Comprehensive genetic and phenotype analysis of 95 individuals with mosaic tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a neurogenetic disorder due to loss-of-function TSC1 or TSC2 variants, characterized by tumors affecting multiple organs, including skin, brain, heart, lung, and kidney. Mosaicism for TSC1 or TSC2 variants occurs in 10%-15% of individuals diagnosed with TSC. Here, we report comprehensive characterization of TSC mosaicism by using massively parallel sequencing (MPS) of 330 TSC samples from a variety of tissues and fluids from a cohort of 95 individuals with mosaic TSC. TSC1 variants in individuals with mosaic TSC are much less common (9%) than in germline TSC overall (26%) (p < 0.0001). The mosaic variant allele frequency (VAF) is significantly higher in TSC1 than in TSC2, in both blood and saliva (median VAF: TSC1, 4.91%; TSC2, 1.93%; p = 0.036) and facial angiofibromas (median VAF: TSC1, 7.7%; TSC2 3.7%; p = 0.004), while the number of TSC clinical features in individuals with TSC1 and TSC2 mosaicism was similar. The distribution of mosaic variants across TSC1 and TSC2 is similar to that for pathogenic germline variants in general TSC. The systemic mosaic variant was not present in blood in 14 of 76 (18%) individuals with TSC, highlighting the value of analysis of multiple samples from each individual. A detailed comparison revealed that nearly all TSC clinical features are less common in individuals with mosaic versus germline TSC. A large number of previously unreported TSC1 and TSC2 variants, including intronic and large rearrangements (n = 11), were also identified.

Prevention of Epilepsy in Infants with Tuberous Sclerosis Complex in the EPISTOP Trial.

OBJECTIVE: Epilepsy develops in 70 to 90% of children with tuberous sclerosis complex (TSC) and is often resistant to medication. Recently, the concept of preventive antiepileptic treatment to modify the natural history of epilepsy has been proposed. EPISTOP was a clinical trial designed to compare preventive versus conventional antiepileptic treatment in TSC infants. METHODS: In this multicenter study, 94 infants with TSC without seizure history were followed with monthly video electroencephalography (EEG), and received vigabatrin either as conventional antiepileptic treatment, started after the first electrographic or clinical seizure, or preventively when epileptiform EEG activity before seizures was detected. At 6 sites, subjects were randomly allocated to treatment in a 1:1 ratio in a randomized controlled trial (RCT). At 4 sites, treatment allocation was fixed; this was denoted an open-label trial (OLT). Subjects were followed until 2 years of age. The primary endpoint was the time to first clinical seizure. RESULTS: In 54 subjects, epileptiform EEG abnormalities were identified before seizures. Twenty-seven were included in the RCT and 27 in the OLT. The time to the first clinical seizure was significantly longer with preventive than conventional treatment [RCT: 364 days (95% confidence interval [CI] = 223-535) vs 124 days (95% CI = 33-149); OLT: 426 days (95% CI = 258-628) vs 106 days (95% CI = 11-149)]. At 24 months, our pooled analysis showed preventive treatment reduced the risk of clinical seizures (odds ratio [OR] = 0.21, p = 0.032), drug-resistant epilepsy (OR = 0.23, p = 0.022), and infantile spasms (OR = 0, p < 0.001). No adverse events related to preventive treatment were noted. INTERPRETATION: Preventive treatment with vigabatrin was safe and modified the natural history of seizures in TSC, reducing the risk and severity of epilepsy. ANN NEUROL 2021;89:304-314.

