Truncating the i-leader open reading frame enhances release of human adenovirus type 5 in glioma cells

Background: The survival of glioma patients with the current treatments is poor. Early clinical trails with replicating adenoviruses demonstrated the feasibility and safety of the use of adenoviruses as oncolytic agents. Antitumor efficacy has been moderate due to inefficient virus replication and spread. Previous studies have shown that truncation of the adenovirus i-leader open reading frame enhanced cytopathic activity of HAdV-5 in several tumor cell lines. Here we report the effect of an i-leader mutation on the cytopathic activity in glioma cell lines and in primary high-grade glioma

No evidence for circulating HuD-specific CD8+ T cells in patients with paraneoplastic neurological syndromes and Hu antibodies

Aim: In paraneoplastic neurological syndromes (PNS) associated with small cell lung cancer (SCLC) and Hu antibodies (Hu-PNS), Hu antigens expressed by the tumour hypothetically trigger an immune response that also reacts with Hu antigens in the nervous system, resulting in tumour suppression and neuronal damage. To gain more insight into the hypothesized CD8+T cell-mediated immune pathogenesis of these syndromes, we searched for circulating HuD-specific CD8+T cells in a large cohort of Hu-PNS patients and controls. Patients and methods: Blood was tested from 43 Hu-PNS patients, 31 Hu antibody negativ

Metabolic changes related to the IDH1 mutation in gliomas preserve TCA-cycle activity: An investigation at the protein level

The discovery of the IDH1 R132H (IDH1 mut) mutation in low-grade glioma and the associated change in function of the IDH1 enzyme has increased the interest in glioma metabolism. In an earlier study, we found that changes in expression of genes involved in the aerobic glycolysis and the TCA cycle are associated with IDH1 mut. Here, we apply proteomics to FFPE samples of diffuse gliomas with or without IDH1 mutations, to map changes in protein levels associated with this mutation. We observed significant changes in the enz

Paraneoplastic cerebellar degeneration associated with antineuronal antibodies: analysis of 50 patients

Paraneoplastic cerebellar degeneration (PCD) is a heterogeneous group of disorders characterized by subacute cerebellar ataxia, specific tumour types and (often) associated antineuronal antibodies. Nine specific antineuronal antibodies are associated with PCD. We examined the relative frequency of the antineuronal antibodies associated with PCD and compared the neurological symptoms and signs, associated tumours, disability and survival between groups of PCD with different antibodies. Also, we attempted to identify patient-, tumour- and treatment-related characteristics associated with functional outcome and survival. In a 12-year period, we examined >5000 samples for the presence of antineuronal antibodies. A total of 137 patients were identified with a paraneoplastic neurological syndrome and high titre (> or =400) antineuronal antibodies. Fifty (36%) of these patients had antibody-associated PCD, including 19 anti-Yo, 16 anti-Hu, seven anti-Tr, six anti-Ri and two anti-mGluR1. Because of the low number, the anti-mGluR1 patients were excluded from the statistical analysis. While 100% of patients with anti-Yo, anti-Tr and anti-mGluR1 antibodies suffered PCD, 86% of anti-Ri and only 18% of anti-Hu patients had PCD. All patients presented with subacute cerebellar ataxia progressive over weeks to months and stabilized within 6 months. The majority of patients in all antibody groups had both truncal and appendicular ataxia. The frequency of nystagmus and dysarthria was lower in anti-Ri patients (33 and 0%). Later in the course of the disease, involvement of non-cerebellar structures occurred most frequently in anti-Hu patients (94%). In 42 patients (84%), a tumour was detected. The most commonly associated tumours were gynaecological and breast cancer (anti-Yo and anti-Ri), lung cancer (anti-Hu) and Hodgkin's lymphoma (anti-Tr and anti-mGluR1). In one anti-Hu patient, a suspect lung lesion on CT scan disappeared while the PCD evolved. Seven patients improved by at least 1 point on the Rankin scale, while 16 remained stable and 27 deteriorated. All seven patients that improved received antitumour treatment for their underlying cancer, resulting in complete remission. The functional outcome was best in the anti-Ri patients, with three out of six improving neurologically and five were able to walk at the time of last follow-up or death. Only four out of 19 anti-Yo and four out of 16 anti-Hu patients remained ambulatory. Also, survival from time of diagnosis was significantly worse in the anti-Yo (median 13 months) and anti-Hu (median 7 months) patients compared with anti-Tr (median >113 months) and anti-Ri (median >69 months). Patients receiving antitumour treatment (with or without immunosuppressive therapy) lived significantly longer [hazard ratio (HR) 0.3; 95% confidence interval (CI) 0.1-0.6; P = 0.004]. Patients > or =60 years old lived somewhat shorter from time of diagnosis, although statistically not significant (HR 2.9; CI 1.0-8.5; P = 0.06)

