In drug-resistant epilepsy, Stereo-Electroencephalography (SEEG) monitoring is one of the most effective techniques to identify the Epileptogenic Zone (EZ), the fundamental prerequisite for epilepsy surgery. Despite recent technological advances, SEEG recordings remain difficult to interpret, and SEEG-guided surgery still achieves success rates below 70%. In this work, we develop a novel computational framework for SEEG analysis, with the ultimate aim of improving the accuracy in EZ definition. Specifically, we investigate the hypothesis that epileptogenic regions exhibit a tendency to behave independently and thus desynchronize from neighboring brain structures before seizure onset. To this end, we design the Desynchronization Index (DI), an algorithm that identifies the Epileptogenic Zone (EZ) as the subset of channels that disconnect from the SEEG network during the ictal transition. We evaluate the DI algorithm against Epileptogenicity Index (EI), one of the most common tools for EZ definition, on a clinical dataset of 20 patients, considering the channels that were thermocoagulated at the end of SEEG monitoring as the detection target. Our results show that DI overcomes EI in terms of area under the ROC curve (AUC =0.86 vs. AUC =0.83), while combining the two algorithms into a single framework leads to the best performance (AUC =0.88). Overall, the DI algorithm underscores anomalous connectivity patterns that are difficult to detect through visual inspection, improving the accuracy in the EZ definition and providing new insights into the dynamics of seizure generation.

OBJECTIVE:Lafora disease (LD) is a rare and severe form of progressive myoclonus epilepsy characterized by cognitive decline that ultimately leads to dementia. However, its cognitive features and adaptive functioning remain poorly understood. This study aimed to characterize the neuropsychological profile of LD and its progression. METHODS:In this monocentric cohort study, consecutive LD patients were administered a battery including cognitive measures (Leiter-3, Children's Color Trail Test [CCTT], Wechsler Adult Intelligence Scale-IV Vocabulary, and Visual-Motor Integration [VMI] test) and the Vineland Adaptive Behavior Scales, Second Edition (Vineland-II). Longitudinal follow-up data were collected after 12 months. Scores were normalized into z-scores for analysis. RESULTS:Fourteen patients (six females) were included. Mean age at onset was 12.6 ± 2.0 years (range = 10-17), and mean age at baseline assessment was 16.5 ± 2.6 years (range = 13-19). Mean Leiter-3 intelligence quotient (IQ) was 79.8 ± 22.8 (range = 45-120); IQ was impaired in 10 of 14 patients (71%; extremely low, n = 6; borderline, n = 4). Executive dysfunction was the most prominent deficit; impairment was observed in 12 of 14 (86%) on CCTT, nine of 12 (75%) on sustained attention, eight of 12 (67%) on congruent Stroop, and 10 of 12 (83%) on incongruent Stroop. VMI was impaired in 10 of 14 (71%), and verbal abilities in four of seven (57%). Mean Vineland-II Adaptive Behavior Composite score was 82.7 ± 33.7 (range = 20-128), in the moderately low range, mainly driven by reduced communication scores (75.9 ± 36.3, range = 20-120). Nine patients completed 12-month follow-up, showing significant decline in IQ, executive functions, and visuomotor integration (all p < .05), and adaptive functioning showed a downward trend. SIGNIFICANCE:LD is characterized by prominent executive dysfunction accompanied by reduced adaptive communication skills, even in the initial stages. The CCTT was sensitive in detecting early deficits and cognitive decline, supporting its use in clinical practice and research. These findings highlight the need for standardized neuropsychological testing and support its role in tracking disease progression and as an outcome measure in clinical trials.

