Polyclonal lymphoid expansion drives paraneoplastic autoimmunity in neuroblastoma.

Neuroblastoma is a lethal childhood solid tumor of developing peripheral nerves. Two percent of children with neuroblastoma develop opsoclonus myoclonus ataxia syndrome (OMAS), a paraneoplastic disease characterized by cerebellar and brainstem-directed autoimmunity but typically with outstanding cancer-related outcomes. We compared tumor transcriptomes and tumor-infiltrating T and B cell repertoires from 38 OMAS subjects with neuroblastoma to 26 non-OMAS-associated neuroblastomas. We found greater B and T cell infiltration in OMAS-associated tumors compared to controls and showed that both were polyclonal expansions. Tertiary lymphoid structures (TLSs) were enriched in OMAS-associated tumors. We identified significant enrichment of the major histocompatibility complex (MHC) class II allele HLA-DOB∗01:01 in OMAS patients. OMAS severity scores were associated with the expression of several candidate autoimmune genes. We propose a model in which polyclonal auto-reactive B lymphocytes act as antigen-presenting cells and drive TLS formation, thereby supporting both sustained polyclonal T cell-mediated anti-tumor immunity and paraneoplastic OMAS neuropathology.

Younger Age at Onset Is Associated With Worse Long-term Behavioral Outcomes in Anti-NMDA Receptor Encephalitis.

BACKGROUND AND OBJECTIVES: Anti-NMDA receptor encephalitis (anti-NMDARE) is one of the most common causes of encephalitis. It typically presents in adolescence and young adulthood, but little is known about its potential long-term consequences across the lifespan. Adaptive behavior describes an individual's ability to respond and adapt to environmental demands and unanticipated changes in daily routines. In this study, we evaluate the relationship between features from clinical presentation, including age, and long-term adaptive behavior in participants with anti-NMDARE. METHODS: Cross-sectional informant-reported data were collected between 2017 and 2019 from 41 individuals/caregivers of individuals with anti-NMDARE treated at 3 major academic hospitals. Neurologic disability was assessed by record review using the modified Rankin Scale (mRS). Functional outcomes were assessed using the validated Adaptive Behavior Assessment System, Third Edition (ABAS-3). RESULTS: The mean age at the time of study enrollment was 23.4 years (SD 17.0 years), and the mean time from symptom onset to study enrollment was 4.0 years. Seventeen participants were aged <12 years at symptom onset, 19 participants were aged 12-30 years, and 5 participants were aged >30 years. Mean ABAS-3 scores at study enrollment for all participants were in the average range (mean general adaptive composite standard score 92.5, SD 18.7). Individuals aged <12 years at symptom onset had lower mean ABAS-3 scores and were in the below average range compared with those aged 12-30 years at symptom onset, whose mean scores were in the average range (87 vs 99, p < 0.05). Similar differences were seen in 3 of the individual subscales (functional academics, health and safety, and self-care). There were no significant differences in mRS scores between age groups (p > 0.05). DISCUSSION: Although anti-NMDARE is associated with an overall favorable outcome, younger age at onset associates with worse long-term adaptive behavior despite no differences in neurologic disability. These findings suggest that the disease may have distinct consequences on the early developing brain. Future studies should evaluate behavioral recovery and quality of life after anti-NMDARE and identify additional factors associated with differential recovery.

Neurotoxicity after CTL019 in a pediatric and young adult cohort.

