Steven Sparagana

Efficacy and safety of everolimus for subependymal giant cell astrocytomas associated with tuberous sclerosis complex (EXIST-1): a multicentre, randomised, placebo-controlled phase 3 trial

BACKGROUND Tuberous sclerosis complex is a genetic disorder leading to constitutive activation of mammalian target of rapamycin (mTOR) and growth of benign tumours in several organs. In the brain, growth of subependymal giant cell astrocytomas can cause life-threatening symptoms--eg, hydrocephalus, requiring surgery. In an open-label, phase 1/2 study, the mTOR inhibitor everolimus substantially and significantly reduced the volume of subependymal giant cell astrocytomas. We assessed the efficacy and safety of everolimus in patients with subependymal giant cell astrocytomas associated with tuberous sclerosis complex. METHODS In this double-blind, placebo-controlled, phase 3 trial, patients (aged 0-65 years) in 24 centres in Australia, Belgium, Canada, Germany, the UK, Italy, the Netherlands, Poland, Russian Federation, and the USA were randomly assigned, with an interactive internet-response system, in a 2:1 ratio to oral everolimus 4·5 mg/m(2) per day (titrated to achieve blood trough concentrations of 5-15 ng/mL) or placebo. Eligible patients had a definite diagnosis of tuberous sclerosis complex and at least one lesion with a diameter of 1 cm or greater, and either serial growth of a subependymal giant cell astrocytoma, a new lesion of 1 cm or greater, or new or worsening hydrocephalus. The primary endpoint was the proportion of patients with confirmed response--ie, reduction in target volume of 50% or greater relative to baseline in subependymal giant cell astrocytomas. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00789828. FINDINGS 117 patients were randomly assigned to everolimus (n=78) or placebo (n=39). 27 (35%) patients in the everolimus group had at least 50% reduction in the volume of subependymal giant cell astrocytomas versus none in the placebo group (difference 35%, 95% CI 15-52; one-sided exact Cochran-Mantel-Haenszel test, p<0·0001). Adverse events were mostly grade 1 or 2; no patients discontinued treatment because of adverse events. The most common adverse events were mouth ulceration (25 [32%] in the everolimus group vs two [5%] in the placebo group), stomatitis (24 [31%] vs eight [21%]), convulsion (18 [23%] vs ten [26%]), and pyrexia (17 [22%] vs six [15%]). INTERPRETATION These results support the use of everolimus for subependymal giant cell astrocytomas associated with tuberous sclerosis. Additionally, everolimus might represent a disease-modifying treatment for other aspects of tuberous sclerosis. FUNDING Novartis Pharmaceuticals.

Recurrent reciprocal 16p11.2 rearrangements associated with global developmental delay, behavioural problems, dysmorphism, epilepsy, and abnormal head size

Background Deletion and the reciprocal duplication in 16p11.2 were recently associated with autism and developmental delay. Method We indentified 27 deletions and 18 duplications of 16p11.2 were identified in 0.6% of all samples submitted for clinical array-CGH (comparative genomic hybridisation) analysis. Detailed molecular and phenotypic characterisations were performed on 17 deletion subjects and ten subjects with the duplication. Results The most common clinical manifestations in 17 deletion and 10 duplication subjects were speech/language delay and cognitive impairment. Other phenotypes in the deletion patients included motor delay (50%), seizures (∼40%), behavioural problems (∼40%), congenital anomalies (∼30%), and autism (∼20%). The phenotypes among duplication patients included motor delay (6/10), behavioural problems (especially attention deficit hyperactivity disorder (ADHD)) (6/10), congenital anomalies (5/10), and seizures (3/10). Patients with the 16p11.2 deletion had statistically significant macrocephaly (p<0.0017) and 6 of the 10 patients with the duplication had microcephaly. One subject with the deletion was asymptomatic and another with the duplication had a normal cognitive and behavioural phenotype. Genomic analyses revealed additional complexity to the 16p11.2 region with mechanistic implications. The chromosomal rearrangement was de novo in all but 2 of the 10 deletion cases in which parental studies were available. Additionally, 2 de novo cases were apparently mosaic for the deletion in the analysed blood sample. Three de novo and 2 inherited cases were observed in the 5 of 10 duplication patients where data were available. Conclusions Recurrent reciprocal 16p11.2 deletion and duplication are characterised by a spectrum of primarily neurocognitive phenotypes that are subject to incomplete penetrance and variable expressivity. The autism and macrocephaly observed with deletion and ADHD and microcephaly seen in duplication patients support a diametric model of autism spectrum and psychotic spectrum behavioural phenotypes in genomic sister disorders.

