment in both study arms (OR [95% CI]; randomized arm: 0.15 [0.02 0.98] p = 0.047; observational arm: 0.29 [0.04 1.42] p = 0.103). The risk of drug-resistant epilepsy was significantly lower with preventive than conservative treatment in the pooled analysis (OR, 0.26; 95% CI, 0.08 0.8; p = 0.013 Appendix 1).
In both study arms, none of the patients who received preventive treatment developed infantile spasms compared to 4 (31%) and 9 (26%) patients on conservative treatment in the randomized and observational arms, respectively (Table 1). In the pooled analysis, preventive treatment was associated with a significantly reduced risk of infantile spams (OR, 0; 95% CI, 0, 0.52; p = 0.003).
The risks of abnormal EEG, hypsarrhythmia, autism, cognitive impairment, language impairment, motor impairment, socio-emotional impairment, and impairment of adaptive behavior were similar with preventive and conservative treatments in both study arms, including in the pooled analysis.
The rates of both all adverse events and serious adverse events were lower with preventive than conservative treatment. There was one death, which was due to cardiac arrest during epilepsy surgery. Compared to children on conservative treatment, children on preventive treatment had lower rates of status epilepticus, febrile seizure, and epilepsy surgery. The rates of other adverse events were similar with preventive and conservative treatment. None of the drugs used in this study were investigational products. The adverse events observed were typical for the antiepileptic drugs used. There were no adverse events related to the preventive treatment.
In conclusion, our findings confirmed that active monitoring with video EEG to start antiepileptic treatment early improves epilepsy-related outcomes in children with TSC. Moreover, preventive treatment, started at the onset of pre-seizure activity, offers an additional benefit.
Molecular study results performed in Work Package 3
The objectives of the molecular studies to be performed on EPISTOP subjects were the following, all with the aim to identify biomarkers of epileptogenesis and epilepsy in TSC patients:
1. Expression (RNA) profiling of serial blood samples by RNA-Seq, with confirmation by Q-RT-PCR.
2. Proteomic profiling of serial serum samples by mass spectroscopy, followed by Western blot or immunoassay confirmation.
3. miRNA profiling of serial serum samples.
4. metabolomic profiling of serial serum samples.
5. Whole Genome Sequencing (WGS) of peripheral blood DNA.
6. 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.
Background:
Tuberous sclerosis complex (TSC) is often a devastating neurodevelopmental syndrome in which seizures, intellectual deficit, autism, and a variety of neuropsychiatric conditions collectively termed TSC-associated neurologic disease (TAND) are seen in the majority of subjects. Seizures occur in over 75% of TSC children prior to the age of 2 years, and early onset and/or poorly controlled seizures are strongly associated with severity of TAND and poor cognitive outcome. Identification of which TSC infants are at risk of seizure development and at which time during their early development (age birth to 2 years) has a high priority, to enable early intervention with anti-epileptic medication to completely prevent or at least significantly modify the trajectory of seizure development.
The EPISTOP cohort of 101 TSC patients has been subjected to detailed molecular studies, as listed above. This detailed molecular analysis makes these TSC infants the most highly studied infants/young children in human history. As such it was our hope and hypothesis at the start of the EPISTOP trial that some combination of these molecular features could be used to predict seizure development in these TSC infants. Here we report the findings of this analysis for each of these molecular studies. We then follow that individual analysis with an integrated analysis of all available data for seizure prediction.
TSC1/TSC2 mutation analysis (item 6 on the list above)
TSC is due to inactivating mutations occurring in either TSC1 or TSC2. Previously it has been known that TSC1 mutations in TSC are associated with a milder phenotype in general than TSC2 mutations. Furthermore, it is known that mosaic mutations in these genes are fairly common in TSC patients, being seen in about 10% overall, and are also associated with a milder clinical phenotype.
One hundred one (101) subjects were enrolled into EPISTOP. Clinical evaluation indicated that 5 of these subjects did not meet diagnostic crieria for TSC, and hence mutation analysis was performed on the remaining 96 EPISTOP subjects with TSC. We used deep massively parallel sequencing (MPS) methods that we have used previously and published to identify single nucleotide variants and small indels, and confirmed each mutation identified by a secondary analysis. Other experimental and computational methods were used to identify large genomic deletions occurring in either TSC1 or TSC2, and such large deletions were also validated by independent methods.
Mutations in TSC1 or TSC2 were identified in 93 of 96 (97%) subjects (Figure 4). 23 (25%) mutations were in TSC1 while 70 (75%) were in TSC2. Eight (of 93, 9%) subjects had mosaic mutations, at allele frequencies ranging from 0.7% to 18%. The types of mutation seen were the following: 8 (9%) insertions, 17 (18%) deletions, 17 (18%) splice, 21 (23%) missense, 21 (23%) nonsense, 7 (7%) large deletions, and 2 (2%) in-frame deletions.
Very few cohorts of infant TSC patients have been analyzed comprehensively for mutations, as we have done here. Our findings of mutations in 97% of subjects is clearly a very high mutation detection rate. It was surprising that 3 subjects had mosaic mutations present at allele frequencies < 5% (0.7%, 3.0%, 4.4%), and yet still met diagnostic criteria with multi-system involvement by age 3 months. This likely reflects that the mosaic allele frequency is different in different tissues and organ systems. The lack of a mutation in 3 subjects is consistent with the possibility (which we have reported in other patients) that these individuals are mosaic with no representation of the mutation in blood DNA.
None of 3 (0%) NMI, 3 of 8 (38%) mosaic, and 56 of 85 (66%) of those with heterozygous TSC1 or TSC2 mutations developed seizures before age 2 years (p=0.0024) indicating that being NMI or mosaic was strongly associated with a reduced risk of seizure development.
Whole Genome Sequencing (WGS) of EPISTOP subjects (item 5 on the list above)
WGS in the EPISTOP cohort provides a wealth of data, including genotype analysis for the 5-10,000,000 common SNPs in the human genome. Although many correlations between SNP alleles and clinical features can be performed, they are vastly underpowered, and a replication population is not available. Consequently, we have chosen to focus our analysis on a panel of 85 SNPs that have been implicated in epilepsy GWAS as having associations with epilepsy risk. We have assessed the association between alleles of these SNPs and epilepsy onset in the EPISTOP cohort, and have identified 3 (of 75) SNPs that show evidence of an association (Table 2).
These findings will be validated in a separate TSC population.
RNA profiling of serial blood samples by RNA-Seq (item 1 on the list above)
Total blood RNA was isolated, globin mRNA depleted, mRNA was isolated by oligo-dT columns, and cDNA libraries prepared by standard methods. Sequencing was performed to a read depth of 36 million reads per sample. After standard processing of read data and alignment to the genome, differential expression analysis was performed using the R package DESeq2, to compare different subsets of samples from EPISTOP subjects at different time points. The primary comparison was made among subjects with different clinical outcomes, considering the RNA-Seq data available from the initial blood samples (V1, V1/VaEEG). Multiple genes were identified that were statistically significantly different between subjects that: had autism spectrum disorder (ASD) or did not at age 2 years; developed refractory epilepsy or did not by age 2 years; etc. In addition, comparison of data for samples obtained at the time of active seizures (VCS, VCSII) with age-matched controls also showed many differences with over 2,000 differentially expressed genes (Figure 5).
Proteomic profiling of serial serum samples (item 2 on the list above)
Serum proteome profiling was performed after depletion of the 14 most abundant proteins by affinity chromatography, followed by digestion with trypsin, and analysis of derived peptides using ultra high performance liquid chromatography (UHPLC; Ultimate 3000) over a 50cm reversed phase column (Dr. Maisch GmbH) online coupled to an Orbitrap mass spectrometer (Orbitrap XL). MaxQuant software was used for peptide identification and quantification. 544 protein groups (462 allowing only Oxidation) from 7405 peptides (6900) were identified.
73 different protein groups were identified by this analysis that varied in one or more comparisons among different subsets of the EPISTOP cohort by age. These protein groups all correlated with age of the EPISTOP subjects (Figure 6).
Beyond these major differences in serum protein levels according to age, there were relatively small differences detected in different clinical groups, e.g. those with seizures versus those without, among the serum protein levels. Nonetheless, some serum proteins may contribute to seizure prediction in the integrated analysis.
miRNA profiling of serial serum samples (item 3 on the list above)
PCR array analysis was performed using miRCURY LNA miRNA Custom PCR panels (Qiagen) on a Roche LightCycler 480 thermocycler to examine the expression of 45 miRNAs in all EPISTOP samples. These 45 miRNAs were chosen based on RNA-seq data performed on a subset of the serum samples, or based on their possible relevance to epilepsy, seizures or cognitive functioning from previous studies.
Seven miRNAs were differentially expressed (all higher) in the TSC subject V1 samples in comparison to controls of age 0-6 months (Figure 7). These miRNAs are candidate biomarkers of TSC which might be useful for diagnosis or monitoring in TSC infants. Within the TSC V1 group, we also compared miRNA values from subjects that later developed clinical seizures (CS) to those EPISTOP subjects who never had seizures (Figure 8). Four miRNAs were differentially expressed, that are candidate biomarkers for seizure development in TSC.
Metabolomic profiling of serial serum samples (item 4 on the list above)
Metabolomic profiling was performed on 274 EPISTOP serum samples and 33 control serum samples, split into seven 50-52 sample batches, on a SCIEX QTRAP 6500 LC-MS/MS mass spectrometry system in Boston, MA. Total Ion Current (TIC) values were determined for each metabolite, which were then scaled within each sample to the total TIC, and then compared among samples within each batch to calculate a normalized value using the median value for all samples in a batch. Eighteen metabolites were identified that differed according to age at P < 10-20, including myo-inositol, kynurenine, hydroxyproline, and phosphocreatine. Furthermore, six metabolites were significantly increased in EPISTOP subjects who were taking vigabatrin (Figure 9). One of these metabolites had been recognized previously to be affected but all the others are novel findings.
These metabolite data were also analyzed to search for metabolites that were different according to TSC status versus controls in an age-matched manner. Interestingly, several cysteine metabolites were somewhat lower in the TSC subject samples in comparison to controls, including cysteine, cystine, and cysteine sulfinate (Figure 10). All of these metabolites are potential biomarkers of TSC for use in diagnosis and prediction of seizure development.
Integrated Analysis of all molecular biomarkers for prediction of seizure risk
An integrated analysis of all data types was performed to generate a series of predictive classifiers of epilepsy risk in infants with TSC.
The following datasets were utilized on V1 samples from EPISTOP subjects:
1) Proteomic dataset - 384 features and 418 samples
2) miRNA dataset - 45 features and 384 samples
3) RNA-Seq data - 63677 features and 339 samples
4) Metabolite dataset - 250 features and 357 samples
5) miRNA sequencing dataset – 20,000 isomir features and 44 samples
6) TSC gene mutation status.
The entire dataset consisted of more than 80 thousand variables characterizing 97 subjects. Hence, there are a very large number of variables, and a relatively small number of patients with outcomes, a typical “curse of dimensionality” problem. To deal with this problem, only variables with an AUC > 0.64 for discrimination of epilepsy status were retained for building the classification models, while all others were discarded. In addition, we limited the number of features from each data type for consideration in the classification model to a maximum of 30, such that altogether 126 variables were considered for classification model development.
We performed logistic regression modelling to build the classification models, since it enables simple interpretation of results, and is relatively efficient from a computational perspective. We also reasoned that inclusion of more than 3 parameters in a model would lead to a greater chance of overfitting with generation of an inaccurate model based on chance alone. Thus, we fit regression models to all 3-way combinations of pre-selected variables, giving 333,501 possible models to evaluate.
Since our classifier was aimed at implementation in clinical practice, we focused on three performance measures for each classifier: Negative Predictive Value (NPV), the chance that classifier is correct when it predicts seizures; Positive Predictive Value (PPV), the chance that classifier is correct when it predicts no seizures; and the Mean Misclassification Error (MME), number of times when the classifier prediction is wrong divided by total number of subjects assessed. We used a strategy in which a model is fitted based on 2/3 of the data and then assessed on the remaining 1/3. Model fitting was done a hundred times for each combination of three markers.
We generated classifiers both with, and without, inclusion of the miRNA sequencing dataset. We considered classifiers without that data set because that analysis had been done on only 44 samples, limiting the sample set available for predictive modeling (Table 3).
Note that the predictors which include the isomiR data (lower set of 10 predictors) are based on a smaller number of samples (because miR sequencing was done on a smaller set of samples), and have corresponding better NPV and PPV on average. Classifiers done without the isomiR data have lower NPV and PPV values, but the larger sample size used for those classifiers may explain those findings.
Hence, this analysis has identified multiple potential classifiers of seizure risk in the EPISTOP cohort, which may have clinical utility for assessing seizure risk in TSC infants through age 2 years. However, since the number of samples analyzed is limited, there is a strong possibility of overfitting this data, so that assessment and validation in a separate population is desired.
Molecular study results performed in Work Package 5
The objectives of this WP were to investigate the biomarkers of epilepsy and molecular targets for novel therapies and validate the peripheral epilepsy biomarkers in human TSC brain tissue.
A systematic evaluation and characterization of specific clinico-pathological subtypes of TSC brain lesions has been performed (see Deliverable no: D5.1). A manuscript on the above reported finding has been published (Novel histopathological pattern in cortical tubers of epilepsy surgery patients with Tuberous Sclerosis, PLOS one, 11(6):e0157396 ; P8 /AMC, P7/UMC, P5/ UHM and P10/ MUW). A study focusing on the white matter pathology in pediatric epilepsy surgery specimens (quantitative histological analysis; P10/ MUW) was carried out and completed. Decreased content of oligodendroglia and missing precursor cells indicated insufficient oligodendroglial development, probably mediated by mTOR, which may ultimately lead to severe myelin loss. A manuscript on the above reported finding has been published (Impaired oligodendroglial turnover is associated with myelin pathology in Focal Cortical Dysplasia and Tuberous Sclerosis Complex. Brain Pathol, 2016; doi: 10.1111/bpa.12452 P8 /AMC and P10/ MUW). We evaluated the pattern of cortical layering disruption observed in brain tissue of TSC patients. A manuscript on the above reported finding has been published (Specific pattern of maturation and differentiation in the formation of cortical tubers in tuberous sclerosis complex (TSC): evidence from layer-specific marker expression., 2016. J Neurodev Disord Apr 1;8:9.; P8 /AMC, P7/UMC, P5/ UHM and P10/ MUW).