OBJECTIVE: To characterize the incidence and clinical characteristics of neurotoxicity in the month following CTL019 infusion in children and young adults, to define the relationship between neurotoxicity and cytokine release syndrome (CRS), and to identify predictive biomarkers for development of neurotoxicity following CTL019 infusion. METHODS: We analyzed data on 51 subjects, 4 to 22 years old, who received CTL019, a chimeric antigen receptor-modified T-cell therapy against CD19, between January 1, 2010 and December 1, 2015 through a safety/feasibility clinical trial (NCT01626495) at our institution. We recorded incidence of significant neurotoxicity (encephalopathy, seizures, and focal deficits) and CRS, and compared serum cytokine levels in the first month postinfusion between subjects who did and did not develop neurotoxicity. RESULTS: Neurotoxicity occurred in 23 of 51 subjects (45%, 95% confidence interval = 31-60%) and was positively associated with higher CRS grade (p < 0.0001) but was not associated with demographic characteristics or prior oncologic treatment history. Serum interleukin (IL)-2, IL-15, soluble IL-4, and hepatocyte growth factor concentrations were higher in subjects with neurotoxicity than those with isolated CRS. Differences in peak levels of select cytokines including IL-12 and soluble tumor necrosis factor receptor-1 within the first 3 days were seen in subjects with neurotoxicity. INTERPRETATION: Neurotoxicity is common after CTL019 infusion in children and young adults, and is associated with higher CRS grade. Differences in serum cytokine profiles between subjects with neurotoxicity and those with isolated CRS suggest unique pathophysiological mechanisms. Serum cytokine profiles in the first 3 days postinfusion may help identify children and young adults at risk for neurotoxicity, and may provide a foundation for investigation into potential mitigation strategies. Ann Neurol 2018;84:537-546.

Monoclonal antibodies from a patient with anti-NMDA receptor encephalitis.

OBJECTIVE: Anti-NMDA receptor encephalitis (ANRE) is a potentially lethal encephalitis attributed to autoantibodies against the N-methyl-D-aspartate receptor (NMDAR). We sought to clone and characterize monoclonal antibodies (mAbs) from an ANRE patient. METHODS: We used a hybridoma method to clone two IgG mAbs from a female patient with ANRE without teratoma, and characterized their binding activities on NMDAR-transfected cell lines, cultured primary rat neurons, and mouse hippocampus. We also assessed their effects on voluntary locomotor activity in mice and binding to NMDAR in vivo. RESULTS: The mAbs are structurally distinct and arose from distinct B-cell lineages. They recognize different epitopes on the GluN1 amino terminal domain (ATD), yet both require amino acids important for post-translational modification. Both mAbs bind subsets of GluN1 on cultured rat hippocampal neurons. The 5F5 mAb binds mouse brain hippocampal tissues, and the GluN1 recognized on cultured rat neurons was substantially extra-synaptic. Antibody binding to primary hippocampal neurons induced receptor internalization. The NMDAR inhibitor MK-801 inhibited internalization without preventing mAb binding; AP5 inhibited both mAb binding and internalization. Exposure of mice to the mAbs following permeabilization of the blood brain barrier increased voluntary wheel running activity, similar to low doses of the NMDAR inhibitor, MK-801. INTERPRETATION: These mAbs recapitulate features demonstrated in previous studies of ANRE patient CSF, and exert effects on NMDAR in vitro and in vivo consistent with modulation of NMDAR activity.

Anti-NMDA receptor encephalitis and nonencephalitic HSV-1 infection.

OBJECTIVE: To determine whether there is an association between nonencephalitic herpes simplex virus 1 (HSV-1) infection and anti-NMDA receptor encephalitis (anti-NMDARE). METHODS: Antibody testing was performed using samples from 2 cohorts in a case-control observational study. The cohort "Philadelphia" included 16 serum samples of pediatric anti-NMDARE cases and 42 age-matched controls with other neuroinflammatory disorders studied at the Children's Hospital of Philadelphia and University of Pennsylvania. The cohort "Barcelona" contained 23 anti-NMDARE patient samples and 26 age-matched participants with other neuroinflammatory disorders studied at IDIBAPS-Hospital Clinic, University of Barcelona. The presence of HSV-1 IgG antibodies was examined by ELISA. As an additional control, IgG antibodies to cytomegalovirus (CMV) and Epstein-Barr virus viral capsid antigen (EBV-VCA) were determined. RESULTS: In each cohort, more participants with anti-NMDARE than controls had anti-HSV-1 IgG antibodies. In the Philadelphia cohort (58 participants), 44% of anti-NMDARE cases had antibodies to HSV-1 compared with 14% controls (OR 4.67, 95% CI 1.3-17.3, p = 0.031). In the Barcelona cohort (49 participants), 52% of participants with anti-NMDARE had antibodies to HSV-1 compared with 31% of controls (OR 2.45, 95% CI 0.7-7.9, p = 0.155). Overall, 49% of anti-NMDARE cases have antibodies to HSV-1 in these 2 combined cohorts compared with 21% of controls (Mantel-Haenszel OR 3.21, 95% CI 1.3-7.7, p = 0.007). CONCLUSION: Past HSV-1 infection was found in significantly more anti-NMDARE cases than controls. This suggests a meaningful association between nonencephalitic HSV-1 infection and development of anti-NMDARE.

