Cognition and health-related quality of life in long-term survivors of high-grade glioma: an interactive perspective from patient and caregiver.

BACKGROUND: The health-related quality of life (HRQoL) and cognition are important indicators for the quality of survival in patients with high-grade glioma (HGG). However, data on long-term survivors and their caregivers are scarce. We aim to investigate the interaction between cognition and HRQoL in long-term survivors, their caregivers' evaluations, and the effect on caregiver strain and burden. METHODS: 21 long-term HGG (8 WHO grade III and 13 WHO grade IV) survivors (survival ≥ 5 years) and 15 caregivers were included. Cognition (verbal memory, attention, executive functioning, and language), HRQoL, anxiety and depression, caregiver strain, and caregiver burden were assessed with standardized measures. Questionnaires were completed by patients and/or their caregivers. RESULTS: Mean survival was 12 years (grade III) and 8 years (grade IV). Cognition was significantly impaired with a large individual variety. Patients' general HRQoL was not impaired but all functioning scales were deviant. Patient-proxy agreement was found in most HRQoL subscales. Three patients (14%) showed indications of anxiety or depression. One-third of the caregivers reported a high caregiver strain or a high burden. Test scores for attention, executive functioning, language, and/or verbal memory were correlated with perceived global health status, cognitive functioning, and/or communication deficits. Caregiver burden was not related to cognitive deficits. CONCLUSIONS: In long-term HGG survivors maintained HRQoL seems possible even when cognition is impaired in a large variety at the individual level. A tailored approach is therefore recommended to investigate the cognitive impairments and HRQoL in patients and the need for patient and caregiver support.

Antibodies Contributing to Focal Epilepsy Signs and Symptoms Score.

OBJECTIVE: Diagnosing autoimmune encephalitis (AIE) is difficult in patients with less fulminant diseases such as epilepsy. However, recognition is important, as patients require immunotherapy. This study aims to identify antibodies in patients with focal epilepsy of unknown etiology, and to create a score to preselect patients requiring testing. METHODS: In this prospective, multicenter cohort study, adults with focal epilepsy of unknown etiology, without recognized AIE, were included, between December 2014 and December 2017, and followed for 1 year. Serum, and if available cerebrospinal fluid, were analyzed using different laboratory techniques. The ACES score was created using factors favoring an autoimmune etiology of seizures (AES), as determined by multivariate logistic regression. The model was externally validated and evaluated using the Concordance (C) statistic. RESULTS: We included 582 patients, with median epilepsy duration of 8 years (interquartile range = 2-18). Twenty patients (3.4%) had AES, of whom 3 had anti-leucine-rich glioma inactivated 1, 3 had anti-contactin-associated protein-like 2, 1 had anti-N-methyl-D-aspartate receptor, and 13 had anti-glutamic acid decarboxylase 65 (enzyme-linked immunosorbent assay concentrations >10,000IU/ml). Risk factors for AES were temporal magnetic resonance imaging hyperintensities (odds ratio [OR] = 255.3, 95% confidence interval [CI] = 19.6-3332.2, p < 0.0001), autoimmune diseases (OR = 13.31, 95% CI = 3.1-56.6, p = 0.0005), behavioral changes (OR 12.3, 95% CI = 3.2-49.9, p = 0.0003), autonomic symptoms (OR = 13.3, 95% CI = 3.1-56.6, p = 0.0005), cognitive symptoms (OR = 30.6, 95% CI = 2.4-382.7, p = 0.009), and speech problems (OR = 9.6, 95% CI = 2.0-46.7, p = 0.005). The internally validated C statistic was 0.95, and 0.92 in the validation cohort (n = 128). Assigning each factor 1 point, an antibodies contributing to focal epilepsy signs and symptoms (ACES) score ≥ 2 had a sensitivity of 100% to detect AES, and a specificity of 84.9%. INTERPRETATION: Specific signs point toward AES in focal epilepsy of unknown etiology. The ACES score (cutoff ≥ 2) is useful to select patients requiring antibody testing. ANN NEUROL 2021;89:698-710.

Novel Antibody-Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma.