We performed a systematic evaluation on a large cohort of TSC brain lesions (n=42; n=9 perituberal cortex; n=17 controls). We analyzed the expression of NeuN, GFAP, pS6, vimentin, MBP, MOBP (myelin-associated oligodendrocyte basic protein), olig2, CD3, CD34, Cr3/43 (MHC-II), PLP and SMI32 separately for grey and white matter in each tuber compared to the perituberal samples and age-matched controls. Immunohistochemistry was digitalized and semi-automated histological quantified. These results were in a first step compared between groups (lesional – perilesional – control). As previously published there is an increase of inflammatory markers in grey and white matter as well as an overall reduction of neurons in the cortex. Furthermore, there is loss of myelin in the white matter. Interestingly, the number of oligodendroglial cells is reduced in the grey and white matter. MBP reflects the oligodendroglial count, nevertheless showing only a slightly reduced myelin content in the lesional cortex without showing significant differences in the amount of axons present (SMI31). Additionally, the results were correlated to TSC tuber subtype (as identified before, see publication above) and clinical parameters as seizure frequency, MRI data, surgery outcome, age at surgery, duration of epilepsy, drug load and more. The reduction of oligodendroglial cells in the grey matter seems to be a rather global phenomenon than comparable with the situation in the white matter (as previously published, see above). The number of oligodendroglia is negatively correlated with the amount of T-cells and microglia (corrected for age). The duration of epilepsy does not influence these findings. The early myelin protein MOBP suggests even dysmyelination and interestingly MOBP and the number oligodendroglial cells in white matter showed a negative correlation the presence of autism, whereas the content of MOBP and number oligodendroglial cells in grey matter showed with the presence of intellectual disability. In the last 6 months data analysis has been finalized and a manuscript is in preparation (A. Mühlebner EPISTOP-WP leaders et al. White matter abnormalities in TSC cortical tubers). We further elucidated the relationship between white matter pathology and inflammation utilizing the previously obtained mRNA sequencing data. Subsequently, we studied the relationship between micro-vascular density and cortical developmental abnormalities. In the last 6 months the data analysis has been finalized and the results indicate an increase in microvasculature in white matter of TSC cortical tubers compared to controls, which is linked to an inflammatory response (Abstract/poster Dutch Neuroscience 2017); a manuscript has been prepared and is presently under revision.
Evaluation of a number of molecular targets for anti-epileptogenic therapy emerging from previous gene expression studies in TSC and different animal models of epileptogenesis (including inflammation, glutamate and adenosine homeostasis and GABAergic transmission and new potential targets) has been performed.
In TSC, overexpression of numerous genes associated with inflammation has been observed. Among different proinflammatory cytokines, interleukin-1β (IL-1β) has been shown to be significantly involved in epileptogenesis and maintenance of seizures. Our results provide the first evidence of epigenetic modulation of the IL-1β signaling in TSC. This study has been finalized and published (Hypomethylation Correlates with IL-1β Overexpression in Tuberous Sclerosis Complex J. Mol. Neuroscience, 59(4):464-70). Large scale DNA methylation profiling and copy number variation analyses (using Illumina Human Methylation 450 array) have been performed in the same cohort of TSC samples used to evaluate small non-coding RNA and mRNA profiles (see below). Data analysis of tubers with different histopathological patterns (A versus B, see PLOS one, 11(6):e0157396) has been also performed.
We investigated the expression pattern of constitutive (β1, β5) and (immune)proteasome (β1i, β5i) subunits using immunohistochemistry in malformations of cortical development. Our observations support the dysregulation of the proteasome system in TSC and provide new insights on the mechanism of regulation of the (immuno)proteasome in astrocytes and the molecular links between inflammation, mTOR activation and epilepsy. Two manuscripts on the above reported finding have been published (Dysregulation of the (immuno)proteasome pathway in malformations of cortical development. J Neuroinflammation 2016; 13(1):202; P8 /AMC, P7/UMC, P5/ UHM and P10/ MUW; Increased expression of (immuno)proteasome subunit during epileptogenesis is attenuated by inhibition of the mammalian target of rapamycin pathway. Epilepsia 58(8):1462-1472. doi: 10.1111/epi.13823) and these findings have been presented during the last 6 months in different international meetings (see dissemination).
Voltage-clamp recordings were performed on Xenopus oocytes “micro-transplanted” with membranes from cortical tubers and age matched control brain tissue. Our data indicate that in human TSC cortical tubers the pattern of GABAAR and GLUA1/GLUA2 function retains features that are typical of an immature brain. A manuscript on the above reported finding has been published (Functional aspects of early brain development are preserved in tuberous sclerosis complex (TSC) epileptogenic lesions. Neurobiol Dis, 2016; 95:93-101; P8 /AMC, P7/UMC, P5/ UHM and P10/ MUW; see dissemination). The effect of C1q and C3 complement components (upregulated in TSC brain) on GABAA evoked current has been evaluated, using oocytes microtransplanted with TSC cortical membranes. The results of these experiments (validated during the last 6 months) indicate that acute co-application with C3 or C1q fraction complements did not show any effect, while incubations higher than 40 min with C3 were effective in decreasing GABAA evoked current reaching the maximum effect after one hour. The decrease of GABAA currents was completely reversible after 20 min of wash with Ringer solution.
Moreover, we transplanted in Xenopus oocytes cell membranes obtained from brain tissues of autopsies of both TSC and Dravet syndrome patients as a pathological comparison, and age-matched controls and evaluated the effect of of cannabidiol on GABAA current . Additionally, experiments were performed on oocytes expressing human α1β2γ2 and α1β2 GABAA receptors. GABAA currents were recorded using the two-microelectrodes voltage-clamp technique. We found an increase of GABAA currents induced by low doses of cannabidiol both TSC and in Dravet syndrome, comparable to that induced by a classical benzodiazepine, flunitrazepam, that still persists in γ-less GABAA receptors. This study has been finalized and published (G. Ruffolo, et al., . Epilepsia, 59(11):2106-2117, 2018).
Collaboration has been established with P14/GenomeScan to characterize small non-coding RNAs and mRNAs in tubers and age matched control cortex, using the Illumina Next Generation Sequencing (NGS) Technology. A weighted correlation network analysis (WGCNA) identified innate immune response, extracellular matrix organisation, neurogenesis and glutamate receptor signalling as important modules in TSC. A manuscript including these results has been published (Coding and small non-coding transcriptional landscape of tuberous sclerosis complex cortical tubers: implications for pathophysiology and treatment. Scientific Reports, 7(1):8089. doi: 10.1038/s41598-017-06145-8 2017).
We further analyzed the expression profile in tubers with different histopathological patterns (A versus B, see PLOS one, 11(6):e0157396). We found a significant difference between A and B. There were 246 genes up-regulated in the type B tubers and 33 up-regulated in the type A tubers. The enriched GO terms and Pathways for the genes up-regulated in the type B tubers indicate a higher inflammatory and immune response in the type B tubers, amongst the pathways the MAPK signalling pathway, TNF signalling pathway and ECM-receptor interaction are enriched and these findings will be included in a manuscript (Angelika Mühlebner et al., in preparation).
Further, we identified the transcription factor Spi-1 proto-oncogene (SPI1) (up-regulated 3-fold in TSC) that may regulate 209/269 of the overexpressed gene in TSC, including the cytokines/chemokines (CCL2, CCL3, CCL4 and IL1β) and complement components (C1QB, C1QC and C3). SPI1 also appears to play a key role in the cellular response to oxidative stress. SPI1 expression is activated in a primary fetal astrocyte culture after stimulation with hydrogen peroxide in a dose dependent manner. Emerging evidence suggests that oxidative stress plays a major role in TSC pathology and the subsequent seizure development. We hypothesize that SPI1 is a master regulator of genes involved in TSC pathology. Interestingly, in line with the findings in human TSC brain tissue genes related to inflammation and immune response, along with both SPI1 homologs (spi1a and spi1b) were also up-regulated in the brain of homozygous tsc2-/- mutant zebrafish (P04/VUB; P09/KUL) (mTOR-related neuropathology in mutant tsc2 zebrafish: Phenotypic, transcriptomic and pharmacological analysis. Neurobiology of Disease, 108:225-237, doi: 10.1016/j.nbd.2017.09.004). In the last 6 months, we are continuing to investigate the role of SPI1 in oxidative stress and inflammation in human cell-lines and experimental models of TSC.
To provide evidence of the role of inflammation-related microRNAs in TSC, we employed real-time PCR and in situ hybridization to characterize the expression of miR21, miR146a and miR155 in TSC lesions (cortical tubers and subependymal giant cell astrocytomas, SEGAs). Our study provides supportive evidence of the role of inflammation-related microRNAs in TSC. In particular, miR146a and miR155 appear to be key players in the regulation of astrocyte-mediated inflammatory response, with miR146a as most interesting anti-inflammatory therapeutic candidate. This study has been finalized and published (P1/IPCZD; P5/ UHM; P7/UMC; P11/IIMCB; P10/ MUW; Expression of microRNAs miR21, miR146a and miR155 in tuberous sclerosis complex cortical tubers and their regulation in human astrocytes and SEGA-derived cell cultures, Glia 64(6):1066-82, 2016; see dissemination). In collaboration with P14/GenomeScan we performed small RNA sequencing in the same cohort used for mRNA profiling. Analysis of data revealed 48 miRNAs which were up-regulated and 10 miRNAs which were down-regulated in TSC tubers compared to control cortex. Unsupervised hierarchical cluster analysis showed a majority of TSC tubers clustering together. Increased expression of miR34a-5p, miR34b-5p, miR34c-5p, miR302a-3p, miR577 and miR21-5p was observed in TSC tubers compared to control cortex. We further investigated the effect of overexpression of miR34b-5p on neurite outgrowth in mouse hippocampal neurons. Neurons transfected with miR34b-5p mimic showed increased number of longer neurites as compared to Scr transfected cultures. A manuscript including these results has been published (Scientific Reports, 7(1):8089. doi: 10.1038/s41598-017-06145-8). In the last six months, we further investigated the role of the miR34 family in TSC. We have found that overexpression of miR34b induces IL-1β pathway activation in human astrocytes. We are also investigating the role of miR34a-b and miR155 in the molecular mechanisms involved in the switch to an inflammatory state after oxidative stress in both neuronal and glial cells in culture. Functional studies focusing on the role of miR34a-5p amd miR34b-5p during corticogenesis using an in utero electroporation model have been also performed and indicate a negative regulation of neuronal migration by both miR34a-5p and miR34b-5p. Further, we have confirmed that Contactin 3 (CNTN3, also known as BIG-1) a gene down-regulated in TSC and involved in neurogenesis is a target of both miR34a-5p and miR34b-5p. More specifically CNTN3 is down-regulated in cortical tubers resected from young patients (<10 years) to levels below those seen in control fetal tissue. Further experiments have been carried out to elucidate the functional relevance of CNTN3 and include over-expression studies by the CRISPR-dCas9-VP64 activation system, and inhibition by siRNAs in the human neuronal cell line SH-SY5Y, coupled with differentiation experiments by treating the cells with retinoic acid supplemented with brain derived neurotrophic factor. This research project is ongoing, and the data produced was presented at the Amsterdam Annual Neuroscience meeting (The altered expression of cell adhesion molecule contactin-3 in tuberous sclerosis complex, Korotov, et al. 2018) and results are included in a manuscript in preparation (A. Korotov, J. Mills et al., in preparation).
llumina NGS was also employed to identify small non-coding RNAs and mRNAs related to the IL-1β pathway in human astrocytes. 54 mRNAs were detected as differentially expressed in human astrocytes stimulated with IL-1β compared to controls (unstimulated cells). We detected a strong up-regulation of both miR146a and mir147b. We further evaluated the function of miR147, a microRNA that is induced by IL-1β - receptor stimulation. The functional effect of overexpression of miRNAs on inflammatory signaling was studied by transfection of astrocyte cultures with miRNA mimic oligonucleotides. Overexpression of miR147b inhibited IL-6 and COX-2 up-regulation after IL-1β stimulation. Moreover, miR147b overexpression down-regulated its predicted targets DEPTOR and ADAM15. To determine whether miRNA transfection could restore altered proliferation and generation of astrocytes seen in inflammatory related pathologies, immunocytochemical Ki67 staining and flow cytometric analysis of propidium iodide staining was assessed. Indeed, transfection of astrocytes with miR146a and miR147b both decreased proliferation of astrocytes. The effect on cell fate determination in neuronal stem cells (NSCs) derived from fetal brain tissue was examined by immunocytochemical staining of transfected, differentiated stem cells. Transfection with both miR146a and miR147 promoted neuronal differentiation of NSCs, supporting the role of these miRNAs deregulated in TSC in NSC differentiation. (miR147b: A novel key regulator of interleukin1 beta-mediated inflammation in human astrocytes. Glia, 66(5): 1082-1097, doi: 10.1002/glia.23302 2017). These findings have been presented during the last 6 months in different international meetings (see dissemination).
Based on earlier findings in by the Jaworski’s group (IIMCB) in blood cell derived RNA from TSC patients and controls, a collaborative pilot study was carried out to assay the expression of 6 genes (RAB11A, PAK2, GPRC5A, SF3A1, GOS2 and IL8) in TSC cortical tubers and control cortex by RT-qPCR. In validation study, GPRC5A showed significantly increased expression (P=0.006) in TSC tubers compared to control cortex, whereas RAB11A showed a significant decrease (P=0.02) and the other genes remained unchanged. GPRC5B encodes an orphan G protein-coupled receptor (which has been suggested to play a role in neuronal fate determination of cortical progenitors) and will be further evaluated and validated within the EPISTOP cohort. Furthermore, based on our previous results and sequencing data from TSC brain tissue and serum of experimental models of epileptogenesis a set of inflammatory genes including cytokines/chemokines (CCL2, CCL3, CCL4, IL1β, IL6 and COX2) and complement components (C1QA, C3, and C4B) have been assayed in TSC brain tissue. We also evaluated the matrix metalloproteinase genes (MMP2, MMP3, MMP9 and MMP14) showing significantly increased expression of MMP9 and MMP14 but not of MMP2 and MMP3 in the TSC tissue. Using immunohistochemistry, we found up-regulation of MMP2, MMP3 and MMP14 in glial cells. High immunoreactivity of all mentioned MMPs was seen in dysmorphic neurons and giant cells. We also studied the regulation of MMPs by miRNA in astrocytes, showing a modulation of MMP3 by miR132, miR146a, miR155 in human astrocytes. These findings have been included in a manuscript that has been submitted and is presently under revision (D. Broekaart, J. van Scheppingen et al.; Increased expression of matrix metalloproteinases associated with tuberous sclerosis complex can be attenuated by microRNAs 146a and 147b).