A Mild PUM1 Mutation Is Associated with Adult-Onset Ataxia, whereas Haploinsufficiency Causes Developmental Delay and Seizures.

Certain mutations can cause proteins to accumulate in neurons, leading to neurodegeneration. We recently showed, however, that upregulation of a wild-type protein, Ataxin1, caused by haploinsufficiency of its repressor, the RNA-binding protein Pumilio1 (PUM1), also causes neurodegeneration in mice. We therefore searched for human patients with PUM1 mutations. We identified eleven individuals with either PUM1 deletions or de novo missense variants who suffer a developmental syndrome (Pumilio1-associated developmental disability, ataxia, and seizure; PADDAS). We also identified a milder missense mutation in a family with adult-onset ataxia with incomplete penetrance (Pumilio1-related cerebellar ataxia, PRCA). Studies in patient-derived cells revealed that the missense mutations reduced PUM1 protein levels by ∼25% in the adult-onset cases and by ∼50% in the infantile-onset cases; levels of known PUM1 targets increased accordingly. Changes in protein levels thus track with phenotypic severity, and identifying posttranscriptional modulators of protein expression should identify new candidate disease genes.

Anti-NMDA Receptor Encephalitis: Clinical Features and Basic Mechanisms.

In slightly more than 10 years, anti-NMDA receptor (NMDAR) encephalitis has changed from a rare paraneoplastic syndrome to the most common cause of nonviral encephalitis. It presents fulminantly with progressive psychosis, seizures, and autonomic dysfunction, leading to death if untreated. However, rapid recognition and treatment can lead to survival and a return to baseline levels of functioning in many patients. While initially associated with ovarian teratomas, it is now associated with other tumors and can reflect a postviral event. The antibodies to the NMDAR made in this syndrome are pathogenic and are directed at the extracellular domain of the GluN1 subunit. Such antibodies lead to internalization of NMDARs in model systems, leading to a physiological state characterized by NMDAR hypofunction. Analogous disorders, characterized by antibodies to other synaptic receptors, present with neurological and psychiatric dysfunction and also appear to reflect antibody-induced internalization of receptors. However, this simple pathophysiology may be too simplistic to reflect the complexity of events in anti-NMDAR encephalitis. Future scientific investigations may allow a more complete understanding of this disorder and improve treatment of anti-NMDAR encephalitis.

Intravenous immunoglobulin with prednisone and risk-adapted chemotherapy for children with opsoclonus myoclonus ataxia syndrome associated with neuroblastoma (ANBL00P3): a randomised, open-label, phase 3 trial.

Purpose: No previous clinical trial has been conducted for patients with neuroblastoma associated opsoclonus myoclonus ataxia syndrome (OMA), and current treatment is based on case reports. To evaluate the OMA response to prednisone and risk-adapted chemotherapy and determine if the addition of intravenous gammaglobulin (IVIG) further improves response, the Children's Oncology Group designed a randomized therapeutic trial. Patient and Methods: Eligible subjects were randomized to receive twelve cycles of IVIG (IVIG+) or no IVIG (NO-IVIG) in addition to prednisone and neuroblastoma risk-adapted chemotherapy. All low-risk patients were treated with cyclophosphamide. The severity of OMA symptoms was evaluated at 2, 6, and 12 months using a scale developed by Mitchell and Pike and baseline versus best response scores were compared. A single patient who did not undergo neurologic assessment was excluded from OMA response analysis. This study is registered with Clinical Trials.gov (identifier NCT00033293). Results: Of the 53 patients enrolled in the study, 62% (33/53) were female. There were 44 low-risk, 7 intermediate-risk, and 2 high-risk neuroblastoma patients. Twenty-six subjects were randomized to receive IVIG+ and 27 were randomized to NO-IVIG. The neuroblastoma 3-year event-free survival (95% confidence interval (CI)) was 94.1% (87.3%, 100%) and overall survival was 98.0% (94.1%, 100%). Significantly higher rates of OMA response were observed in patients randomized to IVIG+ compared to NO-IVIG [21/26=80.8% for IVIG+; 11/27=40.7% for NO-IVIG (odds ratio=6.1; 95% CI: (1.5, 25.9), p=0.0029)]. For the majority of patients, the IVIG+ OMA regimen combined with cytoxan or other risk-based chemotherapy was well tolerated, although there was one toxic death in a high-risk subject. Conclusion: This is the only randomized prospective therapeutic clinical trial in children with neuroblastoma-associated OMA. The addition of IVIG to prednisone and risk-adapted chemotherapy significantly improves OMA response rate. IVIG+ constitutes a back-bone upon which to build additional therapy.