We investigated the feasibility of detecting the presence of specific autoantibodies against potential tumor-associated peptide antigens by enriching these antibody-peptide complexes using Melon Gel resin and mass spectrometry. Our goal was to find tumor-associated phospho-sites that trigger immunoreactions and raise autoantibodies that are detectable in plasma of glioma patients. Such immunoglobulins can potentially be used as targets in immunotherapy. To that aim, we describe a method to detect the presence of antibodies in biological samples that are specific to selected clinically relevant peptides. The method is based on the formation of antibody-peptide complexes by mixing patient plasma with a glioblastoma multiforme (GBM) derived peptide library, enrichment of antibodies and antibody-peptide complexes, the separation of peptides after they are released from immunoglobulins by molecular weight filtration and finally mass spectrometric quantification of these peptides. As proof of concept, we successfully applied the method to dinitrophenyl (DNP)-labeled α-casein peptides mixed with anti-DNP. Further, we incubated human plasma with a phospho-peptide library and conducted targeted analysis on EGFR and GFAP phospho-peptides. As a result, immunoaffinity against phospho-peptide GSHQIS[+80]LDNPDYQQDFFPK (EGFR phospho-site S1166) was detected in high-grade glioma (HGG) patient plasma but not in healthy donor plasma. For the GFAP phospho-sites selected, such immunoaffinity was not observed.

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 enzyme abundance associated with aerobic glycolysis, glutamate metabolism, and the TCA cycle in IDH1 mut gliomas. Specifically, the enzymes involved in the metabolism of glutamate, lactate, and enzymes involved in the conversion of α-ketoglutarate were increased in IDH1 mut gliomas. In addition, the bicarbonate transporter (SLC4A4) was increased in IDH1 mut gliomas, supporting the idea that a mechanism preventing intracellular acidification is active. We also found that enzymes that convert proline, valine, leucine, and isoleucine into glutamate were increased in IDH1 mut glioma. We conclude that in IDH1 mut glioma metabolism is rewired (increased input of lactate and glutamate) to preserve TCA-cycle activity in IDH1 mut gliomas.

The expanded clinical spectrum of anti-GABABR encephalitis and added value of KCTD16 autoantibodies.

In this study we report the clinical features of 32 patients with gamma aminobutyric acid B receptor (GABABR) antibodies, identify additional autoantibodies in patients with anti-GABABR encephalitis that mark the presence of an underlying small cell lung carcinoma and optimize laboratory methods for the detection of GABABR antibodies. Patients (n = 3225) were tested for the presence of GABABR antibodies using cell-based assay, immunohistochemistry and live hippocampal neurons. Clinical data were obtained retrospectively. Potassium channel tetramerization domain-containing (KCTD)16 antibodies were identified by immunoprecipitation, mass spectrometry analysis and cell-based assays. KCTD16 antibodies were identified in 23/32 patients with anti-GABABR encephalitis, and in 1/26 patients with small cell lung carcinoma and Hu antibodies, but not in 329 healthy subjects and disease controls. Of the anti-GABABR encephalitis patients that were screened sufficiently, 18/19 (95%) patients with KCTD16 antibodies had a tumour versus 3/9 (33%) anti-GABABR encephalitis patients without KCTD16 antibodies (P = 0.001). In most cases this was a small cell lung carcinoma. Patients had cognitive or behavioural changes (97%) and prominent seizures (90%). Thirteen patients developed a refractory status epilepticus with intensive care unit admittance (42%). Strikingly, 4/32 patients had a rapidly progressive dementia. The addition of KCTD16 to the GABABR cell-based assay improved sensitivity of the in-house fixed cell-based assay, without loss of specificity. Twenty-two of 26 patients improved (partially) to immunotherapy or chemotherapy. Anti-GABABR encephalitis is a limbic encephalitis with prominent, severe seizures, but patients can also present with rapidly progressive dementia. The co-occurrence of KCTD16 antibodies points towards a paraneoplastic origin. The addition of KCTD16 improves the sensitivity of the cell-based assay.

Lack of B and T cell reactivity towards IDH1R132H in blood and tumor tissue from LGG patients.