To evaluate the long-term efficacy of vagus nerve stimulation (VNS) in reducing tonic-clonic seizures (TCS), drop attacks, and seizure clusters in adults with drug-resistant epilepsy (DRE). This retrospective, single-center study included adults with DRE who received VNS and had ≥ 12 months of follow-up. Data were collected pre-implantation (T0), at 12 months (T1), and last follow-up (T2). Outcomes included reduction in total seizure frequency and severity, frequency of TCS and drop attacks, and frequency/duration of seizure clusters. Battery replacement and tolerability were also assessed. Eighty-seven subjects (51 males, median age 33 at T0) with a mean follow-up of 8 years were analyzed. At T2, 54

Although mass spectrometry-based approaches are increasingly used for protein biotherapeutic bioanalysis, validated workflows remain limited for fusion proteins across multiple clinical matrices, including cerebrospinal fluid (CSF). We developed a microflow liquid chromatography-high-resolution mass spectrometry (HRMS) bottom-up proteomics assay for VAL-1221, an investigational fusion protein (cell-penetrating antibody Fab linked to recombinant human acid α-glucosidase). HRMS data were acquired in data-dependent acquisition (DDA) mode to support untargeted characterization of the digest and select a linker-containing signature peptide. Quantification was performed by extracted-ion chromatogram (XIC) integration of a quantifier signal, with confirmation by a qualifier signal. The method was validated in plasma and cerebrospinal fluid, including assessment of selectivity, linearity, precision and accuracy, matrix effects, recovery, and stability. The assay was linear over 68-1700 nM in plasma and 6.8-1700 nM in CSF (coefficients of determination ≥ 0.998), with lower limits of quantification of 55.8 and 5.2 nM, respectively, and precision and accuracy were within 15%. The matrix factor was 80.5% in plasma and 102.4% in cerebrospinal fluid. Applied to compassionate-use samples from five patients with Lafora disease receiving intravenous VAL-1221 (20 mg/kg every other week), VAL-1221 was detected in plasma up to 4 h post-infusion, whereas cerebrospinal fluid concentrations were below the method detection limit under the tested regimen. Overall, this workflow provides a validated, sequence-specific approach for exposure and biodistribution assessment of fusion-protein biotherapeutics in human matrices.

OBJECTIVE:The AMPA study (Study 501; NCT04257604) was a multicenter, prospective, 12-month observational study in Italy that evaluated the effectiveness and safety of adjunctive perampanel in patients with focal-onset seizures (FOS), with or without focal to bilateral tonic-clonic seizures (FBTCS). METHODS:Patients aged ≥12 years with insufficiently controlled FOS, with or without FBTCS, receiving 1-3 anti-seizure medications (ASMs) were prescribed adjunctive perampanel per the approved indication. The primary endpoint was the median percent change in total seizure frequency per 28 days from baseline at Month 6. Baseline seizure frequency per 28 days was calculated using seizure diaries and/or medical records of seizures occurring in the 8 weeks prior to the baseline visit while patients were receiving 1-3 ASMs. Treatment-emergent adverse events (TEAEs), including serious TEAEs, were monitored for up to 12 months. RESULTS:Of the 240 patients enrolled in the study, 234 were included in the Full and Safety Analysis Sets. Median age (minimum, maximum) was 36.0 years (12, 84) and 51.3% (n = 120/234) were female. The majority of patients (77.8% [n = 182/234]) received ≥2 concomitant ASMs at baseline, with the most common being carbamazepine (33.8% [n = 79/234]). The median percent reduction in total seizure frequency per 28 days from baseline (95% confidence interval) was 55.4% (46.7%-66.7%) at Month 6. Overall, the retention rate was 57.3% (n = 134/234) following 12 months of treatment. During the study, the overall incidence of TEAEs was 56.4% (n = 132/234), with the most frequently reported TEAE being dizziness/vertigo (21.8% [n = 51/234]). Serious TEAEs were experienced by 6.0% (n = 14/234) of patients and no deaths were reported during the 12-month treatment period. SIGNIFICANCE:Data from the AMPA study suggest that adjunctive perampanel is associated with improvement in seizure control and with good retention rates and tolerability in a real-world clinical setting. These findings further support the use of adjunctive perampanel as a suitable treatment option for adolescent and adult patients with epilepsy. PLAIN LANGUAGE SUMMARY:Our study looked at teenage and adult patients with epilepsy in Italy who took the study drug, called perampanel, as well as the epilepsy treatments they had already been prescribed. After 12 months, 134 out of 234 patients were still using perampanel. Patients taking perampanel had fewer seizures than they did before they started taking perampanel. Side effects occurred in 132 patients (most commonly dizziness/vertigo, irritability, and sleepiness) and caused 45 of them to withdraw from the study. Perampanel was a suitable treatment option for teenage and adult patients with epilepsy.