Transverse myelitis as an unexpected complication following treatment with dinutuximab in pediatric patients with high-risk neuroblastoma: A case series.

Immunotherapy with the anti-GD2 monoclonal antibody ch14.18, or dinutuximab, represents an important therapeutic advance in the treatment of pediatric high-risk neuroblastoma and is now considered part of standard of care in this patient population. To date, transverse myelitis as a result of dinutuximab therapy has not been reported in clinical trials or in the published literature. We describe three patients with clinical symptoms of transverse myelitis, confirmed via magnetic resonance imaging, shortly following initiation of dinutuximab. All patients were discontinued from dinutuximab treatment and received urgent treatment, with rapid improvement in symptoms and resultant functional recovery.

Variants Within TSC2 Exons 25 and 31 Are Very Unlikely to Cause Clinically Diagnosable Tuberous Sclerosis.

Inactivating mutations in TSC1 and TSC2 cause tuberous sclerosis complex (TSC). The 2012 international consensus meeting on TSC diagnosis and management agreed that the identification of a pathogenic TSC1 or TSC2 variant establishes a diagnosis of TSC, even in the absence of clinical signs. However, exons 25 and 31 of TSC2 are subject to alternative splicing. No variants causing clinically diagnosed TSC have been reported in these exons, raising the possibility that such variants would not cause TSC. We present truncating and in-frame variants in exons 25 and 31 in three individuals unlikely to fulfil TSC diagnostic criteria and examine the importance of these exons in TSC using different approaches. Amino acid conservation analysis suggests significantly less conservation in these exons compared with the majority of TSC2 exons, and TSC2 expression data demonstrates that the majority of TSC2 transcripts lack exons 25 and/or 31 in many human adult tissues. In vitro assay of both exons shows that neither exon is essential for TSC complex function. Our evidence suggests that variants in TSC2 exons 25 or 31 are very unlikely to cause classical TSC, although a role for these exons in tissue/stage specific development cannot be excluded.

Antibodies to dendritic neuronal surface antigens in opsoclonus myoclonus ataxia syndrome.

Opsoclonus myoclonus ataxia syndrome (OMAS) is an autoimmune disorder characterized by rapid, random, conjugate eye movements (opsoclonus), myoclonus, and ataxia. Given these symptoms, autoantibodies targeting the cerebellum or brainstem could mediate the disease or be markers of autoimmunity. In a subset of patients with OMAS, we identified such autoantibodies, which bind to non-synaptic puncta on the surface of live cultured cerebellar and brainstem neuronal dendrites. These findings implicate autoimmunity to a neuronal surface antigen in the pathophysiology of OMAS. Identification of the targeted antigen(s) could elucidate the mechanisms underlying OMAS and provide a biomarker for diagnosis and response to therapy.

Pediatric anti-NMDA receptor encephalitis is seasonal.

In the majority of pediatric anti-N-methyl-d-aspartate receptor encephalitis (NMDARe) cases, the underlying cause of antibody production and subsequent disease remains unknown. We aimed to characterize this poorly understood population, investigating epidemiological factors potentially related to disease etiology, particularly season of onset. In this retrospective case review study, we analyzed data from the 29 pediatric subjects with anti-NMDAR antibodies and found that symptoms were first reported in the warm months of April-September in 78% of non-tumor-related NMDARe (NT-NMDARe) cases. These findings support further investigation into a possible seasonal trigger of NT-NMDARe.

