David M. Mirsky

Erratum to: Clinical metagenomic identification of Balamuthia mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing

Author details Department of Laboratory Medicine, University of California, 185 Berry Street, San Francisco 94107 CA, USA. UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco 91407 CA, USA. Children’s Hospital Colorado and University of Colorado School of Medicine, Aurora, CO, USA. California Department of Public Health, Richmond, CA, USA. Kaiser Permanente Hospital, Oakland, CA, USA. John Muir Hospital, Walnut Creek, CA, USA. Department of Medicine, Division of Infectious Diseases, University of California, San Francisco 94107 CA, USA.

Autophagy inhibition improves chemosensitivity in BRAF(V600E) brain tumors.

UNLABELLED Autophagy inhibition is a potential therapeutic strategy in cancer, but it is unknown which tumors will benefit. The BRAF(V600E) mutation has been identified as important in pediatric central nervous system (CNS) tumors and is known to affect autophagy in other tumor types. We evaluated CNS tumor cells with BRAF(V600E) and found that mutant (but not wild-type) cells display high rates of induced autophagy, are sensitive to pharmacologic and genetic autophagy inhibition, and display synergy when the clinically used autophagy inhibitor chloroquine was combined with the RAF inhibitor vemurafenib or standard chemotherapeutics. Importantly, we also demonstrate that chloroquine can improve vemurafenib sensitivity in a resistant ex vivo primary culture and provide the first demonstration in a patient harboring the V600E mutation treated with vemurafenib that the addition of chloroquine can improve clinical outcomes. These findings suggest that CNS tumors with BRAF(V600E) are autophagy-dependent and should be targeted with autophagy inhibition in combination with other therapeutic strategies. SIGNIFICANCE Autophagy inhibition may improve cancer therapy, but it is unclear which tumors will benefit. We found that BRAF mutations cause brain tumor cells to depend on autophagy and display selective chemosensitization with autophagy inhibition. We present a pediatric case in which deliberate autophagy inhibition halted tumor growth and overcame acquired BRAF-inhibition resistance.

Pediatric Brain: Repeated Exposure to Linear Gadolinium-based Contrast Material Is Associated with Increased Signal Intensity at Unenhanced T1-weighted MR Imaging

Purpose To determine whether repeated exposure of the pediatric brain to a linear gadolinium-based contrast agent (GBCA) is associated with an increase in signal intensity (SI) relative to that in GBCA-naive control subjects at unenhanced T1-weighted magnetic resonance (MR) imaging. Materials and Methods This single-center, retrospective study was approved by the institutional review board and compliant with HIPAA. The authors evaluated 46 pediatric patients who had undergone at least three GBCA-enhanced MR examinations (30 patients for two-group analysis and 16 for pre- and post-GBCA exposure comparisons) and 57 age-matched GBCA-naive control subjects. The SI in the globus pallidus, thalamus, dentate nucleus, and pons was measured at unenhanced T1-weighted MR imaging. Globus pallidus-thalamus and dentate nucleus-pons SI ratios were calculated and compared between groups and relative to total cumulative gadolinium dose, age, sex, and number of and mean time between GBCA-enhanced examinations. Analysis included the Wilcoxon signed rank test, Wilcoxon rank sum test, and Spearman correlation coefficient. Results Patients who underwent multiple GBCA-enhanced examinations had increased SI ratios within the dentate nucleus (mean SI ratio ± standard error of the mean for two-group comparison: 1.007 ± 0.0058 for GBCA-naive group and 1.046 ± 0.0060 for GBCA-exposed group [P < .001]; mean SI ratio for pre- and post-GBCA comparison: 0.995 ± 0.0062 for pre-GBCA group and 1.035 ± 0.0063 for post-GBCA group [P < .001]) but not the globus pallidus (mean SI ratio for two-group comparison: 1.131 ± 0.0070 for GBCA-naive group and 1.014 ± 0.0091 for GBCA-exposed group [P = .21]; mean SI ratio for pre- and post-GBCA comparison: 1.068 ± 0.0094 for pre-GBCA group and 1.093 ± 0.0134 for post-GBCA group [P = .12]). There was a significant correlation between dentate nucleus SI and total cumulative gadolinium dose (r = 0.4; 95% confidence interval [CI]: 0.03, 0.67; P = .03), but not between dentate nucleus SI and patient age (r = 0.23; 95% CI: -0.15, 0.56; P = .22), sex (mean SI ratio: 1.046 ± 0.0072 for boys and 1.045 ± 0.0110 for girls; P = .88), number of contrast-enhanced examinations (r = 0.13; 95% CI: -0.25, 0.48; P = .49), or time between contrast-enhanced examinations (r = -0.06; 95% CI: -0.42, 0.32; P = .75). Conclusion SI in the pediatric brain increases on unenhanced T1-weighted MR images with repeated exposure to a linear GBCA.