Distinct DNA Methylation Patterns of Subependymal Giant Cell Astrocytomas in Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is a monogenic disorder caused by mutations in either the TSC1 or TSC2 gene, two key regulators of the mechanistic target of the rapamycin complex pathway. Phenotypically, this leads to growth and formation of hamartomas in several organs, including the brain. Subependymal giant cell astrocytomas (SEGAs) are low-grade brain tumors commonly associated with TSC. Recently, gene expression studies provided evidence that the immune system, the MAPK pathway and extracellular matrix organization play an important role in SEGA development. However, the precise mechanisms behind the gene expression changes in SEGA are still largely unknown, providing a potential role for DNA methylation. We investigated the methylation profile of SEGAs using the Illumina Infinium HumanMethylation450 BeadChip (SEGAs n = 42, periventricular control n = 8). The SEGA methylation profile was enriched for the adaptive immune system, T cell activation, leukocyte mediated immunity, extracellular structure organization and the ERK1 & ERK2 cascade. More interestingly, we identified two subgroups in the SEGA methylation data and show that the differentially expressed genes between the two subgroups are related to the MAPK cascade and adaptive immune response. Overall, this study shows that the immune system, the MAPK pathway and extracellular matrix organization are also affected on DNA methylation level, suggesting that therapeutic intervention on DNA level could be useful for these specific pathways in SEGA. Moreover, we identified two subgroups in SEGA that seem to be driven by changes in the adaptive immune response and MAPK pathway and could potentially hold predictive information on target treatment response.

Evolution of electroencephalogram in infants with tuberous sclerosis complex and neurodevelopmental outcome: a prospective cohort study.

AIM: To describe the evolution of electroencephalogram (EEG) characteristics in infants with tuberous sclerosis complex (TSC) and the relationship with neurodevelopmental outcome at 24 months. METHOD: Eighty-three infants were enrolled in the EPISTOP trial and underwent serial EEG follow-up until the age of 24 months (males n=45, females n=37, median age at enrolment 28d, interquartile range 14-54d). Maturation of the EEG background and epileptiform discharges were compared between the TSC1 and TSC2 variants and between preventive and conventional groups respectively. RESULTS: Children with TSC2 more frequently had a slower posterior dominant rhythm (PDR) at 24 months (51% vs 11%, p=0.002), a higher number of epileptiform foci (median=8 vs 4, p=0.003), and a lower fraction of EEGs without epileptiform discharges (18% vs 61%, p=0.001) at follow-up. A slower PDR at 24 months was significantly associated with lower cognitive (median=70 vs 80, p=0.028) and motor developmental quotients (median=70 vs 79, p=0.008). A higher fraction of EEGs without epileptiform discharges was associated with a lower probability of autism spectrum disorder symptoms (odds ratio=0.092, 95% confidence interval=0.009-0.912, p=0.042) and higher cognitive (p=0.004), language (p=0.002), and motor (p=0.001) developmental quotients at 24 months. INTERPRETATION: TSC2 is associated with more abnormal EEG characteristics compared to TSC1, which are predictive for neurodevelopmental outcome.

MicroRNA Expression Profile in TSC Cell Lines and the Impact of mTOR Inhibitor.

The aim of this study was to assess the potential implication of microRNA on tuberous sclerosis (TSC) pathogenesis by performing microRNA profiling on cell lines silencing TSC1 or TSC2 genes using qPCR panels, before and after incubation with rapamycin. Significant differences in expression were observed between samples before and after rapamycin treatment in nineteen miRNAs in TSC1, five miRNAs in TSC2 and seven miRNAs in controls. Of miRNAs dysregulated before rapamycin treatment, three normalized after treatment in the TSC1 group (miR-21-3p, miR-433-3p, let-7g-3p) and one normalized in the TSC2 group (miR-1224-3p). Of the miRNAs dysregulated before rapamycin treatment in the TSC1 and TSC2 groups, two did not normalize after treatment (miR-33a-3p, miR-29a-3p). The results of the possible targets indicated that there are four common genes with seed regions susceptible to regulation by those miRNAs: ZBTB20, PHACTR2, PLXNC1 and ATP1B4. Our data show no changes in mRNA expression of these targets after rapamycin treatment. In conclusion, results of our study indicate the involvement of miRNA dysregulation in the pathogenesis of TSC. Some of the miRNA might be used as markers of treatment efficacy and autonomic miRNA as a target for future therapy.