PURPOSE: Mutations in the isocitrate dehydrogenase-1 gene (IDH1) occur at high frequency in grade II-III gliomas (LGGs). IDH1 mutations are somatic, missense and heterozygous affecting codon 132 in the catalytic pocket of the enzyme. In LGG, most mutations (90%) result in an arginine to histidine substitution (IDH1R132H) providing a neo-epitope that is expressed in all tumor cells. To assess the immunogenic nature of this epitope, and its potential use to develop T cell treatments, we measured IDH1R132H-specific B and T cell reactivity in blood and tumor tissue of LGG patients. METHODS: Sera from IDH1R132H-mutated LGG patients (n = 27) were assayed for the presence of a neo-specific antibody response using ELISA. In addition, PBMCs (n = 36) and tumor-infiltrating lymphocytes (TILs, n = 10) were measured for T cell activation markers and IFN-γ production by flow cytometry and ELISA. In some assays, frequencies of CD4 T cells specific for mutated peptide presented by HLA-DR were enriched prior to T cell monitoring assays. RESULTS: Despite high sensitivity of our assay, we failed to detect IDH1R132H-specific IgG in sera of LGG patients. Similarly, we did not observe CD4 T cell reactivity towards IDH1R132H in blood, neither did we observe such reactivity following pre-enrichment of frequencies of IDH1R132H-specific CD4 T cells. Finally, we did not detect IDH1R132H-specific CD4 T cells among TILs. CONCLUSIONS: The absence of both humoral and cellular responses in blood and tumors of LGG patients indicates that IDH1R132H is not sufficiently immunogenic and devaluates its further therapeutic exploitation, at least in the majority of LGG patients.

Anti-LGI1 encephalitis is strongly associated with HLA-DR7 and HLA-DRB4.

Leucine-rich glioma-inactivated1 (LGI1) encephalitis is an antibody-associated inflammation of the limbic area. An autoimmune etiology is suspected but not yet proven. We performed human leukocyte antigen (HLA) analysis in 25 nontumor anti-LGI1 patients and discovered a remarkably strong HLA association. HLA-DR7 was present in 88% compared to 19.6% in healthy controls (p = 4.1 × 10-11 ). HLA-DRB4 was present in all patients and in 46.5% controls (p = 1.19 × 10-7 ). These findings support the autoimmune hypothesis. An exploratory analysis was performed in a small group of 4 tumor-LGI1 patients. The strong HLA association seems not applicable in these patients. Therefore, the absence of HLA-DR7 or HLA-DRB4 could raise tumor suspicion in anti-LGI1 patients. Ann Neurol 2017;81:193-198.

Predictive value of electroencephalography in anti-NMDA receptor encephalitis.

OBJECTIVES: Anti-N-methyl-D-aspartate receptor encephalitis (anti-NMDARE) is a severe, but treatable disease. This study aims to give a detailed description of electroencephalogram (EEG) results in paediatric and adult patients to improve disease recognition, and analyses the predictive value of the first EEG for the final clinical outcome. METHODS: This nationwide cohort study includes patients with N-methyl-D-aspartate receptor antibodies confirmed with cell-based assay and immunohistochemistry in serum and cerebrospinal fluid. EEG recordings were re-evaluated by two experienced neurophysiologists, mixed with control EEGs for blinding. Initial EEG as well as follow-up registrations were analysed. RESULTS: 35 adults and 18 children were included. Only two patients (4%) had a normal EEG. During the first recording, the majority of the patients had normal posterior rhythm (71%), which was associated with better modified Rankin Scale at final outcome (OR 4.74; 95% CI 1.56 to 14.47; p=0.006). In addition, EEGs showed focal (73%) or diffuse (67%) slowing. The first EEG was severely abnormal in 26%. However, 8 of 14 patients with a severely abnormal first EEG still had a favourable outcome. During the course of the disease, extreme delta brushes (EDBs) were present in 6 of 53(11%)patients. CONCLUSIONS: The first EEG commonly shows normal posterior rhythm with focal or diffuse slowing. Although the sensitivity of an abnormal EEG is high (96%), normal EEG does not exclude anti-NMDARE. EDBs are only present in severely affected patients. The first EEG recording is predictive of the final clinical outcome.

Adherence to Analgesics in Oncology Outpatients: Focus on Taking Analgesics on Time.