Vaccine strain varicella-zoster virus-induced central nervous system vasculopathy as the presenting feature of DOCK8 deficiency.

To the Editor: In contrast to autosomal dominant forms of hyper-IgE syndrome resulting from mutations in the STAT3 gene, autosomal recessive dedicator of cytokinesis 8 (DOCK8) deficiency, results in susceptibility to cutaneous viral infections, eosinophilia, and allergic disease. CNS manifestations have been reported in patients with DOCK8 deficiency, but progressive multifocal leukoencephalopathy caused by JC virus has been considered the only known viral etiology to date (1, 2). A 6 year-old boy with atopy and recurrent peripheral blood eosinophilia developed acute intermittent vomiting, diarrhea, headache, and dizziness. Magnetic resonance imaging (MRI) of the brain was normal. One day later, he began giggling inappropriately, experienced left leg paresthesias, urinary incontinence, and fell upon attempting to stand. Repeat MRI with axial diffusion-weighted images revealed multiple areas of acute infarction in areas supplied by both anterior cerebral arteries, a branch of the anterior cerebral artery, the right posterior cerebral artery, and the left middle cerebral artery (Figure 1A). A complete blood count revealed a total eosinophil count of 2,160/μL, but no other abnormalities. He was treated with acetylsalicylic acid and intravenous methylprednisolone followed by oral prednisolone, 2 mg/kg for 3 days after which prednisolone was maintained at 1 mg/kg daily. Figure 1 Manifestation of vaccine strain varicella zoster virus-induced central nervous system vasculopathy On presentation to us one month later, he had developed paresthesias of his hands. Neurological examination showed only mild weakness in the hands. Computed tomography angiogram revealed diffuse vasculopathy. Subsequent 3-dimensional time of flight magnetic resonance angiographic imaging of the circle of Willis revealed vascular narrowing and post-stenotic dilatation (Figure 1B). Post-contrast T1W black blood arterial wall imaging illustrated avid enhancement and thickening of the distal supraclinoid internal carotid arterial walls bilaterally (Figure 1C). Cerebral spinal fluid (CSF) was obtained. While awaiting results of testing for viral CNS infections, the patient was treated with acyclovir, 30 mg/kg intravenous daily, and maintained on oral prednisolone, 2 mg/kg/day. The CSF at the time of diagnosis of vasculopathy contained 21 mononuclear cells and no red blood cells; protein was 39 mg/dL and glucose was 72 mg/dL. Quantitative PCR (Focus Diagnostics Reference Laboratory, Cypress, CA) amplified 7,116 copies of VZV DNA/mL in the CSF. To determine the VZV genotype, Forster Resonance Energy Transfer PCR was used to identify specific VZV DNA sequence polymorphisms within VZV open reading frames 38, 54, and 62, which distinguishes vaccine VZV from wild-type virus (Online Supplement Table 1) (3). This confirmed Oka varicella vaccine strain in the CSF. The CSF also contained anti-VZV IgG antibodies, but no antibodies to HSV-1 or HSV-2, enterovirus, cytomegalovirus, or Epstein-Barr virus were detected and the respective PCR analysis were similarly negative. The serum to CSF ratio of anti-VZV IgG antibody was markedly decreased (ratio 2.3) compared to albumin (ratio 149). Two weeks after treatment with acyclovir, the neurologic examination was completely normal, the CSF was acellular, and PCR was negative for VZV DNA. The patient received routine immunizations including VZV vaccination at 12 months of age. He had 3 episodes of vesicular rashes at ages 3, 4, and 5 years, which always occurred after completing a course of oral corticosteroids. The first rash tested positive for HSV. The second rash was identical. The third rash at age 5 years with vesicles on the lower thigh was diagnosed clinically as zoster. All rashes resolved on oral acyclovir. Past history included early-onset atopic dermatitis, food allergies often with anaphylaxis, biopsy-confirmed eosinophilic esophagitis, asthma and recurrent upper respiratory tract infections. Peripheral blood eosinophilia peaked at 9,000 eosinophils/μL and