Subependymal giant cell astrocytomas are characterized by mTORC1 hyperactivation, a very low somatic mutation rate, and a unique gene expression profile.

Subependymal giant-cell astrocytomas (SEGAs) are slow-growing brain tumors that are a hallmark feature seen in 5-10% of patients with Tuberous Sclerosis Complex (TSC). Though histologically benign, they can cause serious neurologic symptoms, leading to death if untreated. SEGAs consistently show biallelic loss of TSC1 or TSC2. Herein, we aimed to define other somatic events beyond TSC1/TSC2 loss and identify potential transcriptional drivers that contribute to SEGA formation. Paired tumor-normal whole-exome sequencing was performed on 21 resected SEGAs from 20 TSC patients. Pathogenic variants in TSC1/TSC2 were identified in 19/21 (90%) SEGAs. Copy neutral loss of heterozygosity (size range: 2.2-46 Mb) was seen in 76% (16/21) of SEGAs (44% chr9q and 56% chr16p). An average of 1.4 other somatic variants (range 0-7) per tumor were identified, unlikely of pathogenic significance. Whole transcriptome RNA-sequencing analyses revealed 190 common differentially expressed genes in SEGA (n = 16, 13 from a prior study) in pairwise comparison to each of: low grade diffuse gliomas (n = 530) and glioblastoma (n = 171) from The Cancer Genome Atlas (TCGA) consortium, ganglioglioma (n = 10), TSC cortical tubers (n = 15), and multiple normal tissues. Among these, homeobox transcription factors (TFs) HMX3, HMX2, VAX1, SIX3; and TFs IRF6 and EOMES were all expressed >12-fold higher in SEGAs (FDR/q-value < 0.05). Immunohistochemistry supported the specificity of IRF6, VAX1, SIX3 for SEGAs in comparison to other tumor entities and normal brain. We conclude that SEGAs have an extremely low somatic mutation rate, suggesting that TSC1/TSC2 loss is sufficient to drive tumor growth. The unique and highly expressed SEGA-specific TFs likely reflect the neuroepithelial cell of origin, and may also contribute to the transcriptional and epigenetic state that enables SEGA growth following two-hit loss of TSC1 or TSC2 and mTORC1 activation.

Fetal Brain Magnetic Resonance Imaging Findings Predict Neurodevelopment in Children with Tuberous Sclerosis Complex.

OBJECTIVE: To correlate fetal brain magnetic resonance imaging (MRI) findings with epilepsy characteristics and neurodevelopment at 2 years of age in children with tuberous sclerosis complex (TSC) to improve prenatal counseling. STUDY DESIGN: This retrospective cohort study was performed in a collaboration between centers of the EPISTOP consortium. We included children with definite TSC, fetal MRIs, and available follow-up data at 2 years of age. A pediatric neuroradiologist masked to the patient's clinical characteristics evaluated all fetal MRIs. MRIs were categorized for each of the 10 brain lobes as score 0: no (sub)cortical lesions or doubt; score 1: a single small lesion; score 2: more than one small lesion or at least one large lesion (>5 mm). Neurologic manifestations were correlated to lesion sum scores. RESULTS: Forty-one children were included. Median gestational age at MRI was 33.3 weeks; (sub)cortical lesions were detected in 97.6%. Mean lesion sum score was 4.5. At 2 years, 58.5% of patients had epilepsy and 22% had drug-resistant epilepsy. Cognitive, language, and motor development were delayed in 38%, 81%, and 50% of patients, respectively. Autism spectrum disorder (ASD) was diagnosed in 20.5%. Fetal MRI lesion sum scores were significantly associated with cognitive and motor development, and with ASD diagnosis, but not with epilepsy characteristics. CONCLUSIONS: Fetal cerebral lesion scores correlate with neurodevelopment and ASD at 2 years in children with TSC.

MicroRNA-34a activation in tuberous sclerosis complex during early brain development may lead to impaired corticogenesis.