Recent studies indicate that administration of antioxidant drugs reduced neuroinflammation and improved disease outcomes in a rat model epilepsy and oxidative stress related molecules may be potential biomarkers of epileptogenesis. We will further study the role of oxidative stress in TSC evaluating the induction and cellular distribution of oxidative stress markers (i.e. HMGB1, iNOS, Xct and Nrf2; and DNA damage markers) in TSC specimens, as well as the molecular mechanisms involved in the switch to an inflammatory state after oxidative stress (Task 5.4). This research has resulted in one accepted publication (Oxidative stress and inflammation in a spectrum of epileptogenic cortical malformations: molecular insights into their interdependence, Brain Pathology, 2019 May;29(3):351-365. doi: 10.1111/bpa.12661) and one manuscript in preparation (Modulation of astrocyte antioxidant signaling by microRNA-155 and its implication in epileptogenic malformation of cortical development, manuscript in preparation). Additionally, human TSC tissue was used to validate potential peripheral epileptogenesis biomarkers such as inflammatory markers (IL-1β and immunoproteasome subunits; J Neuroinflammation 2016; 13(1):202) J. Mol. Neuroscience, 59(4):464-70, 2016; see dissemination) and miRNAs, including the miR34 family (Scientific Reports, 7(1):8089, 2017; see dissemination) and inflammation-related miRNAs (miR146a and miR147 Glia 64(6):1066-82, 2016; Glia, 66(5): 1082-1097, 2017; see dissemination). Based on our previous results of small RNA-Sequencing of data from TSC brain tissue and TSC serum samples (first stage analysis) we identified miRNAs of interest to be assayed in the entire cohort. Inflammatory related miRNAs (miR21, miR146a, miR147 and miR155; as potential peripheral epilepsy biomarkers) were validated in TSC cortical tubers (see also Task 5.4). These findings have been presented during the last 6 months at different international meetings (see dissemination).
Potential Impact:
The potential impact
EPISTOP was the first study to address the unmet need for prevention of epilepsy and its comorbidities in clinical settings. Epilepsy affects over 6 million people in Europe is associated with an annual societal and healthcare cost of 20 billion Euro. Although new antiepileptic drugs continue to be introduced into clinical practice, freedom from seizures is not achieved in about 30% of patients. Furthermore about 60% of children with epilepsy suffer from chronic and irreversible neuropsychiatric comorbidities, including learning disabilities and autism. Thus, prevention of epilepsy is widely recognized as a “holy grail” in the field of epilepsy care.
Tuberous Sclerosis Complex (TSC) is a major cause of early onset epilepsy, occurring in the first year of life. Given the incidence of TSC, we estimate this disorder affects approximately 100,000 Europeans and one million people worldwide. Before EPISTOP, it was reported that about 90% of TSC patients suffered from epilepsy and over 60% had some form of intellectual disability. In more than 65% of patients, epilepsy begins in childhood. About 50% of children with epilepsy suffer from psychiatric and behavioural comorbidities, such as learning disabilities, and autism spectrum disorders (Ono, 2012). Prevention of epilepsy and its comorbidities in patients at risk presents a major unmet need in clinical medicine, but there were no strategies aimed to prevent of modify the natural course of epilepsy development. EPISTOP results can be exploited directly by using prevention protocols in clinical practice, and indirectly, by stimulation of new research projects, including ones targeted towards validation of EPISTOP results in other types of epilepsy.
In the EPISTOP clinical trial, we developed a set of defined changes on EEG to serve as a non-invasive biomarker of epileptogenesis. With this novel development, we were able to identify infants at high risk of epilepsy. Indeed EEG surveillance has recently been proposed by EPISTOP investigators as an important element of routine clinical care for TSC infants, enabling early diagnosis (before clinical seizures) of epilepsy development and preventive treatment (Curatolo et al. EJPN 2018). With EPISTOP we showed that this approach enables identification and treatment of these earliest forms of seizures, whether clinical or electrographic, and, in consequence, reduces epilepsy severity and the risk of intellectual disability and autism. Recently many other investigators have reported the beneficial effect of EEG survaillance implementation (Whitney et al. Ped.Neurol 2017, Benova et al. EJPN 2018, Chung et al, Ped.Neurol 2017). A key part of the EPISTOP study was to investigate the outcome of TSC children who received treatment based on EEG findings. In a randomized clinical trial we found that preventive treatment with vigabatrin reduced the risk of clinical seizures and drug-resistant epilepsy when compared to treatment after clinical or electrographic seizures.
These findings have major significance for children with TSC and their families. EPISTOP provides evidence that early EEG monitoring and preventive treatment reduce the risk of severe epilepsy, and its neurodevelopmental co-morbidities. It is established that the societal burden of epilepsy and its comorbidities depends on their severity and is higher in patients receiving more than 1 antiepileptic drug or presenting with intellectual disabilities (Jones et al, 2019; Riechmann et al, 2015). Thus management of TSC according to EPISTOP protocol including early intervention should improve the quality of life of many individuals and families coping with TSC. Our new treatment is not associated with additional costs for the patients or healthcare system, as we used a standard antiepileptic drug, vigabatrin. Furthermore, in patients in whom epilepsy is prevented, antiepileptic treatment might be withdrawn at a younger age, reducing their lifetime exposure to treatment and total costs of medication in comparison to patients presenting with clinical seizures. The results of preventive treatment in EPISTOP are being disseminated to TSC and epilepsy communities who are direct beneficiaries of the project.
Neuropsychological analyses in EPISTOP showed for the first time with that the results of tests performed at youngest ages: 6 and 12 months, the risk of autism and learning disability can be predicted. These findings enable the identification of patients at risk of neurodevelopmental comorbidities of epilepsy and implementation of early intervention. Moreover, the risk of severe epilepsy and neurodevelopmental comorbidities of epilepsy can be predicted by using prenatal brain MRI results, which enables more precise indication for early intervention. All these findings are being submitted for publication in professional journals and disseminated to TSC and epilepsy communities.
EPISTOP is a strong, multidisciplinary and comprehensive consortium, which was built to perform the first prospective human study of biomarkers of epileptogenesis beginning from its latent phase, through active epilepsy and chronic disease. Four out of 8 WP leaders were female. Thus, EPISTOP contributed to the development of sustainable and gender-equal research community.
With a comprehensive study of biomarkers, EPISTOP was able to identify the risk factors of the development of epilepsy and its comorbidities. Early recognition of patients at risk will allow personalized early interventions, including prevention. Moreover, clinical and molecular characterization of epilepsy comorbidities in children might contribute to the future studies aimed to identify the targets for innovative and personalized medications, especially in rare and complex disorders of the nervous system.
Given that EPISTOP has generated interest in preventive strategies broadly in the epilepsy community, it has influenced research directions in epileptology. This includes the ongoing randomized controlled trial PREVENT in the US (NCT02849457) started in 2016. The study design in PREVENT is similar to EPISTOP: it is based on the prevention trial in TSC patients. The differences include the use of placebo in PREVENT trial and the inclusion of biomarker studies in EPISTOP. Preliminary results of EPISTOP have been presented at multiple international meetings with various stakeholders, including clinicians, researchers, patients’ advocacy groups and organizations, research funding agencies since 2015. EPISTOP operated closely with other EU-funded projects on epilepsy (EPImiRNA, EPITARGET, EPIPGX, DESIRE) within the EU EpiXchange initiative, as well as with European Reference Network (ERN) for rare and complex epilepsies (EPICARE) and EC and NIH to promote epilepsy research and indicate biomarker and prevention studies as priorities in EU and US research policies.
The main dissemination activities and exploitation of results
At the consortium level
Meetings were organized on a regular basis (every 6 months). Steering Committee meetings were attended by all WP leaders and sometimes the EC representative. These were alternated with General Assembly meetings which were attended by all EPISTOP collaborators and the members of the Scientific Advisory Board (SAB).
At the kick-off meeting, representatives of the Polish minister of Health and local media were present as well. The kick-off meeting took place in Warsaw. All consortium members, representatives of Ethics Board and SAB participated in the meeting. The kick-off meeting started with an opening session for public and invited guests. Lectures about the project were given by prof. Sergiusz Jóźwiak, prof. Lieven Lagae, and prof. David Kwiatkowski. The audience was addressed by Mr. Igor Radziewicz-Winnicki, Undersecretary of State of the Ministry of Health, prof. Małgorzata Syczewska, Director of The Children's Memorial Health Institute, prof. Jacek Kuźnicki, The International Institute of Molecular and Cell Biology, and Anita Kucharska, European Commission. Prof. Jerzy Buzek addressed words of support to the scientists. There was also a press conference (reports and information about the project were published in number of newspapers, magazines, radio and TV programs). After official part followed sessions with all consortium partners with lectures of all WPs’ leaders and representatives of EC, including Anita Kucharska, Małgorzata Urbaniak. At the same time also SAB members and members of the Ethics Board met in Warsaw. The meeting was organized by IPCZD. Pictures are attached.
The closing conference was also organized in Warsaw. The conference started with an opening session for public and invited guests. There were also journalists from television, internet, radio and magazines. The leaders presented a summary of the results achieved and work done in each of the work packages. There were external experts invited by IPCZD, including members of SAB (Annamaria Vezzani, Marcin Balcerzak, Leszek Kaczmarek), and members of the Ethics Board (Katarzyna Łukasiuk, Andrzej Chaberka). Mr. Grzegorz Owsianik, EC representative, attended the final conference and the final meeting. During the second day of the meeting, two different events took place. The clinical partners discussed their results during an educational meeting for TSC clinicians and patients, organized in collaborations with e-TSC, the European TSC patient organization. At the same time partners from the molecular group took part in the workshop entitled "Current challenges in epileptics and TSC molecular biomarkers development" organized by IIMCB (partner in the EPISTOP project). This workshop was also attended by scientists form different Institutes from Poland and other countries. It was a unique possibility to discuss the results obtained in the EPISTOP project with different scientists and present the molecular and computational methods used during the project. In addition, this workshop allowed further dissemination and exploitation of the results obtained by the EPISTOP scientists. Pictures are attached.
The EPISTOP website (www.epistop.eu) was regularly updated (all EPISTOP-related documents per work-package on the password-protected section, EPISTOP-related publications, announcement of consortium meetings through the banner, news updates, overview of internal (General Assembly and Steering Committee meetings) and external meetings (meetings attended by EPISTOP WP leaders) were published). Information about the project was published in English, Polish, Dutch, German, Czech, French and Italian.
At the public level
The project lifespan the progress of study has been discussed in over 40 lectures at national or international conferences or educational meetings. The EPISTOP project was referred to in more than 60 publications and posters published in peer reviewed international journals or presented at national and international meetings.
Details on the EPISTOP initiative (Part 1) as well as on the results (Part 2) were summarized in two publications in the IMPACT journal which were distributed to all consortium members and IMPACT journal readers EU-wide.
The EPISTOP website was used as a tool to communicate with the broader TSC community as well, using especially the NEWS section to share the progress and initiatives of the consortium. An English and Polish Facebook page have been launched (https://www.facebook.com/epistop.TSC/ and https://www.facebook.com/projektepistop/) for education and dissemination purposes. Updates were done regularly throughout the project, with announcements and feedback of events, educational information as well as daily facts on TSC during the month of May, TSC awareness month. Twitter and Instagram accounts were used as well to regularly update the general public and TSC research community on EPISTOP project activities. Publications were shared and highlighted through a project page on ResearchGate.
At the start of the project, flyers were developed to inform the community about the EPISTOP project and to promote patient recruitment for the clinical trial. These flyers were translated in all EPISTOP consortium languages and distributed through all participating clinical trial centers and affiliated patient organizations.
In the beginning of the project a calendar was made to improve visualization of the project. It was handed out to all EPISTOP partners and referring centers. We also prepared promotional gadgets to enhance visualization of the project including t-shirts, sunglasses, shopping bags, and note-blocks
The set-up of the EPISTOP project was explained in a first animated movie which was prepared at the beginning of the project. This movie has been viewed over 4.500 times through the EPISTOP Facebook page. It has been subtitled in all languages of the consortium members and was used to highlight the project during multiple lectures worldwide. A special edition with Japanese subtitles was prepared for the International Research Conference on TSC in Tokyo in 2018. A second movie summarizing the results of the EPISTOP project was finalized at the end of the project and was presented during the Closing Meeting in Warsaw as well as the International TSC Research Conference in Toronto, Canada in 2019. Below you will find links to mentioned movies:
http://epistop.eu/index.php/news/31-epistop-promotional-film
https://www.facebook.com/epistop.TSC/videos/599485826841755/
http://epistop.eu/index.php/news/198-epistop-results-summarized-in-animation-movie
EPISTOP was part of the EPIXCHANGE conference organized on May 23rd 2018 in Brussels by seven large EU-funded projects (including EPISTOP) to bring together Europe’s best brains to pave the way for future epilepsy research. Prof. S. Jozwiak presented the EPISTOP project during the plenary session and the symposium was attended by several EPISTOP Steering Committee members. The results of the EPIXCHANGE meeting led to the publication of a manuscript by Pitkänen et al entitled ‘Advancing research toward faster diagnosis, better treatment, and end of stigma in epilepsy’ published in Epilepsia in 2019. Below you will find the interview with Prof. Sergiusz Jóżwiak:
https://www.youtube.com/watch?v=ynW-JeIyrvs
Two educational meetings were organized to increase awareness on TSC and educate the general TSC and medical community on the key-aspects of the EPISTOP project, including early diagnosis, regular EEG monitoring, preventive treatment of epilepsy, and early monitoring and intervention for developmental delay and autism. Both meetings were co-organized by e-TSC, the European Tuberous Sclerosis Association. The first took place in Leuven, Belgium in the fall of 2018, the second in Warsaw following the closing conference.
During the project two PhD students publicly defended their PhD thesis. Chloë Scheldeman presented her work on ‘Zebrafish-based modeling of human genetic epilepsies: FHF1 and TSC2 genes’ and Jackelien van Scheppingen presented her research on ‘Astrocytes as mediators of inflammation in epilepsy: focus on tuberous sclerosis complex’.
