Smitt, P.A.

Elevated numbers of regulatory T cells, central memory T cells and class-switched B cells in cerebrospinal fluid of patients with anti-Hu antibody associated paraneoplastic neurological syndromes (Article)

2013-04-08T00:00:00Z

Multi-parametric flow cytometry was used to study lymphocyte subsets and dendritic cells in paired blood and CSF samples from 11 newly diagnosed patients with progressive anti-Hu antibody associated paraneoplastic neurological syndromes (Hu-PNS), 9 patients with other inflammatory neurologic disorders (IND), and 12 patients with other non-inflammatory neurologic disorders (OND). Hu-PNS patients had elevated numbers of regulatory T cells, central memory T cells, class-switched B cells and dendritic cells in their CSF. These findings support the hypothesis that the immune system is locally activated in Hu-PNS, and suggests common etiological pathways between Hu-PNS and other inflammatory central nervous system disorders.

Multiplex serology of paraneoplastic antineuronal antibodies (Article)

2013-03-21T00:00:00Z

Paraneoplastic neurological syndromes (PNS) are devastating neurological disorders secondary to cancer, associated with onconeural autoantibodies. Such antibodies are directed against neuronal antigens aberrantly expressed by the tumor. The detection of onconeural antibodies in a patient is extremely important in diagnosing a neurological syndrome as paraneoplastic (70% is not yet known to have cancer) and in directing the search for the underlying neoplasm. At present six onconeural antibodies are considered 'well characterized' and recognize the antigens HuD, CDR62 (Yo), amphiphysin, CRMP-5 (CV2), NOVA-1 (Ri), and Ma2. The gold standard of detection is the characteristic immunohistochemical staining pattern on brain tissue sections combined with confirmation by immunoblotting using recombinant purified proteins. Since all six onconeural antibodies are usually analyzed simultaneously and objective cut-off values for these analyses are warranted, we developed a multiplex assay based on Luminex technology. Reaction of serial dilutions of six onconeural standard sera with microsphere-bound antigens showed lower limits of detection than with Western blotting. Using the six standard sera at a dilution of 1:200, the average within-run coefficient of variation (CV) was 4% (range 1.9-7.3%). The average between-run within-day CV was 5.1% (range 2.9-6.7%) while the average between-day CV was 8.1% (range 2.8-11.6%). The shelf-life of the antigen coupled microspheres was at least two months. The sensitivity of the multiplex assay ranged from 83% (Ri) to 100% (Yo, amphiphysin, CV2) and the specificity from 96% (CV2) to 100% (Ri). In conclusion, Luminex-based multiplex serology is highly reproducible with high sensitivity and specificity for the detection of onconeural antibodies. Conventional immunoblotting for diagnosis of onconeural antibodies in the setting of a routine laboratory may be replaced by this novel, robust technology.

Flow cytometric characterization of cerebrospinal fluid cells (Article)

2011-09-01T00:00:00Z

Flow cytometry facilitates the detection of a large spectrum of cellular characteristics on a per cell basis, determination of absolute cell numbers and detection of rare events with high sensitivity and specificity. White blood cell (WBC) counts in cerebrospinal fluid (CSF) are important for the diagnosis of many neurological disorders. WBC counting and differential can be performed by microscopy, hematology analyzers, or flow cytometry. Flow cytometry of CSF is increasingly being considered as the method of choice in patients suspected of leptomeningeal localization of hematological malignancies. Additionally, in several neuroinflammatory diseases such as multiple sclerosis and paraneoplastic neurological syndromes, flow cytometry is commonly performed to obtain insight into the immunopathogenesis of these diseases. Technically, the low cellularity of CSF samples, combined with the rapidly declining WBC viability, makes CSF flow cytometry challenging. Comparison of flow cytometry with microscopic and molecular techniques shows that each technique has its own advantages and is ideally combined. We expect that increasing the number of flow cytometric parameters that can be simultaneously studied within one sample, will further refine the information on CSF cell subsets in low-cellular CSF samples and enable to define cell populations more accurately.

First-line temozolomide chemotherapy in progressive low-grade astrocytomas after radiotherapy: Molecular characteristics in relation to response (Article)