AIMS: Tuberous sclerosis complex (TSC) is a genetic disorder associated with dysregulation of the mechanistic target of rapamycin complex 1 (mTORC1) signalling pathway. Neurodevelopmental disorders, frequently present in TSC, are linked to cortical tubers in the brain. We previously reported microRNA-34a (miR-34a) among the most upregulated miRs in tubers. Here, we characterised miR-34a expression in tubers with the focus on the early brain development and assessed the regulation of mTORC1 pathway and corticogenesis by miR-34a. METHODS: We analysed the expression of miR-34a in resected cortical tubers (n = 37) compared with autopsy-derived control tissue (n = 27). The effect of miR-34a overexpression on corticogenesis was assessed in mice at E18. The regulation of the mTORC1 pathway and the expression of the bioinformatically predicted target genes were assessed in primary astrocyte cultures from three patients with TSC and in SH-SY5Y cells following miR-34a transfection. RESULTS: The peak of miR-34a overexpression in tubers was observed during infancy, concomitant with the presence of pathological markers, particularly in giant cells and dysmorphic neurons. miR-34a was also strongly expressed in foetal TSC cortex. Overexpression of miR-34a in mouse embryos decreased the percentage of cells migrated to the cortical plate. The transfection of miR-34a mimic in TSC astrocytes negatively regulated mTORC1 and decreased the expression of the target genes RAS related (RRAS) and NOTCH1. CONCLUSIONS: MicroRNA-34a is most highly overexpressed in tubers during foetal and early postnatal brain development. miR-34a can negatively regulate mTORC1; however, it may also contribute to abnormal corticogenesis in TSC.

Early epileptiform EEG activity in infants with tuberous sclerosis complex predicts epilepsy and neurodevelopmental outcomes.

OBJECTIVE: To study the association between timing and characteristics of the first electroencephalography (EEG) with epileptiform discharges (ED-EEG) and epilepsy and neurodevelopment at 24 months in infants with tuberous sclerosis complex (TSC). METHODS: Patients enrolled in the prospective Epileptogenesis in a genetic model of epilepsy - Tuberous sclerosis complex (EPISTOP) trial, had serial EEG monitoring until the age of 24 months. The timing and characteristics of the first ED-EEG were studied in relation to clinical outcome. Epilepsy-related outcomes were analyzed separately in a conventionally followed group (initiation of vigabatrin after seizure onset) and a preventive group (initiation of vigabatrin before seizures, but after appearance of interictal epileptiform discharges [IEDs]). RESULTS: Eighty-three infants with TSC were enrolled at a median age of 28 days (interquartile range [IQR] 14-54). Seventy-nine of 83 patients (95%) developed epileptiform discharges at a median age of 77 days (IQR 23-111). Patients with a pathogenic TSC2 variant were significantly younger (P-value .009) at first ED-EEG and more frequently had multifocal IED (P-value .042) than patients with a pathogenic TSC1 variant. A younger age at first ED-EEG was significantly associated with lower cognitive (P-value .010), language (P-value .001), and motor (P-value .013) developmental quotients at 24 months. In the conventional group, 48 of 60 developed seizures. In this group, the presence of focal slowing on the first ED-EEG was predictive of earlier seizure onset (P-value .030). Earlier recording of epileptiform discharges (P-value .019), especially when multifocal (P-value .026) was associated with higher risk of drug-resistant epilepsy. In the preventive group, timing, distribution of IED, or focal slowing, was not associated with the epilepsy outcomes. However, when multifocal IEDs were present on the first ED-EEG, preventive treatment delayed the onset of seizures significantly (P-value <.001). SIGNIFICANCE: Early EEG findings help to identify TSC infants at risk of severe epilepsy and neurodevelopmental delay and those who may benefit from preventive treatment with vigabatrin.

The coding and non-coding transcriptional landscape of subependymal giant cell astrocytomas.

Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited neurocutaneous disorder caused by inactivating mutations in TSC1 or TSC2, key regulators of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. In the CNS, TSC is characterized by cortical tubers, subependymal nodules and subependymal giant cell astrocytomas (SEGAs). SEGAs may lead to impaired circulation of CSF resulting in hydrocephalus and raised intracranial pressure in patients with TSC. Currently, surgical resection and mTORC1 inhibitors are the recommended treatment options for patients with SEGA. In the present study, high-throughput RNA-sequencing (SEGAs n = 19, periventricular control n = 8) was used in combination with computational approaches to unravel the complexity of SEGA development. We identified 9400 mRNAs and 94 microRNAs differentially expressed in SEGAs compared to control tissue. The SEGA transcriptome profile was enriched for the mitogen-activated protein kinase (MAPK) pathway, a major regulator of cell proliferation and survival. Analysis at the protein level confirmed that extracellular signal-regulated kinase (ERK) is activated in SEGAs. Subsequently, the inhibition of ERK independently of mTORC1 blockade decreased efficiently the proliferation of primary patient-derived SEGA cultures. Furthermore, we found that LAMTOR1, LAMTOR2, LAMTOR3, LAMTOR4 and LAMTOR5 were overexpressed at both gene and protein levels in SEGA compared to control tissue. Taken together LAMTOR1-5 can form a complex, known as the 'Ragulator' complex, which is known to activate both mTORC1 and MAPK/ERK pathways. Overall, this study shows that the MAPK/ERK pathway could be used as a target for treatment independent of, or in combination with mTORC1 inhibitors for TSC patients. Moreover, our study provides initial evidence of a possible link between the constitutive activated mTORC1 pathway and a secondary driver pathway of tumour growth.

mTOR controls endoplasmic reticulum-Golgi apparatus trafficking of VSVg in specific cell types.

BACKGROUND: Mammalian/mechanistic target of rapamycin (mTOR) complexes are essential for cell proliferation, growth, differentiation, and survival. mTORC1 hyperactivation occurs in the tuberous sclerosis complex (TSC). mTORC1 localizes to the surface of lysosomes, where Rheb activates it. However, mTOR was also found on the endoplasmic reticulum (ER) and Golgi apparatus (GA). Recent studies showed that the same inputs regulate ER-to-GA cargo transport and mTORC1 (e.g., the level of amino acids or energy status of the cell). Nonetheless, it remains unknown whether mTOR contributes to the regulation of cargo passage through the secretory pathway. METHODS: The retention using selective hooks (RUSH) approach was used to image movement of model cargo (VSVg) between the ER and GA in various cell lines in which mTOR complexes were inhibited. We also investigated VSVg trafficking in TSC patient fibroblasts. RESULTS: We found that mTOR inhibition led to the overall enhancement of VSVg transport through the secretory pathway in PC12 cells and primary human fibroblasts. Also, in TSC1-deficient cells, VSVg transport was enhanced. CONCLUSIONS: Altogether, these data indicate the involvement of mTOR in the regulation of ER-to-GA cargo transport and suggest that impairments in exocytosis may be an additional cellular process that is disturbed in TSC.

New Insights into Red Blood Cell Microcytosis upon mTOR Inhibitor Administration.

The aim of this study was to evaluate the effect of everolimus, a mammalian target of rapamycin (mTOR) inhibitor, on red blood cell parameters in the context of iron homeostasis in patients with tuberous sclerosis complex (TSC) and evaluate its effect on cell size in vitro. Everolimus has a significant impact on red blood cell parameters in patients with TSC. The most common alteration was microcytosis. The mean MCV value decreased by 9.2%, 12%, and 11.8% after 3, 6, and 12 months of everolimus treatment. The iron level declined during the first 3 months, and human soluble transferrin receptor concentration increased during 6 months of therapy. The size of K562 cells decreased when cultured in the presence of 5 µM everolimus by approximately 8%. The addition of hemin to the cell culture with 5 µM everolimus did not prevent any decrease in cell size. The stage of erythroid maturation did not affect the response to everolimus. Our results showed that the mTOR inhibitor everolimus caused red blood cell microcytosis in vivo and in vitro. This effect is not clearly related to a deficit of iron and erythroid maturation. This observation confirms that mTOR signaling plays a complex role in the control of cell size.