BACKGROUND: Inadequate adherence to prescribed analgesics may be one of the reasons why patients with cancer experience unrelieved pain. Adherence is directly influenced by patients' barriers about pain management. Patient pain education programs (PEPs) have been developed to reduce patients' barriers and increase patients' adherence to their analgesics. The purpose of this article was to evaluate patients' adherence in patients receiving a pain consult and patient pain education in comparison with patients receiving standard pain treatment (standard care [SC]), to better explore the difficulties in medication adherence in cancer-related pain and the effects of PEP. METHODS: In 54 adult outpatients with cancer-related pain, patients' adherence to the prescribed around-the-clock analgesics was measured with a Medication Event Monitoring System, in the following time intervals: weeks 1 and 2, weeks 3 and 4, and weeks 7 and 8 after randomization. Adherence was differentiated into taking adherence, taking the correct dose, and taking analgesics at the right time intervals. RESULTS: Taking adherence increased in the intervention group compared to baseline (from 91% to 93%) and decreased in the SC group (from 85% to 78%; P < 0.05). At the end of the study, more patients in the intervention group took their analgesics at the right intervals (78%) than did patients in the SC group (64%, P < 0.05). During the study, patients were more adherent to opioids than to World Health Organization step 1 analgesics. CONCLUSION: The combined intervention can increase adherence. The true problem in pain management is that patients do not take their prescribed analgesics at the right time intervals. With the detailed adherence information from this study, it is possible to further tailor patient education to the individual patient.

Tumor-specific mutations in low-frequency genes affect their functional properties.

Causal genetic changes in oligodendrogliomas (OD) with 1p/19q co-deletion include mutations in IDH1, IDH2, CIC, FUBP1, TERT promoter and NOTCH1. However, it is generally assumed that more somatic mutations are required for tumorigenesis. This study aimed to establish whether genes mutated at low frequency can be involved in OD initiation and/or progression. We performed whole-genome sequencing on three anaplastic ODs with 1p/19q co-deletion. To estimate mutation frequency, we performed targeted resequencing on an additional 39 ODs. Whole-genome sequencing identified a total of 55 coding mutations (range 8-32 mutations per tumor), including known abnormalities in IDH1, IDH2, CIC and FUBP1. We also identified mutations in genes, most of which were previously not implicated in ODs. Targeted resequencing on 39 additional ODs confirmed that these genes are mutated at low frequency. Most of the mutations identified were predicted to have a deleterious functional effect. Functional analysis on a subset of these genes (e.g. NTN4 and MAGEH1) showed that the mutation affects the subcellular localization of the protein (n = 2/12). In addition, HOG cells stably expressing mutant GDI1 or XPO7 showed altered cell proliferation compared to those expressing wildtype constructs. Similarly, HOG cells expressing mutant SASH3 or GDI1 showed altered migration. The significantly higher rate of predicted deleterious mutations, the changes in subcellular localization and the effects on proliferation and/or migration indicate that many of these genes functionally may contribute to gliomagenesis and/or progression. These low-frequency genes and their affected pathways may provide new treatment targets for this tumor type.

Treatment of large low-grade oligodendroglial tumors with upfront procarbazine, lomustine, and vincristine chemotherapy with long follow-up: a retrospective cohort study with growth kinetics.

We treated patients with newly diagnosed and large low-grade oligodendroglial tumors with upfront procarbazine, CCNU and vincristine (PCV) in order to delay radiotherapy. Patients were treated with PCV for a maximum of 6 cycles. The response to treatment was defined according to the RANO criteria; in addition change over time of mean tumor diameters (growth kinetics) was calculated. Thirty-two patients were treated between 1998 and 2006, 18 of which were diagnosed with 1p/19q co-deleted tumors. Median follow-up duration was 8 years (range 0.5-13 years). The median overall survival (mOS) was 120 months and the median progression-free survival (mPFS) was 46 months. Growth kinetics showed an ongoing decrease of the mean tumor diameter after completion of chemotherapy, during a median time of 35 months, but an increase of the mean tumor diameter did not herald progression as detected by RANO criteria. 1p/19q co-deletion was associated with a significant increase in OS (mOS 83 months versus not reached for codeleted tumors; p = 0.003)) and PFS (mPFS 35 months versus 67 months for codeleted tumors; p = 0.024). Patients with combined 1p/19q loss had a 10 year PFS of 34 % and the radiotherapy in these patients was postponed for a median period of more than 6 years. This long-term follow-up study indicates that upfront PCV chemotherapy is associated with long PFS and OS and delays radiotherapy for a considerable period of time in patients with low-grade oligodendroglial tumors, in particular with combined 1p/19q loss.

A proteome comparison between physiological angiogenesis and angiogenesis in glioblastoma.