2011-02-01T00:00:00Z

Only a few studies examined the effect of temozolomide (TMZ) in recurrent low-grade astrocytoma (LGA) after surgery, none of which included a homogeneous and sufficiently sized group of patients with progression after radiotherapy (RT). We evaluated a cohort of 58 patients treated with TMZ for progression after RT of a previous LGA and investigated the relation between outcome and mutations in the IDH1, IDH2, and TP53 genes, O6-methylguanine- methyltransferase (MGMT) promoter methylation, trisomy of chromosome 7, and loss of chromosomes 1p and 19q. All patients received first-line TMZ 200 mg/m2/day on days 1-5 every 4 weeks for a progressive LGA with a contrast-enhancing lesion on MRI after RT. Six months progression-free survival (PFS) was 67%, and the median overall survival was 14 months. An objective response was obtained in 54%. TP53 mutations and loss of chromosome 19q showed a borderline association with PFS, but none of the other molecular characteristics were correlated with the outcome to TMZ. Both a methylated MGMT promoter gene and IDH1 mutations were found in 86% of the tumor samples. A correlation was found between IDH1 mutations and MGMT promoter methyl-ation (P <.001). Neither MGMT promoter methylation nor IDH1 mutations correlated with PFS, but the interval between the very first symptom of the LGA and the start of the TMZ was significantly longer in the patients with IDH1 mutations (P =.01) and a methylated MGMT promoter (P =.02). We conclude that MGMT promoter methylation and IDH1 mutations seem to predict survival from the time of diagnosis, but not PFS to TMZ.

HLA-DQ2+ individuals are susceptible to Hu-Ab associated paraneoplastic neurological syndromes (Article)

2010-09-01T00:00:00Z

Background: Hypothetically, T cells are involved in the pathogenesis of paraneoplastic neurological syndromes associated with Hu-antibodies (Hu-PNS). Objective: To identify genetic risk factors for Hu-PNS and investigate the role of T cells. Methods: HLA-A, B, DRB1 and DQB1 alleles were compared in 53 Hu-PNS patients with 24 small-cell lung-cancer (SCLC) patients and 2440 healthy controls (HC). Results: The frequency of both HLA-DQ2 and HLA-DR3 was significantly higher in Hu-PNS patients than in HC. Conclusions: This study indicates an association between Hu-PNS and presence of HLA-DQ2 and HLA-DR3, which supports a role for CD4+T cells in the pathogenesis of Hu-PNS.

Integrated genomic profiling identifies candidate genes implicated in glioma-genesis and a novel LEO1-SLC12A1 fusion gene (Article)

2010-06-01T00:00:00Z

We performed genotyping and exon-level expression profiling on 21 glioblastomas (GBMs) and 19 oligodendrogliomas (ODs) to identify genes involved in glioma initiation and/or progression. Low-copy number amplifications (2.5 < n < 7) and high-copy number amplifications (n > 7) were more frequently observed in GBMs; ODs generally have more heterozygous deletions per tumor. Four high-copy amplicons were identified in more than one sample and resulted in overexpression of the known oncogenes EGFR, MDM2, and CDK4. In the fourth amplicon, RBBP5, a member of the RB pathway, may act as a novel oncogene in GBMs. Not all hCNAs contain known genes, which may suggest that other transcriptional and/or regulatory elements are the target for amplification. Regions with most frequent allelic loss, both in ODs and GBMs, resulted in a reduced expression of known tumor suppressor genes. We identified a homozygous deletion spanning the Pragmin gene in one sample, but direct sequencing of all coding exons in 20 other glioma samples failed to detect additional genetic changes. Finally, we screened for fusion genes by identifying aberrant 5′-3′ expression of genes that lie over regions of a copy number change. A fusion gene between exon 11 of LEO1 and exon 10 of SLC12A1 was identified. Our data show that integrated genomic profiling can identify genes involved in tumor initiation, and/or progression and can be used as an approach to identify novel fusion genes.

Evaluation of the new body fluid mode on the Sysmex XE-5000 for counting leukocytes and erythrocytes in cerebrospinal fluid and other body fluids (Article)

2010-05-01T00:00:00Z

Background: We evaluated the body fluid (BF) mode on the new Sysmex XE-5000 analyzer. Methods: Red (RBC) and white blood cell (WBC) (differential) counts of BFs (139 patient samples and 87 normal samples) were measured and compared to the Fuchs-Rosenthal chamber and stained cytospin slides. Results: Extended cell counting using the BF mode was noted to have an improved WBC detection limit (CV20%) of 10Ã106/L. Excellent agreement with the manual method was observed for most BFs [mean bias +2 to 6Ã106/L for cerebrospinal fluid (CSF) and -1 to 12Ã106/L for other fluids]. In CSF, the BF-mode counted more WBC (polymorphic nuclear cells) compared with the manual method (mean bias +5 to 6Ã106/L), especially in samples with low cell counts (<20Ã 106/L). Carry over was negligible (mostly <0.17%) and linearity was excellent (mean bias <5%). The reference ranges for CSF (n=87) were RBC 0Ã106/L, WBC and mononuclear <7Ã106/L, and polymorph nucleated cells <3Ã106/L. Conclusions: The BF mode on the Sysmex XE-5000 offers rapid and accurate RBC and WBC (differential) counts in clinically relevant concentration ranges in CSF and other fluids. In addition, the exclusion of high fluorescent cells, such as mesothelial cells and macrophages from WBC counting may reduce the number of manual analyses in pleural fluids and ascites.