RENAL LESION GROWTH IN CHILDREN WITH TUBEROUS SCLEROSIS COMPLEX

PURPOSE Renal lesions, including angiomyolipoma, renal cysts (simple and polycystic kidney disease) and renal cell carcinoma, develop in patients with tuberous sclerosis complex. While there is limited information that these lesions may grow in adults with tuberous sclerosis complex, the incidence, characterization and growth rate in children have not been reported. Also, the age at which these lesions first appear, thus providing insight into their natural history, is unknown. We present our data from a longitudinal renal surveillance study of children with tuberous sclerosis complex. MATERIALS AND METHODS Since 1985 children with tuberous sclerosis complex at our hospital have undergone periodic renal imaging by ultrasonography or computerized tomography to monitor renal lesions. A total of 35 girls and 25 boys 1 to 18 years old have undergone at least 2 or more annual renal ultrasounds. RESULTS On initial evaluation 33 of 60 children (55%) (mean age 6.9 years) had an identifiable renal lesion, which increased to 48 of 60 (80%) at followup (mean age 10.5 years). Angiomyolipoma was the most frequent lesion (75%) followed by simple renal cysts (17%). Angiomyolipomas increased in size and/or number in 10 of 18 boys (56%) and 18 of 27 girls (66%). The largest growth rate in 1 year was from 0 to 4 cm. and from 5 to 9 cm. in diameter. The youngest patient demonstrated lesions at age 2 years. The average age at which a normal ultrasound became abnormal was 7.2 years. While a total of 27 patients had a normal ultrasound on entering the study, lesions had developed in 15 at followup (11 with angiomyolipomas, 4 with cysts). Five patients had cysts that had disappeared at followup. A 7-year-old boy had a 9 cm. renal cell carcinoma removed. One patient has renal lesions characteristic of autosomal dominant polycystic kidney disease. CONCLUSIONS Renal involvement in patients with tuberous sclerosis complex begins in infancy, and angiomyolipoma is the most common lesion (75%). Angiomyolipomas are more likely to grow than remain stable, although the rate of growth varies. Simple renal cysts may appear or disappear with time but angiomyolipomas do not disappear. An initially normal renal ultrasound does not rule out future development of lesions. Periodic surveillance is indicated in children with tuberous sclerosis complex.

Tuberous sclerosis complex.

Tuberous sclerosis complex is an autosomal dominant disorder that causes significant complications in multiple organ systems. Both basic science and clinical research on tuberous sclerosis complex have flourished in recent years, improving our understanding of its molecular genetics and pathophysiology. Two tuberous sclerosis complex genes cause nearly identical phenotypes, and great progress has been made towards understanding how each of these genes functions. The recognition of tuberous sclerosis complex improved with revised diagnostic criteria, and the management of many of the complications of tuberous sclerosis complex has improved.Tuberous sclerosis complex (TSC) is a neurocutaneous syndrome that can affect the brain, skin, eyes, kidneys, heart, and lungs. TSC alters cellular proliferation and differentiation, resulting in hamartomas of various organs, tumor formation, and altered neuronal migration. The phenotype is highly variable. Most individuals have seizures, commonly including infantile spasms, and there is variable intellectual disability and autism. Neonates can present with cardiac failure due to intracardiac rhabdomyomas. The likelihood of renal angiomyolipomas increases with age, and renal disease is the most common cause of death in adults with TSC. Pulmonary involvement occurs predominantly in women and carries a high morbidity and mortality. TSC is inherited as an autosomal dominant trait, but spontaneous mutations are common. A mutation of either TSC1 on chromosome 9 or TSC2 on chromosome 16 leads to dysfunction of hamartin or tuberin, respectively. These two proteins form a functional complex that modulates the mammalian target of rapamycin (mTOR) pathway. Medications that inhibit mTOR are being used to treat TSC-related tumors, and current studies are investigating whether these agents could alleviate other TSC complications. Consensus statements guide identification and optimal management of many of the TSC-related complications at diagnosis and throughout the lifespan. A multidisciplinary approach is necessary for optimal management of individuals with TSC.

Genotype/phenotype correlation in 325 individuals referred for a diagnosis of tuberous sclerosis complex in the United States.