BackgroundEpilepsy significantly impacts on morbidity and mortality. Understanding hospitalization and mortality risks in persons with epilepsy (PWE) is essential for improving healthcare strategies. We aimed to investigate the risk and causes of hospitalization and mortality in PWE compared to a matched general population cohort.MethodsThe EpiLink Bologna historical cohort study analyzed adult PWE in the period 2018-2019. A general population control cohort was used for comparison. Clinical data were linked with health administrative data. PWE were grouped into persons with focal epilepsy, idiopathic generalized epilepsy, and developmental and/or epileptic encephalopathy (PDEE). The primary outcome was the hospitalization rate. Emergency department (ED) visit rate and the risk of death for any cause were also assessed.ResultsThe study included 1438 PWE and 14,096 controls. PWE had higher incidence rate ratio (IRR) for ED visit (IRR 1.26, 95% CI 1.20-1.32), hospital admission (IRR 2.05, 95% CI 1.83-2.29), and death (IRR 1.5, 95% CI 1.1-2.2) compared to control cohort. The highest hospitalization risk was in the PDEE group (IRR 4.70; 95% CI 3.28-6.74). The increased hospitalization rate among PWE was due to both their higher ED visit and elective hospital admission rates. PWE on polytherapy were at higher risk of hospitalization for inflammation of jaw, acid-base/electrolyte imbalances, chronic cerebrovascular disease, major traumas and infections.ConclusionsDuring a 2-year-period, PWE in Bologna had a doubled risk of hospitalization and 50% higher risk of death compared to a matched general population cohort. Hospitalization risks varied significantly by epilepsy type and antiseizure therapy.

Lafora disease (LD) is a fatal childhood progressive myoclonus epilepsy and glycogen storage disease that is caused by recessive mutations in either EPM2A or EPM2B. The hallmarks of LD are cytoplasmic, aberrant glycogen-like aggregates, called Lafora bodies (LBs), that drive disease progression. The 9th Annual Lafora Science Symposium was held in San Diego, California and brought together over 70 researchers, clinicians, academic trainees, and friends and family members of patients with LD and 80 attendees joined virtually. This symposium focused primarily on international collaborations for therapeutic development and biomarker identification and strategies for preparing the Lafora community for upcoming clinical trials.

Seizures are a common and challenging symptom in brain tumors, affecting approximately 60% of patients. Tumor-related epilepsy (TRE) in glioma patients requires personalized and dynamic management in a multidisciplinary environment, especially for its intricate pathophysiology and unpredictable disease evolution. This investigation provides an updated overview about the pathophysiological mechanisms and treatment options of TRE associated with gliomas, based on expert contributions belonging to different areas. By combining the most recent discoveries and expert opinions, this study seeks to provide useful advice for TRE management in glioma patients. To improve patient outcomes and quality of life, prospective, standardized, multicentric studies should be promoted to optimize TRE patient care and refine therapeutic approaches.