Dissemination of the results
The EPISTOP project has led to a critical advancement of knowledge in the field of TSC and of epilepsy more in general. The key-findings will be disseminated through publications in international peer-reviewed journals and presentations at scientific and educational meetings. Various novel scientific concepts will lead to for further validation studies and might in a next step result in collaborations with industry.
The need of early TSC diagnosis will be disseminated with scientific publications to be implemented into routine clinical practice. The stakeholders include obstetricians, gynecologists, neonatologists, perinatologists, pediatric cardiologists, pediatric neurologists, paediatricians, and other medical professionals.
Presymptomatic EEG monitoring in all TSC infants will be disseminated with scientific publications to be exploited as a new routine clinical practice. The stakeholders include pediatric neurologists, pediatricians, neurologists, and psychiatrists. They can be informed about the results of the project and its significance using scientific publications and presentations on meetings and conferences of the professional societies. This new treatment concept should also be disseminated among patients’ advocacy groups and organizations, including TSC associations in individual countries, as well as TSC Europe, EURORDIS, and others.
Preventive antiepileptic treatment in TSC infants will also be disseminated with scientific publications to be introduced into clinical practice. The stakeholders include pediatric neurologists, pediatricians, and neurologists. Given that TSC is a rare disorder, the organizations and institutions supporting rare diseases including European Reference Networks, EU DG Santé, ministries of health in individual countries will be subjected to the exploitation of the results of EPISTOP.
The concept of epilepsy prevention in TSC should be validated in other types of epilepsies in future clinical trials. It might be exploited to research policy makers in order to promote calls and funding for such clinical trials. Exploitation will be discussed with epileptology professionals, including International League Against Epilepsy, neurology associations, including European Paediatric Neurology Society, Child Neurology Society, and others. The need for future epilepsy prevention studies should be discussed with EC research officers, NIH, and local research agencies in individual countries. In the US, a similar study aimed to assess the results of preventive antiepileptic treatment in TSC infants, called PREVENT (ClinicalTrials.gov Identifier: NCT02849457), is currently recruiting patients. It will be of utmost interest to compare the findings in both studies and to share experience from both trials. The EPISTOP and PREVENT (when completed) studies results should be exploited together when discussing the future research plans both in EU and in the US. National Institute of Health admitted importance the EPISTOP project inviting Prof. Jozwiak to the by „Accelerating the Development of Therapies for Anti-Disease Modification Workshop” in Rockville, Maryland,United States.
The need for early assessment and intervention for developmental delay and autism in TSC infants and children will be disseminated to patient associations (Tuberous Sclerosis Europe and Autism Europe) in order to allow a broader diffusion of results to help parents with a child with an early diagnosis to search for a prompt referral and a close neurodevelopmental monitoring.
The MRI protocol and lesion segmentation technique used in this study will be disseminated with scientific publications to be exploited as a new routine clinical practice. The stakeholders include pediatric neurologists, pediatricians, and neurologists. The lesion segmentation technique can be considered as a first step towards the development of fully automated lesion segmentation.
The finding that multiple serum proteins, metabolites and microRNAs show marked changes from birth to age two years may be exploited to discover abnormalities in these developmental changes that may provide diagnostic information for the care of children who are not developing properly.
Several biomarkers were identified as being associated with epilepsy development. New research activities that will derive from these findings are replication of the findings in other TSC and epilepsy populations.
Insights in the molecular mechanisms underlying the mammalian target of rapamycin (mTOR)-related epileptogenesis and associated comorbidities (intellectual disability, neurobehavioral and psychiatric disorders) were obtained, identifying a number of potential molecular targets for antiepileptogenesis or disease-modifying therapies (i.e. inflammation, oxidative stress). This can result in new therapeutic strategies, which however need to be further evaluated and validated in preclinical and/or clinical studies in a separate patient cohort. These molecular insights provide important information for the development of novel therapeutic approaches beyond EPISTOP in patients with TSC, but also in other developmental brain diseases that share the same hallmarks of immaturity leading to intractable seizures and associated complex neurological and neuropsychiatric phenotypes.
List of Websites:
http://epistop.eu/
https://www.facebook.com/projektepistop/
https://www.facebook.com/epistop.TSC/
Epilepsy affects 1% of the world’s population. In Europe, 6 million people have epilepsy (World Health Organization, 2010). Despite great progress in the management of epilepsy and increasing numbers of antiepileptic drugs, 30-40% of epilepsy patients are refractory to all available medications and many suffer from epilepsy-related comorbidities (Jensen, 2011).
The EPISTOP project had two major objectives: a clinical trial objective, focused on epilepsy prevention study in infants with Tuberous Sclerosis Complex (TSC); and a laboratory objective, the identification of biomarkers of epilepsy development and its neurodevelopmental comorbidities. EPISTOP was carried out by a multidisciplinary consortium of 10 clinical sites, 5 laboratories, and 1 administrative partner. It was the first prospective study of epileptogenesis in humans and included a unique clinical trial comparing epilepsy and neurodevelopmental outcome in patients receiving either standard or preventive antiepileptic treatment.
The clinical component of EPISTOP has provided first evidence that EEG monitoring of infants at high epilepsy risk and introduction of antiepileptic treatment before the onset of seizures prevents or delays seizures and reduces the risk of drug-resistant epilepsy. It also reduces the risk of infantile spasms and seizure burden measured as the number of days with seizures, indicating that preventive treatment reduces epilepsy severity in TSC children.
One-hundred-one TSC infants participated in the study, and were the primary beneficiaries of the project. TSC affects 1 in 6,000 newborns, 70% of them develop epilepsy within the first year of life, and our project showed that EEG monitoring and early epilepsy management is feasible in the majority of TSC infants (Recommendations: Curatolo et al. 2018). If future studies confirm the benefit of preventive strategies in other types of epilepsy, then early monitoring and treatment might be recommended in as many as 40% of patients with epilepsy, potentially having a major impact in reducing complications and social stigma associated with epilepsy.
The laboratory component of EPISTOP included analysis of a wide range of EEG, neuroimaging, neuropsychological, and molecular data, to identify risk factors and biomarkers of the development of epilepsy and its comorbidities. EPISTOP investigators developed an EEG scoring system, that also may be validated for other types of epilepsy, and used more broadly to enable early preventive treatment or other disease modification strategies.
Project Context and Objectives:
In Europe, 6 million people have epilepsy (World Health Organization, 2010). Despite great progress in the management of epilepsy and increasing numbers of antiepileptic drugs, 30-40% of epilepsy patients are refractory to all available medications and many suffer from epilepsy-related comorbidities (Jensen, 2011). Given these data and the leading position of European epileptology centres, the Consensus Document of European Brain Research in 2011 recommended research focused on preventing epilepsy development to be supported by European funding (Di Luca, 2011).
In more than 65% of patients, epilepsy begins in childhood and the incidence of epilepsy is highest in the first year of life (Hauser, 1993). In children, the problem of epilepsy is far beyond seizures, as about 50% of children with epilepsy suffer from psychiatric and behavioural comorbidities, including developmental delay, learning disabilities, and autism spectrum disorder (Ono, 2012). Furthermore, early onset of seizures is regarded as one of the major risk factors for development of drug-resistant epilepsy. Epilepsy differs from many other neurological conditions due to its extreme heterogeneity in aetiologies and phenotypes. Thus, studies of epilepsy risk factors might be uniquely enabled by analyses in young children where the influence of the external, environmental factors is lower than in adults. Tuberous Sclerosis Complex (TSC) is a genetically determined neurocutaneous syndrome affecting 1 child in 6,000 (Curatolo, 2008). TSC is often considered an excellent clinical model of severe focal epilepsy, as 70 to 90% of patients are affected by epilepsy and in most cases the seizures are drug-resistant. In the majority of patients epilepsy manifests in the first months of life and half of patients develop cognitive impairment, autism spectrum disorder or other neurodevelopmental disturbances (Jozwiak, 1998). Therefore TSC is an excellent model for both focal epilepsy and infantile spasms. Although there is definite clinical heterogeneity, TSC represents a relatively homogenous group of patients for the studies of epileptogenesis, who are at high risk of this disease. Recent studies (Jozwiak, 2011) demonstrated that in infants with paroxysmal discharges on EEG it is possible to modify the outcome of the disease by antiepileptic treatment before the onset of clinical seizures. EPISTOP model gives us a unique opportunity to investigate whether clinically successful epilepsy prevention is possible, and to explore the molecular mechanisms of this effect. EPISTOP is aimed to better understand the complex pathophysiology of epilepsy in order to develop novel preventative strategies in at risk patients, improve diagnostic methods and develop more effective therapeutic strategies. To achieve this aim, the risk factors and biomarkers of epilepsy were identified by a multidisciplinary, systematic approach in three settings:
- a prospective study of epilepsy development in infants with TSC, using a wide range of clinical, neuroimaging, and genetic analyses
- prospective clinical study of TSC infants treated with antiepileptic drugs at the onset of subclinical seizures in comparison to children treated only after clinical seizures appear
- analysis of biomarkers of epileptogenesis and drug-resistant epilepsy in epileptogenic brain specimens obtained from patients with TSC.
EPISTOP project is consisted of eight Work Packages (WP). EPISTOP is divided into two parts: clinical study (WP2, WP4, WP6, WP7) and molecular study (WP3 and WP5). Additionally two horizontal WPs (WP1 and WP8) were established. Each WP was devoted for specified tasks which were related to overall study objectives. The consortium is consisted of 16 partners, all partners were involved in the activities of various WPs. Partners participating in the study came from 10 countries from 3 continents (Europe, USA, Australia). IPCZD was a leader of the project as well as leader of WP6. Samples from nine clinical sites (P1-IPCZD, P2-TVG, P3-INS, P4-VUB, P5-UHM, P6-CUB, P7-UMC, P10-MUW, P16-LCCH) have been sent to, and are stored at the Jaworski laboratory at IIMCB (P11-IIMCB). IIMCB has distributed multiple sets of samples to all laboratories participating in this work project, including: the laboratory of Dr. Kwiatkowski at the BWH (P12-BWH), the laboratory of Dr. Aronica at AMC (P8-AMC), Dr. Lehmann at the Proteome Factory (P13-PFA), and Dr. Janssen at GenomeScan (P14-SXS/GS). Computational and statistical analyses were performed by Transition Technologies S.A. Consortium was also supported by Scientific Advisory Board and Ethics Board members. Scientific Advisory Board included experts in clinical and experimental epileptology and molecular biology, as well as the biostatistician (prof. Annamaria Vezzani, prof. Carl Stafstrom, prof. Leszek Kaczmarek, prof. Renzo Guerrini, dr Marcin Balcerzak). Ethics Board included both medical professionals and representatives of patients organizations (prof. Katarzyna Łukasiuk, prof. Steve Roach, prof. Raili Riikonen, Andrzej Chaberka).
The objective of WP2 was to determine electroencephalographic biomarkers of epileptogenesis, and to randomize patients into group A and B (WP6). All the patients underwent videoEEG recording every 4 weeks in the children under the age of 6 months, then every 6 weeks in children under 12 months, and every 8 weeks thereafter till the last follow-up visit at the age of 24 months. In WP2, an EPISTOP scoring system for EEG changes in TSC infants has been elaborated and used to identify patients at high risk of seizure. This system was also used to determine the randomization timepoint.
In the analysis phase, the systematic database was used to analyze the actual EEGs in detail. It was found that EEG monitoring performed before seizures significantly improves the prognosis of psychomotor development, probably influencing the early diagnosis of clinical and electroencephalographic seizures. EEG monitored children are developing significantly better than any previously published group of children with TSC. WP3 was aimed to identify the clinically useful blood biomarkers of epileptogenesis and risk factors for epilepsy. In all EPISTOP participants, blood samples for molecular biomarkers studies were collected: 1/ at entry to the project, 2/at appearance of paroxysmal activity on videoEEG, 3/ at onset of clinical seizures, 4/ at the age of 24 months. The genetic, epigenetic, transcriptome, proteome, metabolomic and immunological parameters were being compared in children with and without epilepsy as well as between the set time points in children with epilepsy or epileptiform discharges on EEG and patients with drug-resistant epilepsy were being analyzed. All data were subject to statistical analysis. The objective of WP4 was to deliver the neuroimaging biomarkers of epileptogenesis. MRI is a standard procedure in epilepsy and TSC patients. In our project, MRI was performed at baseline and at the end of follow-up and whenever indicated clinically. Apart from standard MRI, DTI and DWI were performed in order to identify neuroimaging biomarkers of epilepsy, autism, and neurodevelopmental delay susceptibility. It was found that brain lesions identified in prenatal MRI correlate with epilepsy and neurodevelopmental outcome in TSC children. Neuroimaging findings were included in a set of clinical and molecular epilepsy biomarkers from the blood samples obtained during the study.
WP5 aimed at discovery and validation the epileptogenesis and drug-resistance biomarkers in brain samples from TSC patients who underwent epilepsy surgery and TSC autopsy cases. The objective of WP5 was to investigate the epileptogenesis and drug-resistance biomarkers in epileptogenic brain samples, in order to validate the findings from blood analysis and to discover brain-specific biomarkers. In the center of the consortium was WP6 proposing multicenter, prospective clinical study assessing epileptogenesis development in infants with TSC. The number of patients enrolled in the project is 101. All children were followed clinically and with serial videoEEG until 24 month of age. The first phase of the study rested on the observation of patients until subclinical seizures appear on videoEEG. Patients with epileptiform discharges on videoEEG prior to clinical seizures entered the randomized phase of the study. They were randomized into two groups: group A - those who receives vigabatrin for subclinical seizures; and B - those who were treated only after the onset of clinical seizures. Patients without clinical and subclinical seizures were followed up without any medication. Patients with clinical seizures were being given standard treatment with vigabatrin. Preventive treatment was implemented after the onset of epileptiform discharges on videoEEG, but before clinical or subclinical seizures. Conventional treatment included treatment after clinical or subclinical seizures. Thus, EPISTOP included two medical interventions: videoEEG monitoring, which applied to all TSC children in the project, and preventive treatment which was compared to conventional one in the settings of clinical trial.