Phase II trial of natalizumab for the treatment of anti-Hu associated paraneoplastic neurological syndromes

BACKGROUND: Paraneoplastic neurological syndromes with anti-Hu antibodies (Hu-PNS) have a very poor prognosis: more than half of the patients become bedridden and median survival is less than 12 months. Several lines of evidence suggest a pathogenic T cell-mediated immune response. Therefore, we conducted a prospective open-label phase II trial with natalizumab. METHODS: Twenty Hu-PNS patients with progressive disease were treated with a maximum of three monthly natalizumab cycles (300 mg). The primary outcome measure was functional improvement, this was defined as at least one point decrease in modified Rankin Scale (mRS) score at the last treatment visit. In addition, treatment response was assessed wherein a mRS score ≤3 after treatment was defined as treatment responsive. RESULTS: The median age at onset was 67.8 years (SD 8.4) with a female predominance (n = 17, 85%). The median time from symptom onset to Hu-PNS diagnosis was 5 months (IQR 2–11). Most patients had subacute sensory neuronopathy (n = 15, 75%), with a median mRS of 4 at baseline. Thirteen patients had a tumor, all small cell lung cancer. After natalizumab treatment, two patients (10%) showed functional improvement. Of the remaining patients, 60% had a stable functional outcome, while 30% showed further deterioration. Treatment response was classified as positive in nine patients (45%). CONCLUSIONS: Natalizumab may ameliorate the disease course in Hu-PNS, but no superior effects above other reported immunosuppressive and immunomodulatory were observed. More effective treatment modalities are highly needed. TRIAL REGISTRATION: https://www.clinicaltrialsregister.eu/ctr-search/trial/2014-000675-13/N

Peptides from the variable region of specific antibodies are shared among lung cancer patients

Late diagnosis of lung cancer is still the main reason for high mortality rates in lung cancer. Lung cancer is a heterogeneous disease which induces an immune response to different tumor antigens. Several methods for searching autoantibodies have been described that are based on known purified antigen panels. The aim of our study is to find evidence that parts of the antigen-binding-domain of antibodies are shared among lung cancer patients. This was investigated by a novel approach based on sequencing antigen-binding- fragments (Fab) of immunoglobulins using proteomic techniques without the need of previously known antigen panels. From serum of 93 participants of the NELSON trial IgG was isolated and subsequently digested into Fab and Fc. Fab was purified from the digested mixture by SDS-PAGE. The Fab containing gel-bands were excised, tryptic digested and measured on a nano-LC-Orbitrap-Mass- spectrometry system. Multivariate analysis of the mass spectrometry data by linear canonical discriminant analysis combined with stepwise logistic regression resulted in a 12-antibody-peptide model which was able to distinguish lung cancer patients from controls in a high risk population with a sensitivity of 84% and specificity of 90%. With our Fab-purification combined Orbitrap-mass-spectrometry approach, we found peptides from the variable-parts of antibodies which are shared among lung cancer patients

Mimics of Autoimmune Encephalitis:Validation of the 2016 Clinical Autoimmune Encephalitis Criteria

BACKGROUND AND OBJECTIVES: The clinical criteria for autoimmune encephalitis (AE) were proposed by Graus et al. in 2016. In this study, the AE criteria were validated in the real world, and common AE mimics were described. In addition, criteria for probable anti-LGI1 encephalitis were proposed and validated. METHODS: In this retrospective cohort study, patients referred to our national referral center with suspicion of AE and specific neuroinflammatory disorders with similar clinical presentations were included from July 2016 to December 2019. Exclusion criteria were pure cerebellar or peripheral nerve system disorders. All patients were evaluated according to the AE criteria. RESULTS: In total, 239 patients were included (56% female; median age 42 years, range 1-85). AE was diagnosed in 104 patients (44%) and AE mimics in 109 patients (46%). The most common AE mimics and misdiagnoses were neuroinflammatory CNS disorders (26%), psychiatric disorders (19%), epilepsy with a noninflammatory cause (13%), CNS infections (7%), neurodegenerative diseases (7%), and CNS neoplasms (6%). Common confounding factors were mesiotemporal lesions on brain MRI (17%) and false-positive antibodies in serum (12%). Additional mesiotemporal features (involvement extralimbic structures, enhancement, diffusion restriction) were observed more frequently in AE mimics compared with AE (61% vs 24%; p = 0.005). AE criteria showed the following sensitivity and specificity: possible AE, 83% (95% CI 74-89) and 27% (95% CI 20-36); definite autoimmune limbic encephalitis (LE), 10% (95% CI 5-17) and 98% (95% CI 94-100); and probable anti-NMDAR encephalitis, 50% (95% CI 26-74) and 96% (95% CI 92-98), respectively. Specificity of the criteria for probable seronegative AE was 99% (95% CI 96-100). The newly proposed criteria for probable anti-LGI1 encephalitis showed a sensitivity of 66% (95% CI 47-81) and specificity of 96% (95% CI 93-98). DISCUSSION: AE mimics occur frequently. Common pitfalls in AE misdiagnosis are mesiotemporal lesions (predominantly with atypical features) and false-positive serum antibodies. As expected, the specificity of the criteria for possible AE is low because these criteria represent the minimal requirements for entry in the diagnostic algorithm for AE. Criteria for probable AE (-LGI1, -NMDAR, seronegative) and definite autoimmune LE are applicable for decisions on immunotherapy in early disease stage, as specificity is high.</p