Background: VAL-1221 is a biotechnological fusion protein that combines the Fab portion of a cell-penetrating antibody with recombinant human acid alpha-glucosidase. Originally used for the treatment of Pompe disease, it has since attracted interest for possible repurposing in Lafora disease (LD), an ultra-rare, fatal form of progressive myoclonus epilepsy characterized by the accumulation of polyglucosan aggregates (Lafora bodies, LBs) within the central nervous system (CNS). Given its design, which includes a cell-penetrating domain, VAL-1221 has been hypothesized to cross the blood-brain barrier (BBB) and target pathogenic glycogen deposits within the CNS in LD. This study aimed to investigate the presence of VAL-1221 in plasma and cerebrospinal fluid (CSF) of LD patients and assess its potential to cross the BBB, using high-resolution mass spectrometry coupled with micro-liquid chromatography (microLC-HRMS/MS). Methods: As part of a compassionate use program, five LD patients received intravenous VAL-1221 (20 mg/kg, every other week). LD untreated patients were included as controls. Plasma samples were collected at multiple time points up to 24 hours post-infusion, and CSF samples were obtained based on concentration profiles. Untargeted-to-targeted bottom-up proteomics were used to detect a unique peptide tag in biological fluids. Method validation included assessments of precision, accuracy, matrix effects, and analyte stability. Results: VAL-1221 was consistently detected in plasma up to 4 hours post-infusion, while no VAL-1221 was detected in CSF samples with the method limit of detection. The validated method showed high sensitivity, precision (RSD <=15%), accuracy (RE <=15%), and acceptable matrix effect. Recovery was optimal for CSF; it was low in plasma (Rec% > +/-20). Conclusion: VAL-1221 was reliably detected in plasma after infusion, but no measurable levels were observed in CSF based on the validated method sensitivity. These findings suggest that the drug, when administered intravenously, may not reach the central nervous system, indicating that this route may not be appropriate for efficacy. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the European Union - Next Generation EU - NRRP M6C2 - Investment 2.1 Enhancement and strengthening of biomedical research in the NHS. Project 'Drug discovEry and repurposing to Find a trEAtmenT for Lafora Disease (DEFEAT-LD)' - PNRR-MR1-2022-12376430 and supported by the Ricerca Corrente funding from the Italian Ministry of Health. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The local ethics committee Comitato Etico Area Vasta Emilia Centro (CE-AVEC) approved the compassionate use program of intravenous VAL-1221 for the five patients with LD (approvals no. 19, 20, 21, 22, 23-2023-COMPASS-AUSLBO) and the proteomics study (protocol number 178-2024-SPER-AUSLBO) in both treated and untreated subjects. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Objectives:Lafora disease (LD) is a fatal progressive myoclonic epilepsy, characterized by disabling myoclonus, intractable seizures, and progressive cognitive decline. At the onset of symptoms, however, distinction from idiopathic generalized epilepsies may be difficult based on EEG because the background activity is typically preserved and the only abnormalities are rare generalized spike-and-wave discharges facilitated by the intermittent light stimulation. This underscores the urgent need for early biomarkers of the disease, particularly as disease-modifying therapies are being developed. Methods:We describe the 24-month course of a patient with LD, whose older brother was similarly affected, followed up from the presymptomatic to overt disease stage. At each time point, the patient underwent neurologic, neuropsychological, and neurophysiologic evaluations, including an assessment of nocturnal sleep. Results:During the first, presymptomatic assessment, when the patient was 13 years old, we documented generalized spike-and-wave discharges during rapid eye movement (REM) sleep, a very atypical finding for generalized idiopathic epilepsies, whereas wake EEG was substantially normal. Discussion:This case study shows that neurophysiologic changes (wake and sleep EEG) showed early alterations even in the absence of motor and cognitive impairment, implying that an early diagnosis in patients with LD might have important implications for targeting future therapeutic strategies.