serum IgE at 472 IU/mL. During flares of respiratory or skin disease, he was treated with oral corticosteroids intermittently for 3 to 5 days, a few times annually. The frequency of such treatment increased and between ages 5½ and 6, he was treated with oral corticosteroids 1 to 2 times monthly during the prior 6 months. Conventional comparative genomic hybridization array analysis revealed a large deletion of exons 1 to 13 in a single allele of the DOCK8 gene. PCR analysis of genomic DNA and DOCK8 gene sequencing identified a single base pair mutation on the opposite allele at exon 12, resulting in a frame shift and premature stop codon: c.1266delC, p.Y423TfsX18, based upon reference sequence NM_00193536.1, isoform 3 (Figure 2A). Western Blot analysis confirmed lack of DOCK8 protein expression (Figure 2B). Parental testing demonstrated that the large exon deletion was inherited from the mother while the point mutation was inherited from the father. Immunological findings are summarized in Supplemental Table 2. Figure 2 DOCK8 molecular analyses The neurological symptoms and signs, imaging and CSF abnormalities, virological studies, and response to antiviral treatment were all features characteristically seen in VZV vasculopathy (4). Evidence of both focal and diffuse CNS disease was corroborated by widespread infarction produced by stenosis of multiple large cerebral arteries. The CSF examination revealed a pleocytosis as found in two-thirds of patients with VZV vasculopathy and both VZV DNA and anti-VZV IgG antibody with reduced serum/CSF ratios of anti-VZV IgG antibody were detected, indicative of intrathecal synthesis of anti-VZV IgG. Analysis of VZV DNA in CSF revealed that the VZV genotype was vaccine strain, demonstrating, for the first time, that VZV reactivation after vaccination in childhood can result in VZV vasculopathy. Although the underlying immunological consequences of DOCK8 deficiency remain to be fully elucidated, multiple immune system abnormalities may account for enhanced susceptibility to viral infection including impaired dendritic cell migration affecting T cell priming, lymphopenia, defective CD8 T cell activation and expansion, decreased production of the anti-viral cytokines IFN-γ and TNF-α, impaired T cell survival, decreased NK cell cytotoxicity, and antibody abnormalities (5-9). Germinal center formation and survival of germinal center B cells are impaired in DOCK8 deficiency, leading to defective long-lived antibody production (8). Responses to protein or polysaccharide-conjugated vaccines are often variable while responses to previously encountered viruses such as HSV and VZV have been normal as demonstrated here for VZV antibodies in serum and CSF. As in some other young patients with DOCK8 deficiency, this patient tended toward the milder spectrum of the disease with a relatively limited history of cutaneous infections, absence of severe systemic infections other than chronic mild otitis, and the modestly elevated serum IgE level. Instead, eosinophilia and moderate-severe eczema, asthma, and food sensitivities predominated. To control these conditions, courses of oral corticosteroids were administered with increased frequency over time. Interestingly, each of the 3 episodes of cutaneous viral infections with HSV or VZV was preceded by a course of oral corticosteroids. It is possible corticosteroids alone enabled the activation of vaccine strain VZV infection. More likely, the use of oral corticosteroids to gain disease control reduced the patient’s “immunologic threshold” and together resulted in reactivation of vaccine strain VZV and subsequent vasculopathy. In summary, a young patient with significant atopic disease and widespread infarction produced by stenosis of multiple large cerebral arteries was shown to express novel mutations on both alleles of the DOCK8 gene. For the first time, VZV vasculopathy was shown to be due to the vaccine strain. This case highlights the importance of considering the possibility of DOCK8 deficiency in the context of severe allergic disease and the potential risks for CNS infection including VZV vaccine-related vasculopathy.