Addition of serum-containing medium to cerebrospinal fluid prevents cellular loss over time

Immediately after sampling, leukocyte counts in native cerebrospinal fluid (CSF) start to decrease rapidly. As the time lapse between CSF collection to analysis is not routinely registered, the clinical significance of decreasing cell counts in native CSF is not known. Earlier data suggest that addition of serum-containing medium to CSF directly after sampling prevents this rapid decrease in leukocyte counts and, thus, may improve the accuracy of CSF cell counting and cell characterization. Here, we prospectively examined the effect of storage time after lumbar puncture on counts of leukocytes and their major subsets in both native CSF and after immediate addition of serum-containing medium, measured by flow cytometry and microscopy. We collected CSF samples of 69 patients in tubes with and tubes without serum-containing medium and determined counts of leukocytes and subsets at 30 minutes, 1 hour, and 5 hours after sampling. Compared to cell counts at 30 minutes, no significant decrease in cell number was observed in CSF with serum-containing medium 1 and 5 hours after sampling, except for the granulocytes at 1 hour. In native CSF, approximately 50% of leukocytes and all their subsets were lost after 1 hour, both in flow cytometric and microscopic counting. In 6/7 (86%) samples with mild pleocytosis (5–15 × 106 leukocytes/l), native CSF at 1 hour was incorrectly diagnosed as normocellular. In conclusion, addition of serum-containing medium to CSF directly after sampling prevents cell loss and allows longer preservation of CSF cells prior to analysis, both for microscopic and flow cytometric enumeration. We suggest that this protocol results in more accurate CSF cell counts and may prevent incorrect conclusions based on underestimated CSF cell counts

The epigenetic evolution of glioma is determined by the IDH1 mutation status and treatment regimen

Tumor adaptation or selection is thought to underlie therapy resistance in glioma. To investigate longitudinal epigenetic evolution of gliomas in response to therapeutic pressure, we performed an epigenomic analysis of 132 matched initial and recurrent tumors from patients with IDH-wildtype (IDHwt) and IDH-mutant (IDHmut) glioma. IDHwt gliomas showed a stable epigenome over time with relatively low levels of global methylation. The epigenome of IDHmut gliomas showed initial high levels of genome-wide DNA methylation that was progressively reduced to levels similar to those of IDHwt tumors. Integration of epigenomics, gene expression, and functional genomics identified HOXD13 as a master regulator of IDHmut astrocytoma evolution. Furthermore, relapse of IDHmut tumors was accompanied by histological progression that was associated with survival, as validated in an independent cohort. Finally, the initial cell composition of the tumor microenvironment varied between IDHwt and IDHmut tumors and changed differentially following treatment, suggesting increased neo-angiogenesis and T-cell infiltration upon treatment of IDHmut gliomas. This study provides one of the largest cohorts of paired longitudinal glioma samples with epigenomic, transcriptomic, and genomic profiling and suggests that treatment of IDHmut glioma is associated with epigenomic evolution towards an IDHwt-like phenotype

Exon expression arrays as a tool to identify new cancer genes

Background: Identification of genes that are causally implicated in oncogenesis is a major goal in cancer research. An estimated 10-20% of cancer-related gene mutations result in skipping of one or more exons in the encoded transcripts. Here we report on a strategy to screen in a global fashion for such exon-skipping events using PAttern based Correlation (PAC). The PAC algorithm has been used previously to identify differentially expressed splice variants between two predefined subgroups. As genetic changes in cancer are sample specific, we tested the ability of PAC to identify aberrantly expressed exons in single samples. Principal Findings: As a proof-of-principle, we tested the PAC strategy on human cancer samples of which the complete coding sequence of eight cancer genes had been screened for mutations. PAC detected all seven exon-skipping mutants among 12 cancer cell lines. PAC also identified exon-skipping mutants in clinical cancer specimens although detection was compromised due to heterogeneous (wild-type) transcript expression. PAC reduced the number candidate genes/exons for subsequent mutational analysis by two to three orders of magnitude and had a substantial true positive rate. Importantly, of 112 randomly selected outlier exons, sequence analysis identified two novel exon skipping events, two novel base changes and 21 previously reported base changes (SNPs). Conclusions: The ability of PAC to enrich for mutated transcripts and to identify known and novel genetic changes confirms its suitability as a strategy to identify candidate cancer genes