Tuberous sclerosis complex is an autosomal dominant neurocutaneous disorder marked by hamartoma growth in multiple organ systems. We performed mutational analyses on 325 individuals with definite tuberous sclerosis complex diagnostic status. We identified mutations in 72% (199/257) of de novo and 77% (53/68) of familial cases, with 17% of mutations in the TSC1 gene and 50% in the TSC2 gene. There were 4% unclassified variants and 29% with no mutation identified. Genotype/phenotype analyses of all observed tuberous sclerosis complex findings in probands were performed, including several clinical features not analyzed in two previous large studies. We showed that patients with TSC2 mutations have significantly more hypomelanotic macules and learning disability in contrast to those with TSC1 mutations, findings not noted in previous studies. We also observed results consistent with two similar studies suggesting that individuals with mutations in TSC2 have more severe symptoms. On performing meta-analyses of our data and the other two largest studies in the literature, we found significant correlations for several features that individual studies did not have sufficient power to conclude. Male patients showed more frequent neurologic and eye symptoms, renal cysts, and ungual fibromas. Correlating genotypes with phenotypes should facilitate the disease management of tuberous sclerosis complex.

Diagnosis of tuberous sclerosis complex.

Tuberous sclerosis complex is a dominantly inherited disorder affecting multiple organs; because of its phenotypic variability, the diagnosis of tuberous sclerosis complex can be difficult in the young or in individuals with subtle findings. Recently revised consensus diagnostic criteria for tuberous sclerosis complex reflect an improved understanding of its clinical manifestations and its genetic and molecular mechanisms. The diagnostic criteria are based on the premise that there are probably no truly pathognomonic clinical signs for tuberous sclerosis complex; signs that were once regarded as specific occur as isolated findings in individuals with no other clinical or genetic evidence of tuberous sclerosis complex. Consequently, the revised criteria require tuberous sclerosis complex—associated lesions of two or more organ systems or at least two dissimilar lesions of the same organ to confirm the diagnosis. The addition of DNA testing complements clinical diagnosis and allows more precise genetic counseling and, in some individuals, prenatal diagnosis. Nevertheless, the 15% false-negative rate for DNA testing and the occurrence of germline mosaicism in about 2% of individuals with tuberous sclerosis complex make it difficult to exclude the diagnosis of tuberous sclerosis complex in family members. (J Child Neurol 2004; 19:643-649).

22q13.3 Deletion Syndrome: Clinical and Molecular Analysis Using Array CGH

The 22q13.3 deletion syndrome results from loss of terminal segments of varying sizes at 22qter. Few genotype–phenotype correlations have been found but all patients have mental retardation and severe delay, or absence of, expressive speech. We carried out clinical and molecular characterization of 13 patients. Developmental delay and speech abnormalities were common to all and comparable in frequency and severity to previously reported cases. Array‐based comparative genomic hybridization showed the deletions to vary from 95 kb to 8.5 Mb. We also carried out high‐resolution 244K array comparative genomic hybridization in 10 of 13 patients, that defined the proximal and distal breakpoints of each deletion and helped determine the size, extent, and gene content within the deletion. Two patients had a smaller 95 kb terminal deletion with breakpoints within the SHANK3 gene while three other patients had a similar 5.5 Mb deletion implying the recurrent nature of these deletions. The two largest deletions were found in patients with ring chromosome 22. No correlation could be made with deletion size and phenotype although complete/partial SHANK3 was deleted in all patients. There are very few reports on array comparative genomic hybridization analysis on patients with the 22q13.3 deletion syndrome, and we aim to accurately characterize these patients both clinically and at the molecular level, to pave the way for further genotype–phenotype correlations.

Everolimus for subependymal giant cell astrocytoma in patients with tuberous sclerosis complex: 2-year open-label extension of the randomised EXIST-1 study

BACKGROUND In the EXIST-1 trial, initiated on Aug 10, 2009, more than 35% of patients with subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis complex had at least 50% reduction in SEGA volume after 9·6 months of treatment with everolimus. In this Article, we report interim data (up to Jan 11, 2013) to support longer-term tolerability and efficacy of everolimus from the continuing 4-year extension phase of EXIST-1. METHODS We assessed data from a prospective, open-label extension of a multicentre, phase 3, randomised, double-blind, placebo-controlled study in patients with tuberous sclerosis complex who had SEGA that was growing and needed treatment. In this extension study, we included all patients who had been assigned everolimus during the double-blind, randomised phase of the trial and those patients who crossed over from the placebo group to receive everolimus during the randomised phase or at the start of the extension phase. All patients received oral everolimus at a starting dose of 4·5 mg/m(2) per day. Everolimus dose was subsequently adjusted subject to tolerability to attain blood trough concentrations of 5-15 ng/mL. An independent central radiology review team assessed SEGA response (at least a 50% reduction from baseline in total volume of all target SEGAs; the primary endpoint) by MRI at 12, 24, and 48 weeks, then every year thereafter in all patients who received at least one dose of everolimus. This study was registered with ClinicalTrials.gov, number NCT00789828. FINDINGS Of the original 117 randomly assigned patients, 111 were given everolimus between Aug 20, 2009, and Jan 11, 2013 (date of data cutoff); we included these patients in our longer-term analysis. Median duration of everolimus exposure was 29·3 months (IQR 19·4-33·8). Median follow-up was 28·3 months (IQR 19·3-33·0). 54 (49%) patients had a response of 50% or greater reduction in SEGA volume (95% CI 39·0-58·3), and duration of response was between 2·1 and 31·1 months (median not reached). SEGA volume was reduced by 50% or more in 39 (37%) of 105 patients at 24 weeks, 48 (46%) of 104 patients at 48 weeks, 36 (47%) of 76 patients at 96 weeks, and 11 (38%) of 29 patients at 144 weeks. Stomatitis (48 [43%] patients) and mouth ulceration (33 [30%] patients) were the most frequent treatment-related adverse events; infections were the most commonly reported treatment-related serious adverse event, occurring in 15 (14%) patients. 35 (32%) patients reported treatment-related grade 3 or 4 adverse events, the most common of which were stomatitis (nine [8%]) and pneumonia (nine [8%]). 18 (16%) patients had treatment-related serious adverse events. Six (5%) patients withdrew because of adverse events. INTERPRETATION These results support the longer-term use of everolimus in patients who have few treatment options and who need continued treatment for tuberous sclerosis complex and its varied manifestations. Reduction or stabilisation of tumour volume with everolimus will hopefully provide long-term clinical benefit in patients with SEGA. FUNDING Novartis Pharmaceuticals.