Segregation of non-p.R132H mutations in IDH1 in distinct molecular subtypes of glioma (Article)

2010-03-01T00:00:00Z

Mutations in the gene encoding the isocitrate dehydrogenase 1 gene (IDH1) occur at a high frequency (up to 80%) in many different subtypes of glioma. In this study, we have screened for IDH1 mutations in a cohort of 496 gliomas. IDH1 mutations were most frequently observed in low grade gliomas with c.395G>A (p.R132H) representing >90% of all IDH1 mutations. Interestingly, non-p.R132H mutations segregate in distinct histological and molecular subtypes of glioma. Histologically, they occur sporadically in classic oligodendrogliomas and at significantly higher frequency in other grade II and III gliomas. Genetically, non-p.R132H mutations occur in tumors with TP53 mutation, are virtually absent in tumors with loss of heterozygosity on 1p and 19q and accumulate in distinct (gene-expression profiling based) intrinsic molecular subtypes. The IDH1 mutation type does not affect patient survival. Our results were validated on an independent sample cohort, indicating that the IDH1 mutation spectrum may aid glioma subtype classification. Functional differences between p.R132H and non-p.R132H mutated IDH1 may explain the segregation in distinct glioma subtypes.

Intrinsic gene expression profiles of gliomas are a better predictor of survival than histology (Article)

2009-12-01T00:00:00Z

Gliomas are the most common primary brain tumors with heterogeneous morphology and variable prognosis. Treatment decisions in patients rely mainly on histologic classification and clinical parameters. However, differences between histologic subclasses and grades are subtle, and classifying gliomas is subject to a large interobserver variability. To improve current classification standards, we have performed gene expression profiling on a large cohort of glioma samples of all histologic subtypes and grades. We identified seven distinct molecular subgroups that correlate with survival. These include two favorable prognostic subgroups (median survival, >4.7 years), two with intermediate prognosis (median survival, 1-4 years), two with poor prognosis (median survival, <1 year), and one control group. The intrinsic molecular subtypes of glioma are different from histologic subgroups and correlate better to patient survival. The prognostic value of molecular subgroups was validated on five independent sample cohorts (The Cancer Genome Atlas, Repository for Molecular Brain Neoplasia Data, GSE12907, GSE4271, and Li and colleagues). The power of intrinsic subtyping is shown by its ability to identify a subset of prognostically favorable tumors within an external data set that contains only histologically confirmed glioblastomas (GBM). Specific genetic changes (epidermal growth factor receptor amplification, IDH1 mutation, and 1p/19q loss of heterozygosity) segregate in distinct molecular subgroups. We identified a subgroup with molecular features associated with secondary GBM, suggesting that different genetic changes drive gene expression profiles. Finally, we assessed response to treatment in molecular subgroups. Our data provide compelling evidence that expression profiling is a more accurate and objective method to classify gliomas than histologic classification. Molecular classification therefore may aid diagnosis and can guide clinical decision making.

Acute CNS white matter lesions in patients with inflammatory bowel disease (Article)

2009-07-17T00:00:00Z

Background: Neurological manifestations in patients with inflammatory bowel disease supposedly are rare, although the exact frequency is not known. Most previous reports involve cerebral venous thrombosis, central nervous system vasculitis, or peripheral nerve inflammation. Methods: Two cases of patients diagnosed with inflammatory bowel disease developing neurological symptoms with corresponding lesions in the white matter of the central nervous system led us to search a neurological database with clinical and radiological data for similar cases. Results: We identified five patients who presented with acute neurological deficits preceding or following a diagnosis of inflammatory bowel disease with evidence of lesions in the central nervous system white matter on magnetic resonance imaging. Ancillary investigations did not provide evidence of systemic infetcion, coagulation disorders, or vasculitis. Conclusions: These cases, together with previous reports, suggest that white matter lesions may be another extraintestinal manifestation of inflammatory bowel disease. Copyright

A systematic review on barriers hindering adequate cancer pain management and interventions to reduce them: A critical appraisal (Article)

2009-05-01T00:00:00Z

The aim of this paper is to identify the major barriers hindering adequate pain management and critically review interventions aiming to overcome them. We searched relevant literature on PubMed published between January 1986 and April 2007. The most frequently mentioned barriers for both patients and professionals were knowledge deficits, inadequate pain assessment and misconceptions regarding pain. Four interventions were identified: patient education, professional education, pain assessment and pain consultation. These interventions were never combined in multidisciplinary study protocols. Most RCTs included small groups of patients and reported no power analysis. Studies on professional education and pain assessment did not evaluate patients' outcomes. In 5 of 11 RCTs on patient education, pain intensity decreased statistically significantly. In two RCTs on pain consultation, patients' pain decreased statistically significantly, although the adequacy of pain treatment did not change. In conclusion, international guidelines on multidisciplinary interventions in pain management are partly substantiated by clinical trials.