VideoEEG monitoring enabled implementation of antiepileptic treatment earlier than in any previously reported TSC cohort. It is well established that delayed treatment is associated with significantly worse neuropsychological outcome of epilepsy in TSC infants. The overall neuropsychological outcome in children participating in EPISTOP was better than in any previously reported TSC cohort, likely due to early recognition of seizures and implementation of antiepileptic treatment. Preventive treatment resulted in a significantly longer time to first seizure when compared to conventional therapy. Children who received preventive treatment had also lower risk of clinical seizures, drug-resistant epilepsy and infantile spasms. Conducted analysis also confirmed beneficial effect of preventive treatment on reduction in number of days with epileptic seizures in children with TSC and epilepsy. In children receiving preventive treatment lower number of anti-epileptic drugs necessary to use has been observed. WP7 aimed to identify the clinical (WP2, WP4) and molecular (WP3, WP5) biomarkers of epilepsy co-morbidities: autism and neurodevelopmental delay. Neurodevelopmental assessment was being performed in every child at baseline, and then at 6, 12 and 18 months of age. Follow-up examination was done at the age of 24 months. The changes in biomarkers between the groups with and without autistic features, as well as with and without neurodevelopmental delay have been studied and analyzed. Clinical prognostic factors of neurodevelopmental outcome and early clinical markers of Autism Spectrum Disorders and developmental delay have been identified. With regards to neurodevelopmental outcome, the combined use of BSID (Bayley Scales of Infant Developments) and ADOS (Autism Diagnostic Observation Schedule) can reliably identify TSC infants with a higher risk for ASD at age 6-12 months. Therefore, all children with a diagnosis of TSC should undergo neurodevelopmental evaluations every 6 months of life. In children with BSID developmental quotients at 6 months below the normal range, an early and intensive behavioral treatment should be started. Similarly, children with abnormal ADOS results at 12 months should be warranted a prompt referral to intensive behavioral interventions. Further investigations in this field are warranted, in order to obtain wider sample and a longer follow-up to be able to draw international clinical recommendations. WP8 generated an integrative dissemination strategy, created the dedicated internet platform, prepared educational materials on TSC for various stakeholders, elaborated an exploitation plan and expedited measures to promote teaching in TSC. Tasks within WP8 were focused on EPISTOP project promotion and dissemination.
Project Results:
Clinical study results
The clinical part of EPISTOP was composed of two arms: a randomized clinical trial and an open-label observational study. It was carried out from March 2014 to October 2018 in 9 sites in Europe and 1 site in Australia. The inclusion criteria were as follows: age ≤ 4 months, definite TSC according to the Roach criteria or with genetic confirmation of the disease, no seizures reported by caregivers and no clinical or subclinical seizures on baseline video EEG. Exclusion criteria were: any epileptic seizure, antiepileptic treatment, contraindications to magnetic resonance imaging, and any condition considered by the investigator to hinder participation in the study. The study was approved by local Ethics Committees at all study sites, and caregivers of all participants signed informed consent before enrolment. The study adhered to the International Conference on Harmonization Guidelines for Good Clinical Practice and was conducted in accordance with the Declaration of Helsinki.
At enrollment, all patients underwent magnetic resonance imaging (MRI) of the brain to assess for TSC-related brain lesions. From enrollment to study completion at the age of 24 months, patients attended follow-up visits at a frequency recommended by the European guidelines for children with TSC: every 4 weeks in children ≤ 6months; every 6 weeks in children ≤ 12 months; and every 8 weeks in children > 12 months. At enrollment and each follow-up visit, video EEGs was performed and data on the occurrence, frequency, and drug-resistance of epileptic seizures were gathered. Epileptic seizures were captured on video EEGs or reported by patients’ parents in seizure diaries. At enrollment and every six months thereafter, patients were assessed by a neuropsychologist on the following scales: Bayley Scales of Infant Development (BSID), to assess cognitive development, and on the Autistic Diagnostic Observation Schedule (ADOS), to assess for autism.
Video EEGs were recorded for ≥ 1 hour and included periods of both wakefulness and sleep (up to sleep stage 2); intermittent photic stimulation with ≥ 4 frequencies and eyes open and closed was applied to assess background rhythm reactivity. Video EEGs were assessed for pre-seizure epileptiform activity, subclinical epileptic seizures, and clinical epileptic seizures. Pre-seizure epileptiform activity was defined as multifocal (≥ 2 areas involving both brain hemispheres) epileptiform activity that occurred for ≥ 1% of recording time or as generalized epileptiform activity, including hypsarrhythmia. Subclinical seizures were defined as ictal EEG activity with no video correlate. Video EEGs were assessed independently by two investigators: a local and a central one.
Patients received either preventive or conservative antiepileptic treatment. Preventive treatment was defined as treatment started after pre-seizure epileptiform activity and before the first subclinical or clinical seizure. Conservative treatment was defined as treatment started after subclinical seizures or clinical seizures (see Fig. 1).
Both preventive and conservative treatments were started with vigabatrin and then modified according to clinical judgment. Patients were allocated to preventive or conservative treatment in two study arms: a randomized arm and an observational arm.
In the randomized arm, 6 sites randomly allocated patients to either preventive or conservative treatment in a 1:1 ratio. Eligible for randomization were only patients with pre-seizure epileptiform activity observed before the first subclinical or clinical seizure and recognized by both the local and central EEG readers. Patients with clinical or subclinical seizures were excluded, and patients with normal EEGs were followed-up until study completion. The central investigator (LL) randomly allocated eligible patients to either preventive or conservative treatment. Randomization was stratified for study sites. The treatment was always started after subclinical or clinical seizures, but these patients were excluded from the randomized arm and transferred to the observational arm. In the randomized arm, the treating physician was blinded to EEG data and thus did not know whether the treatment was randomly allocated after pre-seizure epileptiform activity or started after subclinical seizures. In the observational arm, 4 sites started antiepileptic treatment according to their routine practice: in two sites all patients received conservative treatment, and in another two sites all patients received preventive treatment. In the observational arm, the criteria for preventive treatment were the same as the criteria for randomization in the randomized arm. In the observational arm, we also included patients whose parents did not agree to random treatment allocation. All these patients received preventive treatment, because all sites that assigned treatment randomly used the preventive treatment as their routine practice. Moreover, patients who were not eligible for randomization because of prior subclinical or clinical seizures were also included in the observational arm as patients who received conservative treatment. The observational arm was open, and the treating physician had access to EEG data. In both study arms, we excluded patients whose EEGs were interpreted as displaying pre-seizure epileptiform activity only by one of the two readers, i.e. the central or local reader. These patients, with discordant readings, were excluded from the study.
The primary end point was the time from birth to the first clinical seizure. The secondary end points were assess at the 24th month of life and included:
• proportion of patients with clinical seizures
• proportion of patients with drug resistant epilepsy. Resistance to antiepileptic drugs was defined as failure of adequate trials of two tolerated, appropriately chosen and used antiepileptic drug schedules (whether as monotherapies or in combination) to achieve sustained seizure freedom.
• proportion of patients with infantile spasms
• proportion of patients with EEG abnormalities, defined as EEG without pre-seizure epileptiform activity and subclinical and clinical seizures.
• proportion of patients with hypsarrhythmia
• proportion of patients with autism (low autism risk on ADOS)
• proportions of patients with impaired cognitive function (BSID cognitive score < 70).
The safety of patients in the study was monitored by an independent ethics board supported by a biostatistician. Adverse events were recorded during follow-up visits.
Survival analysis with Kaplan-Meier curves and proportional hazards regression were used to study the primary end point among all patients who received at least one dose of preventive or conservative treatment. Survival curves were compared with the log-rank test. In both study arms, differences in the frequency of secondary end points between patients who completed the study on either preventive or conservative treatment were reported as odds rations, with the Fisher’s test of significance. In addition to separate analyses for the randomized and observational arms, we performed a pooled analysis that included both arms. Statistical significance was set at p < 0.05; two-tailed for the primary end points and one-tailed for the secondary end points. All calculations were completed in the R software (version 3.5.2) with the packages tidyverse, survival, and survminer.
Of 101 patients assessed for eligibility, 4 were excluded due to misdiagnosis (Fig. 2).
Of 97 patients enrolled, 54 were included in the randomized arm and 43 in the observational arm. In the randomized arm, 21 patients were not eligible for randomization: 9 had clinical and 6 had subclinical seizures before pre-seizure epileptiform changes, 3 had normal EEG throughout the study, and 3 dropped out before randomization. In 6 patients the central and local analyses of EEG were discordant. Of 27 patients eligible for randomization, 13 were randomly allocated to preventive treatment and 14 to conservative treatment. Of 14 patients randomly allocated to conservative treatment, 1 patient dropped out.
Of 43 patients in the observational arm, 22 patients were recruited in sites with routine preventive treatment and 21 patients in sites with routine conservative treatment. Of the 43 patients, 4 did not need treatment and 7 dropped out. Twelve patients received preventive treatment and 23 patients received conservative treatment; additional 12 patients were transferred to the observational arm from the randomized arm. These 12 patients were not eligible for randomization due to prior subclinical or clinical seizures and thus received conservative treatment (Fig. 2). Baseline clinical and anthropometric characteristics did not differ significantly in both study arms between the patients who received preventive or conservative treatment.
In both study arms, patients who received preventive treatment were about 3 times more likely to remain seizure-free over the study period than the patients who received conservative treatment (randomized arm: hazard ratio, 3.19; 95% CI, 1.22 to 8.32; P=0.013; observational arm: hazard ratio, 2.89; 95% CI, 1.19 to 7.00; P=0.014; Fig. 3). In the randomized arm, the median time to the first seizure was more than twice as long with preventive than with conservative treatment (297 vs. 124 days). In the observational arm, the median time to the first seizure was more than 5 times longer with preventive than with conservative treatment (397 vs. 73 days). At 24 months, in both study arms, patients who received preventive treatment were less likely to have experienced a clinical seizure than the patients who received conservative treatment (odds ratio [OR, 95% CI]; randomized arm: 0.23 [0.02 1.74]; observational arm: 0.35 [0.07 1.7)]) but this difference did not reach statistical significance (P≥0.158,). However, in the pooled analysis, the reduction of the risk of clinical seizures was significant with preventive treatment (OR, 0.33 95% CI, 0.1 1.03 P=0.036).
At 24 months, the risk of drug-resistant epilepsy was lower with preventive than conservative treatment in both study arms (OR [95% CI]; randomized arm: 0.15 [0.02 0.98] p = 0.047; observational arm: 0.29 [0.04 1.42] p = 0.103). The risk of drug-resistant epilepsy was significantly lower with preventive than conservative treatment in the pooled analysis (OR, 0.26; 95% CI, 0.08 0.8; p = 0.013 Appendix 1).
In both study arms, none of the patients who received preventive treatment developed infantile spasms compared to 4 (31%) and 9 (26%) patients on conservative treatment in the randomized and observational arms, respectively (Table 1). In the pooled analysis, preventive treatment was associated with a significantly reduced risk of infantile spams (OR, 0; 95% CI, 0, 0.52; p = 0.003).
The risks of abnormal EEG, hypsarrhythmia, autism, cognitive impairment, language impairment, motor impairment, socio-emotional impairment, and impairment of adaptive behavior were similar with preventive and conservative treatments in both study arms, including in the pooled analysis.
The rates of both all adverse events and serious adverse events were lower with preventive than conservative treatment. There was one death, which was due to cardiac arrest during epilepsy surgery. Compared to children on conservative treatment, children on preventive treatment had lower rates of status epilepticus, febrile seizure, and epilepsy surgery. The rates of other adverse events were similar with preventive and conservative treatment. None of the drugs used in this study were investigational products. The adverse events observed were typical for the antiepileptic drugs used. There were no adverse events related to the preventive treatment.
In conclusion, our findings confirmed that active monitoring with video EEG to start antiepileptic treatment early improves epilepsy-related outcomes in children with TSC. Moreover, preventive treatment, started at the onset of pre-seizure activity, offers an additional benefit.
Molecular study results performed in Work Package 3
The objectives of the molecular studies to be performed on EPISTOP subjects were the following, all with the aim to identify biomarkers of epileptogenesis and epilepsy in TSC patients:
1. Expression (RNA) profiling of serial blood samples by RNA-Seq, with confirmation by Q-RT-PCR.
2. Proteomic profiling of serial serum samples by mass spectroscopy, followed by Western blot or immunoassay confirmation.
3. miRNA profiling of serial serum samples.
4. metabolomic profiling of serial serum samples.
5. Whole Genome Sequencing (WGS) of peripheral blood DNA.
6. 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.
Background:
Tuberous sclerosis complex (TSC) is often a devastating neurodevelopmental syndrome in which seizures, intellectual deficit, autism, and a variety of neuropsychiatric conditions collectively termed TSC-associated neurologic disease (TAND) are seen in the majority of subjects. Seizures occur in over 75% of TSC children prior to the age of 2 years, and early onset and/or poorly controlled seizures are strongly associated with severity of TAND and poor cognitive outcome. Identification of which TSC infants are at risk of seizure development and at which time during their early development (age birth to 2 years) has a high priority, to enable early intervention with anti-epileptic medication to completely prevent or at least significantly modify the trajectory of seizure development.
The EPISTOP cohort of 101 TSC patients has been subjected to detailed molecular studies, as listed above. This detailed molecular analysis makes these TSC infants the most highly studied infants/young children in human history. As such it was our hope and hypothesis at the start of the EPISTOP trial that some combination of these molecular features could be used to predict seizure development in these TSC infants. Here we report the findings of this analysis for each of these molecular studies. We then follow that individual analysis with an integrated analysis of all available data for seizure prediction.
TSC1/TSC2 mutation analysis (item 6 on the list above)
TSC is due to inactivating mutations occurring in either TSC1 or TSC2. Previously it has been known that TSC1 mutations in TSC are associated with a milder phenotype in general than TSC2 mutations. Furthermore, it is known that mosaic mutations in these genes are fairly common in TSC patients, being seen in about 10% overall, and are also associated with a milder clinical phenotype.
One hundred one (101) subjects were enrolled into EPISTOP. Clinical evaluation indicated that 5 of these subjects did not meet diagnostic crieria for TSC, and hence mutation analysis was performed on the remaining 96 EPISTOP subjects with TSC. We used deep massively parallel sequencing (MPS) methods that we have used previously and published to identify single nucleotide variants and small indels, and confirmed each mutation identified by a secondary analysis. Other experimental and computational methods were used to identify large genomic deletions occurring in either TSC1 or TSC2, and such large deletions were also validated by independent methods.