In this work, we propose a new computational framework to assist neurophysiologists in Stereoelectroencephalography (SEEG) analysis, with the final aim of improving the definition of the Epileptogenic Zone (EZ) in patients with drug-resistant epilepsy. We design a new algorithm, named Desynchronization Index (DI), that classifies as most epileptogenic those SEEG channels that show independent behavior during the seconds preceding the seizure propagation. We test the proposed DI algorithm against the Epileptogenic Index (EI) algorithm on a clinical dataset of 11 patients, considering the neurophysiological evaluation of the EZ as the clinical ground truth. Our results denote that DI overcomes EI in terms of area under the ROC curve (AUC=0.81 vs AUC=0.74) while combining the two algorithms as a unique tool leads to the best performance (AUC=0.87). The DI algorithm underscores connectivity dynamics that can hardly be identified with a pure visual analysis, increasing the accuracy of the EZ definition compared to traditional methods. This technique can lead to the definition of a new effective biomarker of the EZ, reducing the burden required by the SEEG review in the case of extensive implants and improving our understanding of the dynamics leading to the generation of seizures.

Lafora disease (LD) is a fatal progressive myoclonus epilepsy that affects previously healthy adolescents and lacks effective treatments. It is caused by pathogenic variants in EPM2A or NHLRC1, leading to the accumulation of polyglucosan in the brain and other tissues. This study is the first to evaluate the administration of a potentially disease-modifying drug – VAL-1221, a glycogen-degrading antibody-enzyme fusion – in LD patients through a 12-month compassionate use program. Five patients (aged 17–24 years; three females) with intermediate to advanced LD received VAL-1221 intravenous infusions (20 ​mg/kg every other week). Safety was monitored through treatment-emergent adverse events (TEAEs), whereas efficacy was assessed using clinical scales, EEG and neuroimaging. Drug concentration profile was studied via liquid chromatography-high resolution mass spectrometry (LC-HRMS) of plasma and cerebrospinal fluid (CSF), and metabolomics via gas chromatography-MS of CSF. Four patients completed the full treatment course: one discontinued after eight months following status epilepticus. VAL-1221 was well tolerated, with five mild infusion-related TEAEs (skin rash in one, hypotension in four). Efficacy measures showed continued disease progression across patients. LC-HRMS analysis revealed no detectable levels of VAL-1221 in CSF. CSF metabolic profiling revealed no difference between untreated and VAL-1221-treated samples. These findings demonstrate that intravenous VAL-1221 is safe but ineffective, providing an important negative result that prevents further patient exposure to this approach and redirects efforts toward direct central nervous system delivery methods. The study also demonstrates the feasibility of assessing disease progression using clinical and neuroimaging measures, providing a valuable framework for clinical trials in LD.

Objective Stereoelectroencephalography-guided radiofrequency thermocoagulation (SEEG-guided RF-TC) is an invasive procedure based on stereotactic lesioning of cortical targets in the brain using bipolar current through electrode contacts within the SEEG implant. To date, several RF-TC protocols have been described in the literature; however, a consensus has yet to be reached. This work aims to analyze the electrical parameters during RF-TC processes, offering a method to objectively describe and compare different SEEG-guided RF-TC protocols. Methods The study included patients who underwent RF-TC procedures at the IRCCS Istituto delle Scienze Neurologiche di Bologna from February 2022 to May 2023. During each procedure, modifications of the following parameters were measured: voltage, current, impedance, and electric power. An ad-hoc algorithm was implemented to detect abrupt impedance raises, which reflects the occurrence of the thermocoagulation. A two-sample t-test was used to compare parameter curves in RF-TC of different brain structures. Results A total of ninety-two RF-TC procedures were performed in eight patients according to a standardized protocol. During each procedure, impedance levels started at about 700Ω and rose up to 1300Ω, displaying an erratic pattern characterized by one or multiple raises. All measured parameters exhibited similar trends until the first peak, after which changes were influenced by the frequency of impedance raises. No significant correlations were observed between parameter modifications in distinct anatomical sites of RF-TC. Significance The systematic analysis of electrical parameters may represent a reliable tool to compare different RF-TC protocols, paving the way for identifying optimal configurations for SEEG-guided RF-TC procedures in the future.