Nusinersen treatment of Spinal Muscular Atrophy Type 1 - results of expanded access programme in Poland.

AIM OF THE STUDY: This study aimed to evaluate the effects of nusinersen therapy in Polish children with SMA type 1. CLINICAL RATIONALE OF STUDY: Spinal muscular atrophy (SMA) is a neuromuscular disorder that is characterised by the loss of motor neurons, progressive muscle weakness and atrophy, leading to increased disability and mortality. Nusinersen, an antisense oligonucleotide that promotes production of the functional survival motor neuron protein is approved for the treatment of SMA 5q in the European Union. In 2017, an early access programme (EAP) for nusinersen was launched in Poland. In this study, we present the results of nusinersen treatment in Polish patients participating in the EAP. MATERIALS AND METHODS: We collected prospectively clinical data including mutational analysis of SMN1 and SMN2 genes, motor function outcomes as measured on a standardized scales, ventilatory and nutritional status, on SMA type 1 patients receiving nusinersen in three EAP centres in Poland. Scores on the CHOP-INTEND scale after 18-26 months of treatment were compared to baseline. RESULTS: We analysed data from 26 patients with SMA type 1, mean age 4.79 (2-15) years. The mutational analysis revealed two SMN2 gene copies in the majority of patients (61.54%). Three and four copies were found in 34.62% and 3.84%, respectively. Median disease duration was 21 months. Half (n = 13) of the patients required mechanical ventilation at baseline and 57.69% (n = 15) were fed by nasogastric tube or percutaneous endoscopic gastrostomy. No patient worsened during the follow-up. Mean improvement in CHOP-INTEND from baseline to the last follow-up was 7.38 points (p < 0.001). CHOP-INTEND scores did not decline for any patient. Patients with three or more SMN2 gene copies had higher scores than did the patients with two copies (p = 0.013), and they tended to show greater improvement over time, but the difference was not significant (p = 0.324). Shorter disease duration and higher CHOP-INTEND baseline score were associated with a better response (p = 0.015). Patients with a CHOP-INTEND score above the median had higher scores overall than the rest (p < 0.0013), and they improved significantly more than the rest (p = 0.037). Nusinersen was well tolerated, no new safety findings were identified. CONCLUSIONS AND CLINICAL IMPLICATIONS: Our data indicates that nusinersen treatment might be effective in SMA type 1 patients, regardless of their age and functional status.

TSC2 pathogenic variants are predictive of severe clinical manifestations in TSC infants: results of the EPISTOP study.

PURPOSE: To perform comprehensive genotyping of TSC1 and TSC2 in a cohort of 94 infants with tuberous sclerosis complex (TSC) and correlate with clinical manifestations. METHODS: Infants were enrolled at age <4 months, and subject to intensive clinical monitoring including electroencephalography (EEG), brain magnetic resonance imaging (MRI), and neuropsychological assessment. Targeted massively parallel sequencing (MPS), genome sequencing, and multiplex ligation-dependent probe amplification (MLPA) were used for variant detection in TSC1/TSC2. RESULTS: Pathogenic variants in TSC1 or TSC2 were identified in 93 of 94 (99%) subjects, with 23 in TSC1 and 70 in TSC2. Nine (10%) subjects had mosaicism. Eight of 24 clinical features assessed at age 2 years were significantly less frequent in those with TSC1 versus TSC2 variants including cortical tubers, hypomelanotic macules, facial angiofibroma, renal cysts, drug-resistant epilepsy, developmental delay, subependymal giant cell astrocytoma, and median seizure-free survival. Additionally, quantitative brain MRI analysis showed a marked difference in tuber and subependymal nodule/giant cell astrocytoma volume for TSC1 versus TSC2. CONCLUSION: TSC2 pathogenic variants are associated with a more severe clinical phenotype than mosaic TSC2 or TSC1 variants in TSC infants. Early assessment of gene variant status and mosaicism might have benefit for clinical management in infants and young children with TSC.