Mutations in TSC1 or TSC2 were identified in 93 of 96 (97%) subjects (Figure 4). 23 (25%) mutations were in TSC1 while 70 (75%) were in TSC2. Eight (of 93, 9%) subjects had mosaic mutations, at allele frequencies ranging from 0.7% to 18%. The types of mutation seen were the following: 8 (9%) insertions, 17 (18%) deletions, 17 (18%) splice, 21 (23%) missense, 21 (23%) nonsense, 7 (7%) large deletions, and 2 (2%) in-frame deletions.
Very few cohorts of infant TSC patients have been analyzed comprehensively for mutations, as we have done here. Our findings of mutations in 97% of subjects is clearly a very high mutation detection rate. It was surprising that 3 subjects had mosaic mutations present at allele frequencies < 5% (0.7%, 3.0%, 4.4%), and yet still met diagnostic criteria with multi-system involvement by age 3 months. This likely reflects that the mosaic allele frequency is different in different tissues and organ systems. The lack of a mutation in 3 subjects is consistent with the possibility (which we have reported in other patients) that these individuals are mosaic with no representation of the mutation in blood DNA.
None of 3 (0%) NMI, 3 of 8 (38%) mosaic, and 56 of 85 (66%) of those with heterozygous TSC1 or TSC2 mutations developed seizures before age 2 years (p=0.0024) indicating that being NMI or mosaic was strongly associated with a reduced risk of seizure development.
Whole Genome Sequencing (WGS) of EPISTOP subjects (item 5 on the list above)
WGS in the EPISTOP cohort provides a wealth of data, including genotype analysis for the 5-10,000,000 common SNPs in the human genome. Although many correlations between SNP alleles and clinical features can be performed, they are vastly underpowered, and a replication population is not available. Consequently, we have chosen to focus our analysis on a panel of 85 SNPs that have been implicated in epilepsy GWAS as having associations with epilepsy risk. We have assessed the association between alleles of these SNPs and epilepsy onset in the EPISTOP cohort, and have identified 3 (of 75) SNPs that show evidence of an association (Table 2).
These findings will be validated in a separate TSC population.
RNA profiling of serial blood samples by RNA-Seq (item 1 on the list above)
Total blood RNA was isolated, globin mRNA depleted, mRNA was isolated by oligo-dT columns, and cDNA libraries prepared by standard methods. Sequencing was performed to a read depth of 36 million reads per sample. After standard processing of read data and alignment to the genome, differential expression analysis was performed using the R package DESeq2, to compare different subsets of samples from EPISTOP subjects at different time points. The primary comparison was made among subjects with different clinical outcomes, considering the RNA-Seq data available from the initial blood samples (V1, V1/VaEEG). Multiple genes were identified that were statistically significantly different between subjects that: had autism spectrum disorder (ASD) or did not at age 2 years; developed refractory epilepsy or did not by age 2 years; etc. In addition, comparison of data for samples obtained at the time of active seizures (VCS, VCSII) with age-matched controls also showed many differences with over 2,000 differentially expressed genes (Figure 5).
Proteomic profiling of serial serum samples (item 2 on the list above)
Serum proteome profiling was performed after depletion of the 14 most abundant proteins by affinity chromatography, followed by digestion with trypsin, and analysis of derived peptides using ultra high performance liquid chromatography (UHPLC; Ultimate 3000) over a 50cm reversed phase column (Dr. Maisch GmbH) online coupled to an Orbitrap mass spectrometer (Orbitrap XL). MaxQuant software was used for peptide identification and quantification. 544 protein groups (462 allowing only Oxidation) from 7405 peptides (6900) were identified.
73 different protein groups were identified by this analysis that varied in one or more comparisons among different subsets of the EPISTOP cohort by age. These protein groups all correlated with age of the EPISTOP subjects (Figure 6).
Beyond these major differences in serum protein levels according to age, there were relatively small differences detected in different clinical groups, e.g. those with seizures versus those without, among the serum protein levels. Nonetheless, some serum proteins may contribute to seizure prediction in the integrated analysis.
miRNA profiling of serial serum samples (item 3 on the list above)
PCR array analysis was performed using miRCURY LNA miRNA Custom PCR panels (Qiagen) on a Roche LightCycler 480 thermocycler to examine the expression of 45 miRNAs in all EPISTOP samples. These 45 miRNAs were chosen based on RNA-seq data performed on a subset of the serum samples, or based on their possible relevance to epilepsy, seizures or cognitive functioning from previous studies.
Seven miRNAs were differentially expressed (all higher) in the TSC subject V1 samples in comparison to controls of age 0-6 months (Figure 7). These miRNAs are candidate biomarkers of TSC which might be useful for diagnosis or monitoring in TSC infants. Within the TSC V1 group, we also compared miRNA values from subjects that later developed clinical seizures (CS) to those EPISTOP subjects who never had seizures (Figure 8). Four miRNAs were differentially expressed, that are candidate biomarkers for seizure development in TSC.
Metabolomic profiling of serial serum samples (item 4 on the list above)
Metabolomic profiling was performed on 274 EPISTOP serum samples and 33 control serum samples, split into seven 50-52 sample batches, on a SCIEX QTRAP 6500 LC-MS/MS mass spectrometry system in Boston, MA. Total Ion Current (TIC) values were determined for each metabolite, which were then scaled within each sample to the total TIC, and then compared among samples within each batch to calculate a normalized value using the median value for all samples in a batch. Eighteen metabolites were identified that differed according to age at P < 10-20, including myo-inositol, kynurenine, hydroxyproline, and phosphocreatine. Furthermore, six metabolites were significantly increased in EPISTOP subjects who were taking vigabatrin (Figure 9). One of these metabolites had been recognized previously to be affected but all the others are novel findings.
These metabolite data were also analyzed to search for metabolites that were different according to TSC status versus controls in an age-matched manner. Interestingly, several cysteine metabolites were somewhat lower in the TSC subject samples in comparison to controls, including cysteine, cystine, and cysteine sulfinate (Figure 10). All of these metabolites are potential biomarkers of TSC for use in diagnosis and prediction of seizure development.
Integrated Analysis of all molecular biomarkers for prediction of seizure risk
An integrated analysis of all data types was performed to generate a series of predictive classifiers of epilepsy risk in infants with TSC.
The following datasets were utilized on V1 samples from EPISTOP subjects:
1) Proteomic dataset - 384 features and 418 samples
2) miRNA dataset - 45 features and 384 samples
3) RNA-Seq data - 63677 features and 339 samples
4) Metabolite dataset - 250 features and 357 samples
5) miRNA sequencing dataset – 20,000 isomir features and 44 samples
6) TSC gene mutation status.
The entire dataset consisted of more than 80 thousand variables characterizing 97 subjects. Hence, there are a very large number of variables, and a relatively small number of patients with outcomes, a typical “curse of dimensionality” problem. To deal with this problem, only variables with an AUC > 0.64 for discrimination of epilepsy status were retained for building the classification models, while all others were discarded. In addition, we limited the number of features from each data type for consideration in the classification model to a maximum of 30, such that altogether 126 variables were considered for classification model development.
We performed logistic regression modelling to build the classification models, since it enables simple interpretation of results, and is relatively efficient from a computational perspective. We also reasoned that inclusion of more than 3 parameters in a model would lead to a greater chance of overfitting with generation of an inaccurate model based on chance alone. Thus, we fit regression models to all 3-way combinations of pre-selected variables, giving 333,501 possible models to evaluate.
Since our classifier was aimed at implementation in clinical practice, we focused on three performance measures for each classifier: Negative Predictive Value (NPV), the chance that classifier is correct when it predicts seizures; Positive Predictive Value (PPV), the chance that classifier is correct when it predicts no seizures; and the Mean Misclassification Error (MME), number of times when the classifier prediction is wrong divided by total number of subjects assessed. We used a strategy in which a model is fitted based on 2/3 of the data and then assessed on the remaining 1/3. Model fitting was done a hundred times for each combination of three markers.
We generated classifiers both with, and without, inclusion of the miRNA sequencing dataset. We considered classifiers without that data set because that analysis had been done on only 44 samples, limiting the sample set available for predictive modeling (Table 3).
Note that the predictors which include the isomiR data (lower set of 10 predictors) are based on a smaller number of samples (because miR sequencing was done on a smaller set of samples), and have corresponding better NPV and PPV on average. Classifiers done without the isomiR data have lower NPV and PPV values, but the larger sample size used for those classifiers may explain those findings.
Hence, this analysis has identified multiple potential classifiers of seizure risk in the EPISTOP cohort, which may have clinical utility for assessing seizure risk in TSC infants through age 2 years. However, since the number of samples analyzed is limited, there is a strong possibility of overfitting this data, so that assessment and validation in a separate population is desired.
Molecular study results performed in Work Package 5
The objectives of this WP were to investigate the biomarkers of epilepsy and molecular targets for novel therapies and validate the peripheral epilepsy biomarkers in human TSC brain tissue.
A systematic evaluation and characterization of specific clinico-pathological subtypes of TSC brain lesions has been performed (see Deliverable no: D5.1). A manuscript on the above reported finding has been published (Novel histopathological pattern in cortical tubers of epilepsy surgery patients with Tuberous Sclerosis, PLOS one, 11(6):e0157396 ; P8 /AMC, P7/UMC, P5/ UHM and P10/ MUW). A study focusing on the white matter pathology in pediatric epilepsy surgery specimens (quantitative histological analysis; P10/ MUW) was carried out and completed. Decreased content of oligodendroglia and missing precursor cells indicated insufficient oligodendroglial development, probably mediated by mTOR, which may ultimately lead to severe myelin loss. A manuscript on the above reported finding has been published (Impaired oligodendroglial turnover is associated with myelin pathology in Focal Cortical Dysplasia and Tuberous Sclerosis Complex. Brain Pathol, 2016; doi: 10.1111/bpa.12452 P8 /AMC and P10/ MUW). We evaluated the pattern of cortical layering disruption observed in brain tissue of TSC patients. A manuscript on the above reported finding has been published (Specific pattern of maturation and differentiation in the formation of cortical tubers in tuberous sclerosis complex (TSC): evidence from layer-specific marker expression., 2016. J Neurodev Disord Apr 1;8:9.; P8 /AMC, P7/UMC, P5/ UHM and P10/ MUW).
We performed a systematic evaluation on a large cohort of TSC brain lesions (n=42; n=9 perituberal cortex; n=17 controls). We analyzed the expression of NeuN, GFAP, pS6, vimentin, MBP, MOBP (myelin-associated oligodendrocyte basic protein), olig2, CD3, CD34, Cr3/43 (MHC-II), PLP and SMI32 separately for grey and white matter in each tuber compared to the perituberal samples and age-matched controls. Immunohistochemistry was digitalized and semi-automated histological quantified. These results were in a first step compared between groups (lesional – perilesional – control). As previously published there is an increase of inflammatory markers in grey and white matter as well as an overall reduction of neurons in the cortex. Furthermore, there is loss of myelin in the white matter. Interestingly, the number of oligodendroglial cells is reduced in the grey and white matter. MBP reflects the oligodendroglial count, nevertheless showing only a slightly reduced myelin content in the lesional cortex without showing significant differences in the amount of axons present (SMI31). Additionally, the results were correlated to TSC tuber subtype (as identified before, see publication above) and clinical parameters as seizure frequency, MRI data, surgery outcome, age at surgery, duration of epilepsy, drug load and more. The reduction of oligodendroglial cells in the grey matter seems to be a rather global phenomenon than comparable with the situation in the white matter (as previously published, see above). The number of oligodendroglia is negatively correlated with the amount of T-cells and microglia (corrected for age). The duration of epilepsy does not influence these findings. The early myelin protein MOBP suggests even dysmyelination and interestingly MOBP and the number oligodendroglial cells in white matter showed a negative correlation the presence of autism, whereas the content of MOBP and number oligodendroglial cells in grey matter showed with the presence of intellectual disability. In the last 6 months data analysis has been finalized and a manuscript is in preparation (A. Mühlebner EPISTOP-WP leaders et al. White matter abnormalities in TSC cortical tubers). We further elucidated the relationship between white matter pathology and inflammation utilizing the previously obtained mRNA sequencing data. Subsequently, we studied the relationship between micro-vascular density and cortical developmental abnormalities. In the last 6 months the data analysis has been finalized and the results indicate an increase in microvasculature in white matter of TSC cortical tubers compared to controls, which is linked to an inflammatory response (Abstract/poster Dutch Neuroscience 2017); a manuscript has been prepared and is presently under revision.
Evaluation of a number of molecular targets for anti-epileptogenic therapy emerging from previous gene expression studies in TSC and different animal models of epileptogenesis (including inflammation, glutamate and adenosine homeostasis and GABAergic transmission and new potential targets) has been performed.
In TSC, overexpression of numerous genes associated with inflammation has been observed. Among different proinflammatory cytokines, interleukin-1β (IL-1β) has been shown to be significantly involved in epileptogenesis and maintenance of seizures. Our results provide the first evidence of epigenetic modulation of the IL-1β signaling in TSC. This study has been finalized and published (Hypomethylation Correlates with IL-1β Overexpression in Tuberous Sclerosis Complex J. Mol. Neuroscience, 59(4):464-70). Large scale DNA methylation profiling and copy number variation analyses (using Illumina Human Methylation 450 array) have been performed in the same cohort of TSC samples used to evaluate small non-coding RNA and mRNA profiles (see below). Data analysis of tubers with different histopathological patterns (A versus B, see PLOS one, 11(6):e0157396) has been also performed.
We investigated the expression pattern of constitutive (β1, β5) and (immune)proteasome (β1i, β5i) subunits using immunohistochemistry in malformations of cortical development. Our observations support the dysregulation of the proteasome system in TSC and provide new insights on the mechanism of regulation of the (immuno)proteasome in astrocytes and the molecular links between inflammation, mTOR activation and epilepsy. Two manuscripts on the above reported finding have been published (Dysregulation of the (immuno)proteasome pathway in malformations of cortical development. J Neuroinflammation 2016; 13(1):202; P8 /AMC, P7/UMC, P5/ UHM and P10/ MUW; Increased expression of (immuno)proteasome subunit during epileptogenesis is attenuated by inhibition of the mammalian target of rapamycin pathway. Epilepsia 58(8):1462-1472. doi: 10.1111/epi.13823) and these findings have been presented during the last 6 months in different international meetings (see dissemination).