Microdeletions including YWHAE in the Miller-Dieker syndrome region on chromosome 17p13.3 result in facial dysmorphisms, growth restriction, and cognitive impairment

Background: Deletions in the 17p13.3 region are associated with abnormal neuronal migration. Point mutations or deletion copy number variants of the PAFAH1B1 gene in this genomic region cause lissencephaly, whereas extended deletions involving both PAFAH1B1 and YWHAE result in Miller–Dieker syndrome characterised by facial dysmorphisms and a more severe grade of lissencephaly. The phenotypic consequences of YWHAE deletion without deletion of PAFAH1B1 have not been studied systematically. Methods: We performed a detailed clinical and molecular characterization of five patients with deletions involving YWHAE but not PAFAH1B1, two with deletion including PAFAH1B1 but not YWHAE, and one with deletion of YWHAE and mosaic for deletion of PAFAH1B1. Results: Three deletions were terminal whereas five were interstitial. Patients with deletions including YWHAE but not PAFAH1B1 presented with significant growth restriction, cognitive impairment, shared craniofacial features, and variable structural abnormalities of the brain. Growth restriction was not observed in one patient with deletion of YWHAE and TUSC5, implying that other genes in the region may have a role in regulation of growth with CRK being the most likely candidate. Using array based comparative genomic hybridisation and long range polymerase chain reaction, we have delineated the breakpoints of these nonrecurrent deletions and show that the interstitial genomic rearrangements are likely generated by diverse mechanisms, including the recently described Fork Stalling and Template Switching (FoSTeS)/Microhomology Mediated Break Induced Replication (MMBIR). Conclusions: Microdeletions of chromosome 17p13.3 involving YWHAE present with growth restriction, craniofacial dysmorphisms, structural abnormalities of brain and cognitive impairment. The interstitial deletions are mediated by diverse molecular mechanisms.

Early Diagnosis of Subependymal Giant Cell Astrocytoma in Patients With Tuberous Sclerosis

We present 19 patients with tuberous sclerosis complex and subependymal giant cell astrocytoma. The mean age at the time of tumor diagnosis was 9.4 years (range, 1.5 to 21 years). Computed cranial tomography (CT) or cranial magnetic resonance imaging (MRI) identified the lesion which was resected in all cases. Seven patients had hydrocephalus and there was an interval increase in the tumor size or a large tumor without hydrocephalus in 12 patients. Surgical criteria included: (1) presence of hydrocephalus; (2) interval increase in tumor size; (3) new focal neurologic deficit attributable to the tumor; and/or (4) symptoms of increased intracranial pressure. Eight patients were identified through a surveillance program involving annual computed cranial tomography. All of these eight patients had their tumor removed prior to the development of symptoms, none had neurologic deficits which persisted after surgery, and none has so far developed recurrent subependymal giant cell astrocytoma. In contrast, of the 11 patients from the non-surveillance group 7 were symptomatic at tumor diagnosis, 1 had a complicated postoperative course, 2 developed recurrent giant cell astrocytoma, and 1 had an extensive lesion that could not be completely excised. Periodic cranial imaging may help to identify subependymal giant cell astrocytomas in tuberous sclerosis patients before they become symptomatic. Earlier diagnosis and treatment could reduce surgical morbidity and the risk of tumor recurrence. (J Child Neurol 1998;13:173-177).