A shower of second hit events as the cause of multifocal renal cell carcinoma in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a genetic disorder characterized by seizures and tumor formation in multiple organs, mainly in the brain, skin, kidney, lung and heart. Renal cell carcinoma (RCC) occurs in ∼3% of TSC patients, and typically develops at age <50. Here we describe genetic findings in two TSC patients with multiple renal tumors, each of whom had the germline mutation TSC2 p.R905Q. The first (female) TSC patient had a left followed by a right nephrectomy at ages 24 and 27. Both kidneys showed multifocal TSC-associated papillary RCC (PRCC). Targeted, next-generation sequencing (NGS) analysis of TSC2 in five tumors (four from the left kidney, one from the right) showed loss of heterozygosity in one tumor, and four different TSC2 point mutations (p.E1351*, p.R1032*, p.R1713H, c.4178_4179delCT) in the other four samples. Only one of the 11 other tumors available from this patient had one of the TSC2 second hit mutations identified. Whole-exome analysis of the five tumors identified a very small number of additional mutated genes, with an average of 3.4 nonsilent coding, somatic mutations per tumor, none of which were seen in >1 tumor. The second (male) TSC patient had bilateral partial nephrectomies (both at age 36), with similar findings of multifocal PRCC. NGS analysis of TSC2 in two of these tumors identified a second hit mutation c.2355+1G>T in one sample that was not seen in other tumors. In conclusion, we report the first detailed genetic analysis of RCCs in TSC patients. Molecular studies indicate that tumors developed independently due to various second hit events, suggesting that these patients experienced a 'shower' of second hit mutations in TSC2 during kidney development leading to this severe phenotype.

Maintenance therapy with everolimus for subependymal giant cell astrocytoma in patients with tuberous sclerosis (the EMINENTS study).

OBJECTIVE: One of the therapeutic options for patients with tuberous sclerosis (TCS) and subependymal giant cell astrocytoma (SEGA) is everolimus treatment once daily, every day, to attain trough concentrations of 5-15 ng/ml (standard treatment). The aim of this study was to evaluate the efficacy and safety of a reduced dose of everolimus (three times a week with a daily dose as in standard treatment-maintenance therapy) in a group of patients who were previously treated with standard dose for at least 12 months. MATERIALS AND METHODS: Ten patients (six males, four females; median age 14.23 years) with TSC-related SEGA who met inclusion criteria were included into a single-arm, prospective trial. All the patients were followed over at least 12 months (median 12 and range 12-24 months). Tumor volumes from day 0, 90, 180, and 360 were evaluated by an experienced radiologist and an objective computer-based method and compared. Adverse events (AEs) noted during maintenance therapy were compared to the AEs observed during standard everolimus therapy. RESULTS: The differences in SEGA volume between subsequent time points (day 0, 90, 120, and 360) were not statistically significant. No clinical symptoms of tumor regrowth were observed. AEs were significantly less severe and less frequent during maintenance compared with standard therapy. CONCLUSIONS: Maintenance therapy with reduced-dose everolimus is an effective therapeutic option for patients with TSC and SEGA after the completion of standard therapy and may moderate the rates of adverse effects.

What are the true volumes of SEGA tumors? Reliability of planimetric and popular semi-automated image segmentation methods.