Autophagy inhibition overcomes multiple mechanisms of resistance to BRAF inhibition in brain tumors

Kinase inhibitors are effective cancer therapies, but tumors frequently develop resistance. Current strategies to circumvent resistance target the same or parallel pathways. We report here that targeting a completely different process, autophagy, can overcome multiple BRAF inhibitor resistance mechanisms in brain tumors. BRAFV600Emutations occur in many pediatric brain tumors. We previously reported that these tumors are autophagy-dependent and a patient was successfully treated with the autophagy inhibitor chloroquine after failure of the BRAFV600E inhibitor vemurafenib, suggesting autophagy inhibition overcame the kinase inhibitor resistance. We tested this hypothesis in vemurafenib-resistant brain tumors. Genetic and pharmacological autophagy inhibition overcame molecularly distinct resistance mechanisms, inhibited tumor cell growth, and increased cell death. Patients with resistance had favorable clinical responses when chloroquine was added to vemurafenib. This provides a fundamentally different strategy to circumvent multiple mechanisms of kinase inhibitor resistance that could be rapidly tested in clinical trials in patients with BRAFV600E brain tumors. DOI: http://dx.doi.org/10.7554/eLife.19671.001

Cavernous Sinus Thrombosis in Children: Imaging Characteristics and Clinical Outcomes.

Background and Purpose— Cavernous sinus thrombosis (CST) is a rare life-threatening cerebrovascular disease known to cause carotid artery narrowing (CAN) and arterial ischemic stroke. The imaging features of CST and related complications have been reported in adults, but rarely in children. Methods— We performed a retrospective review of children with imaging confirmed CST from 2003 to 2014, describing presenting symptoms, imaging findings, and treatment. Results— Ten patients with CST were identified. All had CAN and 6 of 10 developed infarcts. Of 8 patients treated with anticoagulation therapy, 3 developed new infarcts. None required discontinuation of anticoagulation therapy because of bleeding. Visual impairment secondary to infectious neuritis was common. Imaging characteristics include cavernous sinus expansion, filling defects, restricted diffusion, arterial wall enhancement, empyema, superior ophthalmic vein enlargement and thrombosis, orbital cellulitis, and pituitary inflammation. CAN resolved in 60% of cases. Outcomes were mostly good, with a modified Rankin Scale score of ⩽1 for 7 of 10 patients at discharge and 1 death. Conclusions— CAN and infarcts were common in this modest cohort of children with CST. Despite the high incidence of CAN and infarction, outcomes were often favorable. Although this is the largest cohort of childhood CST reported to date, large multicenter cohorts are needed to confirm our findings and determine the preferred therapeutic strategies for childhood CST.

Pathways for Neuroimaging of Childhood Stroke.

BACKGROUND The purpose of this article is to aid practitioners in choosing appropriate neuroimaging for children who present with symptoms that could be caused by stroke. METHODS The Writing Group members participated in one or more pediatric stroke neuroimaging symposiums hosted by the Stroke Imaging Laboratory for Children housed at the Hospital for Sick Children in Toronto, Ontario, Canada. Through collaboration, literature review, and discussion among child neurologists with expertise diagnosing and treating childhood stroke and pediatric neuroradiologists and neuroradiologists with expertise in pediatric neurovascular disease, suggested imaging protocols are presented for children with suspected stroke syndromes including arterial ischemic stroke, cerebral sinovenous thrombosis, and hemorrhagic stroke. RESULTS This article presents information about the epidemiology and classification of childhood stroke with definitions based on the National Institutes of Health Common Data Elements. The role of imaging for the diagnosis of childhood stroke is examined in depth, with separate sections for arterial ischemic stroke, cerebral sinovenous thrombosis, and hemorrhagic stroke. Abbreviated neuroimaging protocols for rapid diagnosis are discussed. The Writing Group provides suggestions for optimal neuroimaging investigation of various stroke types in the acute setting and suggestions for follow-up neuroimaging. Advanced sequences such as diffusion tensor imaging, perfusion imaging, and vessel wall imaging are also discussed. CONCLUSIONS This article provides protocols for the imaging of children who present with suspected stroke.