The molecular pathways involved in neovascularization of regenerating tissues and tumor angiogenesis resemble each other. However, the regulatory mechanisms of neovascularization under neoplastic circumstances are unbalanced leading to abnormal protein expression patterns resulting in the formation of defective and often abortive tumor vessels. Because gliomas are among the most vascularized tumors, we compared the protein expression profiles of proliferating vessels in glioblastoma with those in tissues in which physiological angiogenesis takes place. By using a combination of laser microdissection and LTQ Orbitrap mass spectrometry comparisons of protein profiles were made. The approach yielded 29 and 12 differentially expressed proteins for glioblastoma and endometrium blood vessels, respectively. The aberrant expression of five proteins, i.e. periostin, tenascin-C, TGF-beta induced protein, integrin alpha-V, and laminin subunit beta-2 were validated by immunohistochemistry. In addition, pathway analysis of the differentially expressed proteins was performed and significant differences in the usage of angiogenic pathways were found. We conclude that there are essential differences in protein expression profiles between tumor and normal physiological angiogenesis.

Molecular subtypes of glioma identified by genome-wide methylation profiling.

Recent studies have indicated a prognostic role for genome-wide methylation in gliomas: Tumors that show an overall increase in DNA methylation at CpG sites (CIMP+; CpG island methylator phenotype) have a more favorable prognosis than CIMP- gliomas. Here, we have determined whether methylation profiling can identify more and clinically relevant molecular subtypes of glioma by performing genome-wide methylation profiling on 138 glial brain tumors of all histological diagnosis. Hopach (Hierarchical ordered partitioning and collapsing hybrid) clustering using the 1,000 most variable CpGs identified three distinct glioma subtypes (C+(1p19q), C+(wt), and C-) and one adult brain subtype. All "C+(1p19q) " and "C+(wt)" tumors were CIMP+ whereas most (50/54) "C-" tumors were CIMP-. The C- subtype gliomas contained many glioblastomas and all pilocytic astrocytomas. 1p19q LOH was frequent in the C+(1p19q) subtype. Other genetic changes (IDH1 mutation and EGFR amplification) and gene-expression based molecular subtypes also segregated in distinct methylation subtypes, demonstrating that these subtypes are also genetically distinct. Each subtype was associated with its own prognosis: median survival for C-, C+(1p19q), and C+(wt) tumors was 1.18, 5.00, and 2.62 years, respectively. The prognostic value of these methylation subtypes was validated on an external dataset from the TCGA. Analysis of recurrences of 14 primary tumors samples indicates that shifts between some C+(wt) and C+(1p/19q) tumors can occur between the primary and recurrent tumor, but CIMP status remained stable. Our data demonstrate that methylation profiling identifies at least three prognostically relevant subtypes of glioma that can aid diagnosis and potentially guide treatment for patients.

Improved detection of tryptic immunoglobulin variable region peptides by chromatographic and gas-phase fractionation techniques.

The polyclonal repertoire of circulating antibodies potentially holds valuable information about an individual's humoral immune state. While bottom-up proteomics is well suited for serum proteomics, the vast number of antibodies and dynamic range of serum challenge this analysis. To acquire the serum proteome more comprehensively, we incorporated high-field asymmetric waveform ion-mobility spectrometry (FAIMS) or two-dimensional chromatography into standard trypsin-based bottom-up proteomics. Thereby, the number of variable region (VR)-related spectra increased 1.7-fold with FAIMS and 10-fold with chromatography fractionation. To match antibody VRs to spectra, we combined de novo searching and BLAST alignment. Validation of this approach showed that, as peptide length increased, the de novo accuracy decreased and BLAST performance increased. Through in silico calculations on antibody repository sequences, we determined the uniqueness of tryptic VR peptides and their suitability as antibody surrogate. Approximately one-third of these peptides were unique, and about one-third of all antibodies contained at least one unique peptide.

Autoimmune Encephalitis and Paraneoplastic Neurologic Syndromes: A Nationwide Study on Epidemiology and Antibody Testing Performance.