OBJECTIVE: To evaluate the reliability of the standard planimetric methodology of volumetric analysis and three different open-source semi-automated approaches of brain tumor segmentation. MATERIALS AND METHODS: The volumes of subependymal giant cell astrocytomas (SEGA) examined by 30 MRI studies of 10 patients from a previous everolimus-related trial (EMINENTS study) were estimated using four methods: planimetric method (modified MacDonald ellipsoid method), ITK-Snap (pixel clustering, geodesic active contours, region competition methods), 3D Slicer (level-set thresholding), and NIRFast (k-means clustering, Markov random fields). The methods were compared, and a trial simulation was performed to determine how the choice of approach could influence the final decision about progression or response. RESULTS: Intraclass correlation coefficient was high (0.95; 95% CI 0.91-0.98). The planimetric method always overestimated the size of the tumor, while virtually no mean difference was found between ITK-Snap and 3D Slicer (P = 0.99). NIRFast underestimated the volume and presented a proportional bias. During the trial simulation, a moderate level of agreement between all the methods (kappa 0.57-0.71, P < 0.002) was noted. CONCLUSION: Semi-automated segmentation can ease oncological follow-up but the moderate level of agreement between segmentation methods suggests that the reference standard volumetric method for SEGA tumors should be revised and chosen carefully, as the selection of volumetry tool may influence the conclusion about tumor progression or response.

Tuberous sclerosis complex: From molecular biology to novel therapeutic approaches.

Tuberous sclerosis complex (TSC) is a rare multi-system disorder, primary manifestations of which are benign tumors and lesions in various organs of the body, including the brain. TSC patients often suffer from epilepsy, mental retardation, and autism spectrum disorder (ASD). Therefore, TSC serves as a model of epilepsy, ASD, and tumorigenesis. TSC is caused by the lack of functional Tsc1-Tsc2 complex, which serves as a major cellular inhibitor of mammalian Target of Rapamycin Complex 1 (mTORC1). mTORC1 is a kinase controlling most of anabolic processes in eukaryotic cells. Consequently, mTORC1 inhibitors, such as rapamycin, serve as experimental or already approved drugs for several TSC symptoms. However, rapalogs, although quite effective, need to be administered chronically and likely for a lifetime, since therapy discontinuation results in tumor regrowth and epilepsy recurrence. Recent studies revealed that metabolism and excitability (in the case of neurons) of cells lacking Tsc1-Tsc2 complex are changed, and these features may potentially be used to treat some of TSC symptoms. In this review, we first provide basic facts about TSC and its molecular background, to next discuss the newest findings in TSC cell biology that can be used to improve existing therapies of TSC and other diseases linked to mTORC1 hyperactivation. © 2016 IUBMB Life, 68(12):955-962, 2016.

Safety of Everolimus in Patients Younger than 3 Years of Age: Results from EXIST-1, a Randomized, Controlled Clinical Trial.

OBJECTIVES: To assess the long-term safety of everolimus in young children with tuberous sclerosis complex (TSC)-associated subependymal giant cell astrocytoma (SEGA). STUDY DESIGN: EXamining everolimus In a Study of Tuberous Sclerosis Complex-1 (EXIST-1) was a multicenter, randomized, double-blind phase 3 study with an open-label extension evaluating the efficacy and tolerability of everolimus in patients with TSC-associated SEGA. Everolimus was initiated at 4.5 mg/m(2)/day and titrated to blood trough levels of 5-15 ng/mL. Post hoc analysis of safety data (adverse events [AEs]) was performed in a subgroup of patients aged <3 years at everolimus initiation. RESULTS: Eighteen patients (median age 1.82 years) were included; 16 were still receiving everolimus at the analysis cut-off date of January 11, 2013. Median everolimus exposure was 31.1 months (range, 11.5-39 months). One patient discontinued treatment because of AEs (ie, Acinetobacter bacteremia, increased blood alkaline phosphatase, and viral infection). AEs were reported in all patients, but events were mostly grade 1/2 in severity; 12 patients (66.7%) experienced grade 3 events, and 2 patients (11.1%) reported grade 4 events. The most common AEs were stomatitis, cough, pharyngitis, and pyrexia; no new safety issues were identified in this population. Serious AEs were reported in 50% of patients; these were suspected to be medication related in 4 patients (22.2%). CONCLUSIONS: Everolimus appears to be a safe therapeutic option for patients aged <3 years with TSC-associated SEGA. The small sample size in this subpopulation limits interpretation of the results; additional studies in the pediatric population are needed and are underway. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00789828.