Voltage-clamp recordings were performed on Xenopus oocytes “micro-transplanted” with membranes from cortical tubers and age matched control brain tissue. Our data indicate that in human TSC cortical tubers the pattern of GABAAR and GLUA1/GLUA2 function retains features that are typical of an immature brain. A manuscript on the above reported finding has been published (Functional aspects of early brain development are preserved in tuberous sclerosis complex (TSC) epileptogenic lesions. Neurobiol Dis, 2016; 95:93-101; P8 /AMC, P7/UMC, P5/ UHM and P10/ MUW; see dissemination). The effect of C1q and C3 complement components (upregulated in TSC brain) on GABAA evoked current has been evaluated, using oocytes microtransplanted with TSC cortical membranes. The results of these experiments (validated during the last 6 months) indicate that acute co-application with C3 or C1q fraction complements did not show any effect, while incubations higher than 40 min with C3 were effective in decreasing GABAA evoked current reaching the maximum effect after one hour. The decrease of GABAA currents was completely reversible after 20 min of wash with Ringer solution.
Moreover, we transplanted in Xenopus oocytes cell membranes obtained from brain tissues of autopsies of both TSC and Dravet syndrome patients as a pathological comparison, and age-matched controls and evaluated the effect of of cannabidiol on GABAA current . Additionally, experiments were performed on oocytes expressing human α1β2γ2 and α1β2 GABAA receptors. GABAA currents were recorded using the two-microelectrodes voltage-clamp technique. We found an increase of GABAA currents induced by low doses of cannabidiol both TSC and in Dravet syndrome, comparable to that induced by a classical benzodiazepine, flunitrazepam, that still persists in γ-less GABAA receptors. This study has been finalized and published (G. Ruffolo, et al., . Epilepsia, 59(11):2106-2117, 2018).
Collaboration has been established with P14/GenomeScan to characterize small non-coding RNAs and mRNAs in tubers and age matched control cortex, using the Illumina Next Generation Sequencing (NGS) Technology. A weighted correlation network analysis (WGCNA) identified innate immune response, extracellular matrix organisation, neurogenesis and glutamate receptor signalling as important modules in TSC. A manuscript including these results has been published (Coding and small non-coding transcriptional landscape of tuberous sclerosis complex cortical tubers: implications for pathophysiology and treatment. Scientific Reports, 7(1):8089. doi: 10.1038/s41598-017-06145-8 2017).
We further analyzed the expression profile in tubers with different histopathological patterns (A versus B, see PLOS one, 11(6):e0157396). We found a significant difference between A and B. There were 246 genes up-regulated in the type B tubers and 33 up-regulated in the type A tubers. The enriched GO terms and Pathways for the genes up-regulated in the type B tubers indicate a higher inflammatory and immune response in the type B tubers, amongst the pathways the MAPK signalling pathway, TNF signalling pathway and ECM-receptor interaction are enriched and these findings will be included in a manuscript (Angelika Mühlebner et al., in preparation).
Further, we identified the transcription factor Spi-1 proto-oncogene (SPI1) (up-regulated 3-fold in TSC) that may regulate 209/269 of the overexpressed gene in TSC, including the cytokines/chemokines (CCL2, CCL3, CCL4 and IL1β) and complement components (C1QB, C1QC and C3). SPI1 also appears to play a key role in the cellular response to oxidative stress. SPI1 expression is activated in a primary fetal astrocyte culture after stimulation with hydrogen peroxide in a dose dependent manner. Emerging evidence suggests that oxidative stress plays a major role in TSC pathology and the subsequent seizure development. We hypothesize that SPI1 is a master regulator of genes involved in TSC pathology. Interestingly, in line with the findings in human TSC brain tissue genes related to inflammation and immune response, along with both SPI1 homologs (spi1a and spi1b) were also up-regulated in the brain of homozygous tsc2-/- mutant zebrafish (P04/VUB; P09/KUL) (mTOR-related neuropathology in mutant tsc2 zebrafish: Phenotypic, transcriptomic and pharmacological analysis. Neurobiology of Disease, 108:225-237, doi: 10.1016/j.nbd.2017.09.004). In the last 6 months, we are continuing to investigate the role of SPI1 in oxidative stress and inflammation in human cell-lines and experimental models of TSC.
To provide evidence of the role of inflammation-related microRNAs in TSC, we employed real-time PCR and in situ hybridization to characterize the expression of miR21, miR146a and miR155 in TSC lesions (cortical tubers and subependymal giant cell astrocytomas, SEGAs). Our study provides supportive evidence of the role of inflammation-related microRNAs in TSC. In particular, miR146a and miR155 appear to be key players in the regulation of astrocyte-mediated inflammatory response, with miR146a as most interesting anti-inflammatory therapeutic candidate. This study has been finalized and published (P1/IPCZD; P5/ UHM; P7/UMC; P11/IIMCB; P10/ MUW; Expression of microRNAs miR21, miR146a and miR155 in tuberous sclerosis complex cortical tubers and their regulation in human astrocytes and SEGA-derived cell cultures, Glia 64(6):1066-82, 2016; see dissemination). In collaboration with P14/GenomeScan we performed small RNA sequencing in the same cohort used for mRNA profiling. Analysis of data revealed 48 miRNAs which were up-regulated and 10 miRNAs which were down-regulated in TSC tubers compared to control cortex. Unsupervised hierarchical cluster analysis showed a majority of TSC tubers clustering together. Increased expression of miR34a-5p, miR34b-5p, miR34c-5p, miR302a-3p, miR577 and miR21-5p was observed in TSC tubers compared to control cortex. We further investigated the effect of overexpression of miR34b-5p on neurite outgrowth in mouse hippocampal neurons. Neurons transfected with miR34b-5p mimic showed increased number of longer neurites as compared to Scr transfected cultures. A manuscript including these results has been published (Scientific Reports, 7(1):8089. doi: 10.1038/s41598-017-06145-8). In the last six months, we further investigated the role of the miR34 family in TSC. We have found that overexpression of miR34b induces IL-1β pathway activation in human astrocytes. We are also investigating the role of miR34a-b and miR155 in the molecular mechanisms involved in the switch to an inflammatory state after oxidative stress in both neuronal and glial cells in culture. Functional studies focusing on the role of miR34a-5p amd miR34b-5p during corticogenesis using an in utero electroporation model have been also performed and indicate a negative regulation of neuronal migration by both miR34a-5p and miR34b-5p. Further, we have confirmed that Contactin 3 (CNTN3, also known as BIG-1) a gene down-regulated in TSC and involved in neurogenesis is a target of both miR34a-5p and miR34b-5p. More specifically CNTN3 is down-regulated in cortical tubers resected from young patients (<10 years) to levels below those seen in control fetal tissue. Further experiments have been carried out to elucidate the functional relevance of CNTN3 and include over-expression studies by the CRISPR-dCas9-VP64 activation system, and inhibition by siRNAs in the human neuronal cell line SH-SY5Y, coupled with differentiation experiments by treating the cells with retinoic acid supplemented with brain derived neurotrophic factor. This research project is ongoing, and the data produced was presented at the Amsterdam Annual Neuroscience meeting (The altered expression of cell adhesion molecule contactin-3 in tuberous sclerosis complex, Korotov, et al. 2018) and results are included in a manuscript in preparation (A. Korotov, J. Mills et al., in preparation).
llumina NGS was also employed to identify small non-coding RNAs and mRNAs related to the IL-1β pathway in human astrocytes. 54 mRNAs were detected as differentially expressed in human astrocytes stimulated with IL-1β compared to controls (unstimulated cells). We detected a strong up-regulation of both miR146a and mir147b. We further evaluated the function of miR147, a microRNA that is induced by IL-1β - receptor stimulation. The functional effect of overexpression of miRNAs on inflammatory signaling was studied by transfection of astrocyte cultures with miRNA mimic oligonucleotides. Overexpression of miR147b inhibited IL-6 and COX-2 up-regulation after IL-1β stimulation. Moreover, miR147b overexpression down-regulated its predicted targets DEPTOR and ADAM15. To determine whether miRNA transfection could restore altered proliferation and generation of astrocytes seen in inflammatory related pathologies, immunocytochemical Ki67 staining and flow cytometric analysis of propidium iodide staining was assessed. Indeed, transfection of astrocytes with miR146a and miR147b both decreased proliferation of astrocytes. The effect on cell fate determination in neuronal stem cells (NSCs) derived from fetal brain tissue was examined by immunocytochemical staining of transfected, differentiated stem cells. Transfection with both miR146a and miR147 promoted neuronal differentiation of NSCs, supporting the role of these miRNAs deregulated in TSC in NSC differentiation. (miR147b: A novel key regulator of interleukin1 beta-mediated inflammation in human astrocytes. Glia, 66(5): 1082-1097, doi: 10.1002/glia.23302 2017). These findings have been presented during the last 6 months in different international meetings (see dissemination).
Based on earlier findings in by the Jaworski’s group (IIMCB) in blood cell derived RNA from TSC patients and controls, a collaborative pilot study was carried out to assay the expression of 6 genes (RAB11A, PAK2, GPRC5A, SF3A1, GOS2 and IL8) in TSC cortical tubers and control cortex by RT-qPCR. In validation study, GPRC5A showed significantly increased expression (P=0.006) in TSC tubers compared to control cortex, whereas RAB11A showed a significant decrease (P=0.02) and the other genes remained unchanged. GPRC5B encodes an orphan G protein-coupled receptor (which has been suggested to play a role in neuronal fate determination of cortical progenitors) and will be further evaluated and validated within the EPISTOP cohort. Furthermore, based on our previous results and sequencing data from TSC brain tissue and serum of experimental models of epileptogenesis a set of inflammatory genes including cytokines/chemokines (CCL2, CCL3, CCL4, IL1β, IL6 and COX2) and complement components (C1QA, C3, and C4B) have been assayed in TSC brain tissue. We also evaluated the matrix metalloproteinase genes (MMP2, MMP3, MMP9 and MMP14) showing significantly increased expression of MMP9 and MMP14 but not of MMP2 and MMP3 in the TSC tissue. Using immunohistochemistry, we found up-regulation of MMP2, MMP3 and MMP14 in glial cells. High immunoreactivity of all mentioned MMPs was seen in dysmorphic neurons and giant cells. We also studied the regulation of MMPs by miRNA in astrocytes, showing a modulation of MMP3 by miR132, miR146a, miR155 in human astrocytes. These findings have been included in a manuscript that has been submitted and is presently under revision (D. Broekaart, J. van Scheppingen et al.; Increased expression of matrix metalloproteinases associated with tuberous sclerosis complex can be attenuated by microRNAs 146a and 147b).
Recent studies indicate that administration of antioxidant drugs reduced neuroinflammation and improved disease outcomes in a rat model epilepsy and oxidative stress related molecules may be potential biomarkers of epileptogenesis. We will further study the role of oxidative stress in TSC evaluating the induction and cellular distribution of oxidative stress markers (i.e. HMGB1, iNOS, Xct and Nrf2; and DNA damage markers) in TSC specimens, as well as the molecular mechanisms involved in the switch to an inflammatory state after oxidative stress (Task 5.4). This research has resulted in one accepted publication (Oxidative stress and inflammation in a spectrum of epileptogenic cortical malformations: molecular insights into their interdependence, Brain Pathology, 2019 May;29(3):351-365. doi: 10.1111/bpa.12661) and one manuscript in preparation (Modulation of astrocyte antioxidant signaling by microRNA-155 and its implication in epileptogenic malformation of cortical development, manuscript in preparation). Additionally, human TSC tissue was used to validate potential peripheral epileptogenesis biomarkers such as inflammatory markers (IL-1β and immunoproteasome subunits; J Neuroinflammation 2016; 13(1):202) J. Mol. Neuroscience, 59(4):464-70, 2016; see dissemination) and miRNAs, including the miR34 family (Scientific Reports, 7(1):8089, 2017; see dissemination) and inflammation-related miRNAs (miR146a and miR147 Glia 64(6):1066-82, 2016; Glia, 66(5): 1082-1097, 2017; see dissemination). Based on our previous results of small RNA-Sequencing of data from TSC brain tissue and TSC serum samples (first stage analysis) we identified miRNAs of interest to be assayed in the entire cohort. Inflammatory related miRNAs (miR21, miR146a, miR147 and miR155; as potential peripheral epilepsy biomarkers) were validated in TSC cortical tubers (see also Task 5.4). These findings have been presented during the last 6 months at different international meetings (see dissemination).
Potential Impact:
The potential impact
EPISTOP was the first study to address the unmet need for prevention of epilepsy and its comorbidities in clinical settings. Epilepsy affects over 6 million people in Europe is associated with an annual societal and healthcare cost of 20 billion Euro. Although new antiepileptic drugs continue to be introduced into clinical practice, freedom from seizures is not achieved in about 30% of patients. Furthermore about 60% of children with epilepsy suffer from chronic and irreversible neuropsychiatric comorbidities, including learning disabilities and autism. Thus, prevention of epilepsy is widely recognized as a “holy grail” in the field of epilepsy care.
Tuberous Sclerosis Complex (TSC) is a major cause of early onset epilepsy, occurring in the first year of life. Given the incidence of TSC, we estimate this disorder affects approximately 100,000 Europeans and one million people worldwide. Before EPISTOP, it was reported that about 90% of TSC patients suffered from epilepsy and over 60% had some form of intellectual disability. In more than 65% of patients, epilepsy begins in childhood. About 50% of children with epilepsy suffer from psychiatric and behavioural comorbidities, such as learning disabilities, and autism spectrum disorders (Ono, 2012). Prevention of epilepsy and its comorbidities in patients at risk presents a major unmet need in clinical medicine, but there were no strategies aimed to prevent of modify the natural course of epilepsy development. EPISTOP results can be exploited directly by using prevention protocols in clinical practice, and indirectly, by stimulation of new research projects, including ones targeted towards validation of EPISTOP results in other types of epilepsy.