BACKGROUND AND OBJECTIVES: Autoimmune encephalitis (AIE) and paraneoplastic neurologic syndromes (PNSs) encompass a heterogeneous group of antibody-associated disorders. Both the number of syndromes and commercially available antibody tests have increased considerably over the past decade. High-quality population-based data on epidemiology of these disorders and real-world performance of antibody tests are needed. METHODS: In this nationwide retrospective cohort study, we identified all serum and CSF samples tested for antibodies against intracellular antigens (IAs: Hu [ANNA1], Yo [PCA1], CV2 [CRMP5], Ri [ANNA2], Ma1, Ma2 [Ta], amphiphysin, GAD65, GFAP, KLHL11, CARP VIII) or extracellular antigens (EAs: NMDAR, LGI1, Caspr2, GABA-B-R, GABA-A-R, AMPAR, DPPX, GlyR, mGluR1, VGCC, IgLON5, Tr [DNER]) between January 2016 and December 2021 in the Netherlands. Nationwide coverage was guaranteed for all antibodies except anti-GAD65 and anti-VGCC. We calculated sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV); obtained clinical information about patients who tested positive; assigned diagnosis of AIE/PNS according to diagnostic criteria; and calculated incidence rates for IA, EA, and individual antibody-associated syndromes. RESULTS: In the study period, 2,877 (9.5%) of 30,246 samples, belonging to 1,228 patients, tested positive. Sensitivity and specificity were high (>95%) to very high (>99%) for most tests in both serum and CSF. PPVs for several tests were moderate to poor, especially for serum testing of IA antibodies (25%-80%). Clinical data were available for 940 (76.5%) of 1,228 patients. A total of 578 AIE/PNS diagnoses were made. The incidence rate for AIE/PNS (per million person-years) increased from 4.70 (95% CI 3.72-5.85) in 2016 to 5.76 (4.69-7.00) in 2021. Overall, the incidence rate was 5.57 (5.13-6.05), 2.96 (2.64-3.31) for the EA and 2.61 (2.31-2.94) for the IA subgroup. The 4 most common AIE/PNS types were anti-NMDAR, anti-LGI1, anti-Hu, and anti-GAD65, together comprising almost two-thirds of all diagnoses (364/578, 63.0%). DISCUSSION: Most commercial antibody tests perform well overall, but important pitfalls remain. Although almost all tests had high specificity, PPV was only modest in the setting of these rare diseases and mass testing. We observe trends toward increasing incidence of antibody-associated AIE/PNS.

Serum proteomics in amnestic mild cognitive impairment.

We have explored proteins related to mild cognitive impairment (MCI). The serum proteome of 35 amnestic MCI patients and 35 cognitively healthy persons was investigated by LC MS. We identified 108 differentially expressed peptides between MCI patients and controls, belonging to 39 proteins. Eight proteins were selected for further investigation by quantitative protein measurements using a MRM assay; apolipoprotein E, carboxypeptidase N subunit 2, complement factor B (CFAB), galectin-3 binding protein (LG3BP), lumican, serum amyloid A-4 protein (SAA4), serum amyloid P-component, and sex hormone binding globulin. Results of the quantitative protein measurements showed significantly decreased levels of carboxypeptidase N subunit 2, CFAB, LG3BP, SAA4, and serum amyloid P-component in serum from amnestic MCI patients compared with cognitive healthy controls (two-sided t-test; p < 0.05). Apolipoprotein E and lumican showed no significant difference in protein levels, sex hormone binding globulin could not be quantified since the MRM assay did not reach the required sensitivity. A model based on the three most significantly decreased proteins (CFAB, LG3BP, and SAA4) showed a sensitivity and specificity of 73 and 66%, respectively, for the initial sample set. A small external validation set yielded 77% sensitivity and 75% specificity.

Comparative study of targeted and label-free mass spectrometry methods for protein quantification.

We compared data acquired on an LTQ-Orbitrap MS used in a typical shotgun proteomics setting (optimized for protein identification) with data from a quadrupole ion trap MS operated in the MRM mode. Six relative abundant proteins were quantified in identical sets of serum and CSF samples by the following methods: a qual/quant method with and without use of internal standards and a quantitative method (MRM with use of internal standards). Comparison of these methods with an antibody-based method in CSF samples showed good linearity for both methods (R(2) of 0.961 and 0.971 for the qual/quant method with use of internal standards and the quantitative method, respectively). Besides its better linearity, the quantitative method was also more reproducible with lower CVs for all samples. Next to these comparisons we also explored why a qual/quant approach had typically a lower reproducibility compared to MRM analyses. We observed that modified peptides, or peptides with a cysteine or a methionine, yielded a significant increase in CV. Furthermore, a positive correlation was found between the length of the peptide and the CV. We conclude that qual/quant is an alternative for the quantification of abundant proteins and that the use of internal standards in qual/quant could be advantageous. Furthermore, the ongoing development in MS techniques increases the possibilities of qual/quant in protein quantification.