Ophthalmic manifestations and outcomes after cavernous sinus thrombosis in children

PURPOSE To review the causes, treatment, and outcomes of cavernous sinus thrombosis (CST) in children. METHODS The medical records of children (<18 years of age) diagnosed with thrombophlebitis of an intracranial venous sinus were reviewed to identify cases of CST presenting to Childrens Hospital Colorado from January 2000 through January 2013. Cases were evaluated for etiology, symptoms, imaging characteristics, treatment, and outcomes. RESULTS A total of 110 children with a venous thrombus of an intracranial sinus were included. Of these, 9 had a CST. All cases were confirmed by magnetic resonance imaging. All 9 had sinusitis, 4 had orbital involvement, and 1 resulted from a nasal septal abscess. Eight cases presented with ophthalmoplegia, and 5 presented with decreased vision. Every patient underwent sinus surgery: 4 underwent orbitotomy for abscess drainage, and 1 required bilateral exenteration. Cultures were inconclusive in 2 cases, and 2 cases were culture positive rhino-orbital mucormycosis. There was 1 case of methicillin-resistant Staphylococcus aureus. Of the 9 cases, 4 returned to normal vision and ocular motility; 5 had permanent ophthalmoplegia and vision loss. There were no cases of mortality. CONCLUSIONS CST is a rare complication of orbital and sinus disease. High clinical suspicion, early neurologic imaging, and a multidisciplinary approach to management are key factors in reducing morbidity and mortality from CST in children.

Predicting Progression of Intracranial Arteriopathies in Childhood Stroke With Vessel Wall Imaging

Background and Purpose— Childhood arterial ischemic stroke is frequently associated with an intracranial arteriopathy that often progresses in the first 3 to 6 months post stroke. We hypothesized that children with enhancing arteriopathies on vessel wall imaging (VWI) would have a higher risk of arteriopathy progression than those without enhancement. Methods— Our institutional radiographic database was searched for cases of childhood stroke with VWI. Inclusion criteria consisted of age ranging from 1 month through 20 years, diagnosis of arterial ischemic stroke, available VWI, and follow-up magnetic resonance angiogram. Imaging was reviewed to systematically describe VWI findings, categorize arteriopathies, steroid therapy, and identify progressive arteriopathies using CACADE definitions. Results— Sixteen cases of childhood stroke at Children’s Hospital Colorado between January 1, 2010 and July 1, 2016 were reviewed. Strong vessel wall enhancement at presentation was associated with progressive arteriopathy in 83% of cases (10/12), when compared with 0% (0/4) without strong enhancement (P=0.008). Conclusions— Our case series demonstrates the potential benefit of VWI in children with stroke because it may identify patients who will have progressive arterial disease.

Predictive validity of severity grading for cerebral steno‐occlusive arteriopathy in recurrent childhood ischemic stroke

Background Cerebral arteriopathy is a risk factor for incident and recurrent childhood AIS. There are no standardized criteria to quantify arteriopathy severity. Aims To evaluate a method of scoring severity of steno-occlusive arteriopathy in childhood arterial ischemic stroke (AIS) and its association with recurrence. Methods In a single-center prospectively enrolled cohort of 49 children with first AIS and arteriopathy, a composite cerebrovascular stenosis score (CVSS) was measured by two independent raters as the sum of stenosis scores in each of 18 intracranial large and medium arteries, where 0 = none; 1 = low-grade, 1–50%; 2 = high-grade, >50–99%; 3 = occlusion, 100%. Cox proportional-hazards models were used to determine the association of CVSS with recurrence. The analysis was stratified by presence or absence of moyamoya arteriopathy (syndrome or disease). Results At a median follow-up period of 2·5 years (range: 0·8–9), 18/49 children (37%) experienced a recurrence. Median time to recurrence was 0·2 (range: 0·02–2·8) years. Interrater agreement was good, with an intraclass correlation coefficient of 0·77 [95% confidence interval (CI) 0·63–0·87, P < 0·001). Higher CVSS was associated with higher recurrence rate [hazard ratio (HR) per point 1·09, 95% CI 1·04–1·16, P = 0·001]. Among those with moyamoya arteriopathy, CVSS was associated with recurrence (HR per point of CVSS 1·11, 95% CI 1·03–1·19, P = 0·004), but there was no association in those without moyamoya arteriopathy (HR per point of CVSS 0·91, 95% CI 0·75–1·09, P = 0·32). Conclusions The CVSS is a reliable measure of severity of steno-occlusive arteriopathy in childhood stroke. This preliminary study suggests that higher CVSS is associated with stroke recurrence in children with moyamoya arteriopathy.