In the EPISTOP clinical trial, we developed a set of defined changes on EEG to serve as a non-invasive biomarker of epileptogenesis. With this novel development, we were able to identify infants at high risk of epilepsy. Indeed EEG surveillance has recently been proposed by EPISTOP investigators as an important element of routine clinical care for TSC infants, enabling early diagnosis (before clinical seizures) of epilepsy development and preventive treatment (Curatolo et al. EJPN 2018). With EPISTOP we showed that this approach enables identification and treatment of these earliest forms of seizures, whether clinical or electrographic, and, in consequence, reduces epilepsy severity and the risk of intellectual disability and autism. Recently many other investigators have reported the beneficial effect of EEG survaillance implementation (Whitney et al. Ped.Neurol 2017, Benova et al. EJPN 2018, Chung et al, Ped.Neurol 2017). A key part of the EPISTOP study was to investigate the outcome of TSC children who received treatment based on EEG findings. In a randomized clinical trial we found that preventive treatment with vigabatrin reduced the risk of clinical seizures and drug-resistant epilepsy when compared to treatment after clinical or electrographic seizures.
These findings have major significance for children with TSC and their families. EPISTOP provides evidence that early EEG monitoring and preventive treatment reduce the risk of severe epilepsy, and its neurodevelopmental co-morbidities. It is established that the societal burden of epilepsy and its comorbidities depends on their severity and is higher in patients receiving more than 1 antiepileptic drug or presenting with intellectual disabilities (Jones et al, 2019; Riechmann et al, 2015). Thus management of TSC according to EPISTOP protocol including early intervention should improve the quality of life of many individuals and families coping with TSC. Our new treatment is not associated with additional costs for the patients or healthcare system, as we used a standard antiepileptic drug, vigabatrin. Furthermore, in patients in whom epilepsy is prevented, antiepileptic treatment might be withdrawn at a younger age, reducing their lifetime exposure to treatment and total costs of medication in comparison to patients presenting with clinical seizures. The results of preventive treatment in EPISTOP are being disseminated to TSC and epilepsy communities who are direct beneficiaries of the project.
Neuropsychological analyses in EPISTOP showed for the first time with that the results of tests performed at youngest ages: 6 and 12 months, the risk of autism and learning disability can be predicted. These findings enable the identification of patients at risk of neurodevelopmental comorbidities of epilepsy and implementation of early intervention. Moreover, the risk of severe epilepsy and neurodevelopmental comorbidities of epilepsy can be predicted by using prenatal brain MRI results, which enables more precise indication for early intervention. All these findings are being submitted for publication in professional journals and disseminated to TSC and epilepsy communities.
EPISTOP is a strong, multidisciplinary and comprehensive consortium, which was built to perform the first prospective human study of biomarkers of epileptogenesis beginning from its latent phase, through active epilepsy and chronic disease. Four out of 8 WP leaders were female. Thus, EPISTOP contributed to the development of sustainable and gender-equal research community.
With a comprehensive study of biomarkers, EPISTOP was able to identify the risk factors of the development of epilepsy and its comorbidities. Early recognition of patients at risk will allow personalized early interventions, including prevention. Moreover, clinical and molecular characterization of epilepsy comorbidities in children might contribute to the future studies aimed to identify the targets for innovative and personalized medications, especially in rare and complex disorders of the nervous system.
Given that EPISTOP has generated interest in preventive strategies broadly in the epilepsy community, it has influenced research directions in epileptology. This includes the ongoing randomized controlled trial PREVENT in the US (NCT02849457) started in 2016. The study design in PREVENT is similar to EPISTOP: it is based on the prevention trial in TSC patients. The differences include the use of placebo in PREVENT trial and the inclusion of biomarker studies in EPISTOP. Preliminary results of EPISTOP have been presented at multiple international meetings with various stakeholders, including clinicians, researchers, patients’ advocacy groups and organizations, research funding agencies since 2015. EPISTOP operated closely with other EU-funded projects on epilepsy (EPImiRNA, EPITARGET, EPIPGX, DESIRE) within the EU EpiXchange initiative, as well as with European Reference Network (ERN) for rare and complex epilepsies (EPICARE) and EC and NIH to promote epilepsy research and indicate biomarker and prevention studies as priorities in EU and US research policies.
The main dissemination activities and exploitation of results
At the consortium level
Meetings were organized on a regular basis (every 6 months). Steering Committee meetings were attended by all WP leaders and sometimes the EC representative. These were alternated with General Assembly meetings which were attended by all EPISTOP collaborators and the members of the Scientific Advisory Board (SAB).
At the kick-off meeting, representatives of the Polish minister of Health and local media were present as well. The kick-off meeting took place in Warsaw. All consortium members, representatives of Ethics Board and SAB participated in the meeting. The kick-off meeting started with an opening session for public and invited guests. Lectures about the project were given by prof. Sergiusz Jóźwiak, prof. Lieven Lagae, and prof. David Kwiatkowski. The audience was addressed by Mr. Igor Radziewicz-Winnicki, Undersecretary of State of the Ministry of Health, prof. Małgorzata Syczewska, Director of The Children's Memorial Health Institute, prof. Jacek Kuźnicki, The International Institute of Molecular and Cell Biology, and Anita Kucharska, European Commission. Prof. Jerzy Buzek addressed words of support to the scientists. There was also a press conference (reports and information about the project were published in number of newspapers, magazines, radio and TV programs). After official part followed sessions with all consortium partners with lectures of all WPs’ leaders and representatives of EC, including Anita Kucharska, Małgorzata Urbaniak. At the same time also SAB members and members of the Ethics Board met in Warsaw. The meeting was organized by IPCZD. Pictures are attached.
The closing conference was also organized in Warsaw. The conference started with an opening session for public and invited guests. There were also journalists from television, internet, radio and magazines. The leaders presented a summary of the results achieved and work done in each of the work packages. There were external experts invited by IPCZD, including members of SAB (Annamaria Vezzani, Marcin Balcerzak, Leszek Kaczmarek), and members of the Ethics Board (Katarzyna Łukasiuk, Andrzej Chaberka). Mr. Grzegorz Owsianik, EC representative, attended the final conference and the final meeting. During the second day of the meeting, two different events took place. The clinical partners discussed their results during an educational meeting for TSC clinicians and patients, organized in collaborations with e-TSC, the European TSC patient organization. At the same time partners from the molecular group took part in the workshop entitled "Current challenges in epileptics and TSC molecular biomarkers development" organized by IIMCB (partner in the EPISTOP project). This workshop was also attended by scientists form different Institutes from Poland and other countries. It was a unique possibility to discuss the results obtained in the EPISTOP project with different scientists and present the molecular and computational methods used during the project. In addition, this workshop allowed further dissemination and exploitation of the results obtained by the EPISTOP scientists. Pictures are attached.
The EPISTOP website (www.epistop.eu) was regularly updated (all EPISTOP-related documents per work-package on the password-protected section, EPISTOP-related publications, announcement of consortium meetings through the banner, news updates, overview of internal (General Assembly and Steering Committee meetings) and external meetings (meetings attended by EPISTOP WP leaders) were published). Information about the project was published in English, Polish, Dutch, German, Czech, French and Italian.
At the public level
The project lifespan the progress of study has been discussed in over 40 lectures at national or international conferences or educational meetings. The EPISTOP project was referred to in more than 60 publications and posters published in peer reviewed international journals or presented at national and international meetings.
Details on the EPISTOP initiative (Part 1) as well as on the results (Part 2) were summarized in two publications in the IMPACT journal which were distributed to all consortium members and IMPACT journal readers EU-wide.
The EPISTOP website was used as a tool to communicate with the broader TSC community as well, using especially the NEWS section to share the progress and initiatives of the consortium. An English and Polish Facebook page have been launched (https://www.facebook.com/epistop.TSC/ and https://www.facebook.com/projektepistop/) for education and dissemination purposes. Updates were done regularly throughout the project, with announcements and feedback of events, educational information as well as daily facts on TSC during the month of May, TSC awareness month. Twitter and Instagram accounts were used as well to regularly update the general public and TSC research community on EPISTOP project activities. Publications were shared and highlighted through a project page on ResearchGate.
At the start of the project, flyers were developed to inform the community about the EPISTOP project and to promote patient recruitment for the clinical trial. These flyers were translated in all EPISTOP consortium languages and distributed through all participating clinical trial centers and affiliated patient organizations.
In the beginning of the project a calendar was made to improve visualization of the project. It was handed out to all EPISTOP partners and referring centers. We also prepared promotional gadgets to enhance visualization of the project including t-shirts, sunglasses, shopping bags, and note-blocks
The set-up of the EPISTOP project was explained in a first animated movie which was prepared at the beginning of the project. This movie has been viewed over 4.500 times through the EPISTOP Facebook page. It has been subtitled in all languages of the consortium members and was used to highlight the project during multiple lectures worldwide. A special edition with Japanese subtitles was prepared for the International Research Conference on TSC in Tokyo in 2018. A second movie summarizing the results of the EPISTOP project was finalized at the end of the project and was presented during the Closing Meeting in Warsaw as well as the International TSC Research Conference in Toronto, Canada in 2019. Below you will find links to mentioned movies:
http://epistop.eu/index.php/news/31-epistop-promotional-film
https://www.facebook.com/epistop.TSC/videos/599485826841755/
http://epistop.eu/index.php/news/198-epistop-results-summarized-in-animation-movie
EPISTOP was part of the EPIXCHANGE conference organized on May 23rd 2018 in Brussels by seven large EU-funded projects (including EPISTOP) to bring together Europe’s best brains to pave the way for future epilepsy research. Prof. S. Jozwiak presented the EPISTOP project during the plenary session and the symposium was attended by several EPISTOP Steering Committee members. The results of the EPIXCHANGE meeting led to the publication of a manuscript by Pitkänen et al entitled ‘Advancing research toward faster diagnosis, better treatment, and end of stigma in epilepsy’ published in Epilepsia in 2019. Below you will find the interview with Prof. Sergiusz Jóżwiak:
https://www.youtube.com/watch?v=ynW-JeIyrvs
Two educational meetings were organized to increase awareness on TSC and educate the general TSC and medical community on the key-aspects of the EPISTOP project, including early diagnosis, regular EEG monitoring, preventive treatment of epilepsy, and early monitoring and intervention for developmental delay and autism. Both meetings were co-organized by e-TSC, the European Tuberous Sclerosis Association. The first took place in Leuven, Belgium in the fall of 2018, the second in Warsaw following the closing conference.
During the project two PhD students publicly defended their PhD thesis. Chloë Scheldeman presented her work on ‘Zebrafish-based modeling of human genetic epilepsies: FHF1 and TSC2 genes’ and Jackelien van Scheppingen presented her research on ‘Astrocytes as mediators of inflammation in epilepsy: focus on tuberous sclerosis complex’.
Dissemination of the results
The EPISTOP project has led to a critical advancement of knowledge in the field of TSC and of epilepsy more in general. The key-findings will be disseminated through publications in international peer-reviewed journals and presentations at scientific and educational meetings. Various novel scientific concepts will lead to for further validation studies and might in a next step result in collaborations with industry.
The need of early TSC diagnosis will be disseminated with scientific publications to be implemented into routine clinical practice. The stakeholders include obstetricians, gynecologists, neonatologists, perinatologists, pediatric cardiologists, pediatric neurologists, paediatricians, and other medical professionals.
Presymptomatic EEG monitoring in all TSC infants will be disseminated with scientific publications to be exploited as a new routine clinical practice. The stakeholders include pediatric neurologists, pediatricians, neurologists, and psychiatrists. They can be informed about the results of the project and its significance using scientific publications and presentations on meetings and conferences of the professional societies. This new treatment concept should also be disseminated among patients’ advocacy groups and organizations, including TSC associations in individual countries, as well as TSC Europe, EURORDIS, and others.
Preventive antiepileptic treatment in TSC infants will also be disseminated with scientific publications to be introduced into clinical practice. The stakeholders include pediatric neurologists, pediatricians, and neurologists. Given that TSC is a rare disorder, the organizations and institutions supporting rare diseases including European Reference Networks, EU DG Santé, ministries of health in individual countries will be subjected to the exploitation of the results of EPISTOP.
The concept of epilepsy prevention in TSC should be validated in other types of epilepsies in future clinical trials. It might be exploited to research policy makers in order to promote calls and funding for such clinical trials. Exploitation will be discussed with epileptology professionals, including International League Against Epilepsy, neurology associations, including European Paediatric Neurology Society, Child Neurology Society, and others. The need for future epilepsy prevention studies should be discussed with EC research officers, NIH, and local research agencies in individual countries. In the US, a similar study aimed to assess the results of preventive antiepileptic treatment in TSC infants, called PREVENT (ClinicalTrials.gov Identifier: NCT02849457), is currently recruiting patients. It will be of utmost interest to compare the findings in both studies and to share experience from both trials. The EPISTOP and PREVENT (when completed) studies results should be exploited together when discussing the future research plans both in EU and in the US. National Institute of Health admitted importance the EPISTOP project inviting Prof. Jozwiak to the by „Accelerating the Development of Therapies for Anti-Disease Modification Workshop” in Rockville, Maryland,United States.
The need for early assessment and intervention for developmental delay and autism in TSC infants and children will be disseminated to patient associations (Tuberous Sclerosis Europe and Autism Europe) in order to allow a broader diffusion of results to help parents with a child with an early diagnosis to search for a prompt referral and a close neurodevelopmental monitoring.
The MRI protocol and lesion segmentation technique used in this study will be disseminated with scientific publications to be exploited as a new routine clinical practice. The stakeholders include pediatric neurologists, pediatricians, and neurologists. The lesion segmentation technique can be considered as a first step towards the development of fully automated lesion segmentation.
The finding that multiple serum proteins, metabolites and microRNAs show marked changes from birth to age two years may be exploited to discover abnormalities in these developmental changes that may provide diagnostic information for the care of children who are not developing properly.
Several biomarkers were identified as being associated with epilepsy development. New research activities that will derive from these findings are replication of the findings in other TSC and epilepsy populations.
Insights in the molecular mechanisms underlying the mammalian target of rapamycin (mTOR)-related epileptogenesis and associated comorbidities (intellectual disability, neurobehavioral and psychiatric disorders) were obtained, identifying a number of potential molecular targets for antiepileptogenesis or disease-modifying therapies (i.e. inflammation, oxidative stress). This can result in new therapeutic strategies, which however need to be further evaluated and validated in preclinical and/or clinical studies in a separate patient cohort. These molecular insights provide important information for the development of novel therapeutic approaches beyond EPISTOP in patients with TSC, but also in other developmental brain diseases that share the same hallmarks of immaturity leading to intractable seizures and associated complex neurological and neuropsychiatric phenotypes.
List of Websites:
http://epistop.eu/
https://www.facebook.com/projektepistop/
https://www.facebook.com/epistop.TSC/