Alternative normalization and analysis pipeline to address systematic bias in NanoString GeoMx Digital Spatial Profiling data.

Spatial transcriptomics is a novel technique that provides RNA-expression data with tissue-contextual annotations. Quality assessments of such techniques using end-user generated data are often lacking. Here, we evaluated data from the NanoString GeoMx Digital Spatial Profiling (DSP) platform and standard processing pipelines. We queried 72 ROIs from 12 glioma samples, performed replicate experiments of eight samples for validation, and evaluated five external datasets. The data consistently showed vastly different signal intensities between samples and experimental conditions that resulted in biased analysis. We evaluated the performance of alternative normalization strategies and show that quantile normalization can adequately address the technical issues related to the differences in data distributions. Compared to bulk RNA sequencing, NanoString DSP data show a limited dynamic range which underestimates differences between conditions. Weighted gene co-expression network analysis allowed extraction of gene signatures associated with tissue phenotypes from ROI annotations. Nanostring GeoMx DSP data therefore require alternative normalization methods and analysis pipelines.

Dose dense 1 week on/1 week off temozolomide in recurrent glioma: a retrospective study.

Alternative temozolomide regimens have been proposed to overcome O(6)-methylguanine-DNA methyltransferase mediated resistance. We investigated the efficacy and tolerability of 1 week on/1 week off temozolomide (ddTMZ) regimen in a cohort of patients treated with ddTMZ between 2005 and 2011 for the progression of a glioblastoma during or after chemo-radiation with temozolomide or a recurrence of another type of glioma after radiotherapy and at least one line of chemotherapy. Patients received ddTMZ at 100-150 mg/m(2)/d (days 1-7 and 15-21 in cycles of 28-days). All patients had a contrast enhancing lesion on MRI and the response was assessed by MRI using the RANO criteria; complete and partial responses were considered objective responses. Fifty-three patients were included. The median number of cycles of ddTMZ was 4 (range 1-12). Eight patients discontinued chemotherapy because of toxicity. Two of 24 patients with a progressive glioblastoma had an objective response; progression free survival at 6 months (PFS-6) in glioblastoma was 29%. Three of the 16 patients with a recurrent WHO grade 2 or 3 astrocytoma or oligodendroglioma or oligo-astrocytoma without combined 1p and 19q loss had an objective response and PFS-6 in these patients was 38%. Four out of the 12 evaluable patients with a recurrent WHO grade 2 or 3 oligodendroglioma or oligo-astrocytoma with combined 1p and 19q loss had an objective response; PFS-6 in these patients was 62%. This study indicates that ddTMZ is safe and effective in recurrent glioma, despite previous temozolomide and/or nitrosourea chemotherapy. Our data do not suggest superior efficacy of this schedule as compared to the standard day 1-5 every 4 weeks schedule.

Quantitative proteomics and metabolomics analysis of normal human cerebrospinal fluid samples.

The analysis of cerebrospinal fluid (CSF) is used in biomarker discovery studies for various neurodegenerative central nervous system (CNS) disorders. However, little is known about variation of CSF proteins and metabolites between patients without neurological disorders. A baseline for a large number of CSF compounds appears to be lacking. To analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples of patients undergoing routine, non-neurological surgical procedures, we determined the variation of various proteins and metabolites by multiple analytical platforms. A total of 126 common proteins were assessed for biological variations between individuals by ESI-Orbitrap. A large spread in inter-individual variation was observed (relative standard deviations [RSDs] ranged from 18 to 148%) for proteins with both high abundance and low abundance. Technical variation was between 15 and 30% for all 126 proteins. Metabolomics analysis was performed by means of GC-MS and nuclear magnetic resonance (NMR) imaging and amino acids were specifically analyzed by LC-MS/MS, resulting in the detection of more than 100 metabolites. The variation in the metabolome appears to be much more limited compared with the proteome: the observed RSDs ranged from 12 to 70%. Technical variation was less than 20% for almost all metabolites. Consequently, an understanding of the biological variation of proteins and metabolites in CSF of neurologically normal individuals appears to be essential for reliable interpretation of biomarker discovery studies for CNS disorders because such results may be influenced by natural inter-individual variations. Therefore, proteins and metabolites with high variation between individuals ought to be assessed with caution as candidate biomarkers because at least part of the difference observed between the diseased individuals and the controls will not be caused by the disease, but rather by the natural biological variation between individuals.