High prevalence of NMDA receptor IgA/IgM antibodies in different dementia types.

OBJECTIVE: To retrospectively determine the frequency of N-Methyl-D-Aspartate (NMDA) receptor (NMDAR) autoantibodies in patients with different forms of dementia. METHODS: Clinical characterization of 660 patients with dementia, neurodegenerative disease without dementia, other neurological disorders and age-matched healthy controls combined with retrospective analysis of serum or cerebrospinal fluid (CSF) for the presence of NMDAR antibodies. Antibody binding to receptor mutants and the effect of immunotherapy were determined in a subgroup of patients. RESULTS: Serum NMDAR antibodies of IgM, IgA, or IgG subtypes were detected in 16.1% of 286 dementia patients (9.5% IgM, 4.9% IgA, and 1.7% IgG) and in 2.8% of 217 cognitively healthy controls (1.9% IgM and 0.9% IgA). Antibodies were rarely found in CSF. The highest prevalence of serum antibodies was detected in patients with "unclassified dementia" followed by progressive supranuclear palsy, corticobasal syndrome, Parkinson's disease-related dementia, and primary progressive aphasia. Among the unclassified dementia group, 60% of 20 patients had NMDAR antibodies, accompanied by higher frequency of CSF abnormalities, and subacute or fluctuating disease progression. Immunotherapy in selected prospective cases resulted in clinical stabilization, loss of antibodies, and improvement of functional imaging parameters. Epitope mapping showed varied determinants in patients with NMDAR IgA-associated cognitive decline. INTERPRETATION: Serum IgA/IgM NMDAR antibodies occur in a significant number of patients with dementia. Whether these antibodies result from or contribute to the neurodegenerative disorder remains unknown, but our findings reveal a subgroup of patients with high antibody levels who can potentially benefit from immunotherapy.

Antigenic and mechanistic characterization of anti-AMPA receptor encephalitis.

OBJECTIVE: Anti-AMPAR encephalitis is a recently discovered disorder characterized by the presence of antibodies in serum or cerebrospinal fluid against the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. Here, we examine the antigenic specificity of anti-AMPAR antibodies, screen for new patients, and evaluate functional effects of antibody treatment of neurons. METHODS: We developed a fusion protein-based western blotting test for anti-AMPAR encephalitis antibodies. Antibody specificity was also evaluated using immunocytochemistry of HEK293 cells expressing deletion mutants of AMPAR subunits. Purified patient IgG or AMPAR antibody-depleted IgG was applied to live neuronal cultures; amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) were measured to evaluate functional effects of antibodies. RESULTS: Using both immunocytochemistry and fusion protein western blots, we defined an antigenic region of the receptor in the bottom lobe of the amino terminal domain. Additionally, we used fusion proteins to screen 70 individuals with neurologic symptoms of unknown cause and 44 patients with no neurologic symptoms or symptoms of known neuroimmunological origin for anti-AMPAR antibodies. Fifteen of the 70 individuals had anti-AMPAR antibodies, with broader antigenic reactivity patterns. Using purified IgG from an individual of the original cohort of anti-AMPAR encephalitis patients and a newly discovered patient, we found that application of IgG from either patient cohort caused an AMPAR antibody-dependent decrease in the amplitude and frequency of mEPSCs in cultured neurons. INTERPRETATION: These results indicate that anti-AMPAR antibodies are widespread and functionally relevant; given the robust response of patients to immunomodulation, this represents a significant treatable patient population.

Immune therapy for pharmacoresistant epilepsy: ready to go?

Despite the introduction of almost 20 new antiseizure drugs in the last several decades, approximately 30% of patients with epilepsy have pharmacoresistant seizures. Although many genetic disorders and cerebral structural lesions are known causes of difficult-to-treat epilepsy, some refractory patients do not fall into these categories. In the search for other etiologic factors, the multiple and close interactions between the immune system and epilepsy have gained substantial interest.(1.)