OBJECTIVE:The STEPPER (Status Epilepticus in Emilia-Romagna) study aimed to investigate the clinical characteristics, prognostic factors, and treatment approaches of status epilepticus (SE) in adults of the Emilia-Romagna region (ERR), Northern Italy. METHODS:STEPPER, an observational, prospective, multicentric cohort study, was conducted across neurology units, emergency departments, and intensive care units of the ERR over 24 months (October 2019-October 2021), encompassing incident cases of SE. Patients were followed up for 30 days. RESULTS:A total of 578 cases were recruited (56% female, mean age = 70 years, 32% with previous diagnosis of epilepsy, 43% with in-hospital onset, 35% stuporous/comatose, 46% with nonconvulsive SE). Etiology was known in 87% (acute 43%, remote 24%, progressive 17%, definite epileptic syndrome 3%). The mean pre-SE Rankin Scale score was 2, the Status Epilepticus Severity Score was ≥4 in 33%, the Epidemiology-Based Mortality Score in Status Epilepticus score was ≥64 in 61%, and 34% were refractory. The sequence of treatments followed current clinical practice guidelines in 63%. Benzodiazepines (BDZs) were underused as first-line therapy (71%), especially in in-hospital onset cases; 15% were treated with continuous intravenous anesthetic drugs. Mortality was 24%; 63% of survivors had functional worsening. At the two-step multivariable analysis, incorrect versus correct treatment sequence with correct BDZ dose was the strongest predictor of failure to resolve SE in the in-hospital group (odds ratio [OR] = 4.42, 95% confidence interval [CI] = 1.86-10.5), with a similar trend in the out-of-hospital group (OR = 2.22, 95% CI = .98-5.02). In turn, failure to resolve was the strongest predictor of 30-day mortality (OR = 11.3, 95% CI = 4.16-30.9, out-of-hospital SE; OR = 6.42, 95% CI = 2.79-14.8, in-hospital SE) and functional worsening (OR = 5.83, 95% CI = 2.05-16.6, out-of-hospital SE; OR = 9.30, 95% CI 2.22-32.3, in-hospital SE). SIGNIFICANCE:The STEPPER study offers insights into real-world SE management, highlighting its significant morbidity and functional decline implications. Although nonmodifiable clinical factors contribute to SE severity, modifiable factors such as optimized first-line therapies and adherence to guidelines can potentially influence prognosis.

AbstractFamilial epilepsy with auditory features (FEAF), previously known as autosomal‐dominant lateral temporal lobe epilepsy (ADLTE) is a genetically heterogeneous syndrome, clinically characterized by focal seizures with prominent auditory symptoms. It is inherited with autosomal‐dominant pattern with reduced penetrance (about 70%). Sporadic epilepsy with auditory features cases are more frequent and clinically indistinguishable from familial cases. One causal gene, MICAL‐1, encodes MICAL‐1, an intracellular multi‐domain enzyme that is an important regulator of filamentous actin (F‐actin) structures. Pathogenic variants in MICAL‐1 account for approximately 7% of FEAF families. Here, we describe a de novo MICAL‐1 pathogenic variant, p.Arg915Cys, in a sporadic case, an affected 21‐year‐old Italian man with no family history of epilepsy. Genetic testing was performed in the patient and his parents, using a next‐generation sequencing panel. In cell‐based assay, this variant significantly increased MICAL‐1 oxidoreductase activity, which likely resulted in dysregulation of F‐actin organization. This finding provides further support for a gain‐of‐function effect underlying MICAL‐1‐mediated epilepsy pathogenesis, as previously seen with other pathogenic variants. Furthermore, the case study provides evidence that de novo MICAL‐1 pathogenic variants can occur in sporadic cases with epilepsy with auditory feature (EAF).Plain Language SummaryIn this study, we report a new MICAL‐1 pathogenic variant in a patient without family history for epilepsy, not inherited from his parents. MICAL‐1 is a protein with enzymatic activity that reorganizes the structure of the cell. We proved the pathological effect of this variant by testing its enzymatic activity and found an increase of this activity. This result suggests that non‐familial cases should be tested to find novel pathogenic variants in this gene.