Quantitative matrix-assisted laser desorption ionization-fourier transform ion cyclotron resonance (MALDI-FT-ICR) peptide profiling and identification of multiple-sclerosis-related proteins (Article)

2009-03-06T00:00:00Z

We introduce a matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FT-ICR) method for quantitative peptide profiling, using peak height as a measure for abundance. Relative standard deviations in peak height of peptides spiked over 3 orders of magnitude in concentration were below 10% and allowed for accurate comparisons between multiple sclerosis and controls. Application on a set of 163 cerebrospinal fluid (CSF) samples showed significantly differential abundant peptides, which were subsequently identified into proteins (e.g., chromogranin A, clusterin, and complement C3). © 2009 American Chemical Society.

The rate of false positive sequence matches of peptides profiled by MALDI MS and identified by MS/MS (Article)

2008-11-01T00:00:00Z

In most MALDI peptide profiling cases, sequencing is required to identify peptides of interest, preferentially by using different mass spectrometry techniques. Using identical samples, we determined the number of false positive matches in sequence of peptide identification using different mass spectrometers. This paper demonstrates that the reliability of the identification phase greatly benefits from concerted MS-technologies and determines the influence of mass accuracy, signal-to-noise and statistical score on peptide identification.

The Lambert-Eaton myasthenic syndrome 1988-2008: A clinical picture in 97 patients (Article)

2008-09-15T00:00:00Z

Background: Neuromuscular symptoms in patients with Lambert-Eaton myasthenic syndrome (LEMS) and a small cell lung cancer (SCLC) develop more rapidly than in LEMS patients without a SCLC. We studied how this clinical information, which is readily available at the first consultation, can be used to predict the presence of SCLC. Patients and methods: In our study we included 52 LEMS patients with SCLC and 45 non-tumor patients (NT-LEMS). We interviewed patients using a structured checklist and reviewed their clinical records. We compared frequency and onset of symptoms during the course of LEMS. Results: In the first six months, over half the SCLC-LEMS patients had developed seven separate symptoms, while NT-LEMS patients developed only two symptoms. Proximal leg weakness and dry mouth were early symptoms in both groups. Rapid involvement of proximal arm muscles (p = 0.0001), distal arm muscles (p = 0.0037), distal leg muscles (p = 0.0002), dysartria (p = 0.0091) and the presence of erectile dysfunction (p = 0.007) were found significantly more often in SCLC-LEMS patients in both cohorts. Cerebellar symptoms, although present in 9% of LEMS patients, were almost exclusively related to SCLC-LEMS. Conclusion: A rapidly progressive course of disease from onset in LEMS patients should raise a high suspicion of SCLC. Special attention should be paid to involvement of upper extremities, involvement of distal arm and distal leg muscles, to erectile dysfunction and probably ataxia in order to discriminate between SCLC-LEMS and NT-LEMS.

Incidence of early pseudo-progression in a cohort of malignant glioma patients treated with chemoirradiation with temozolomide (Article)

2008-07-15T00:00:00Z

BACKGROUND. Radiotherapy (RT) plus concomitant and adjuvant temozolomide (TMZ) is now the standard of care for patients with newly diagnosed glioblastoma. The occurrence of pseudo-progression directly after RT is a recognized phenomenon, but to the authors' knowledge its incidence after combined RT/TMZ is unknown. The occurrence of early pseudo-progression was retrospectively assessed in a cohort of malignant glioma patients treated with RT/TMZ. METHODS. The pre-RT and post-RT brain scans from patients treated with RT/TMZ for a malignant glioma were reviewed. Scans were made before the start of RT, 4 weeks after the end of RT, and every 3 months thereafter. In addition, information was collected regarding clinical signs and symptoms, dexamethasone dose, histology, and survival. RESULTS. Eighty-five patients were identified. In 36 patients (42%) the first followup scan 4 weeks after the end of RT indicated disease progression. Of these 36 patients, 18 (50%) were diagnosed with pseudo-progression. None of the patients received additional treatment other than TMZ. Six of 18 patients with pseudo-progression and 12 of the 18 patients with real tumor progression developed new clinical signs and symptoms during RT or in the first 4 weeks thereafter. CONCLUSIONS. Up to 50% of malignant glioma patients treated with RT/TMZ and progression immediately after RT develop pseudo-progression. The current study data support the idea to continue TMZ in the case of progressive lesions immediately after RT/TMZ. Surgery should be considered in symptomatic cases. The inclusion of patients with progressive lesions developing direcdy after chemoradiation in studies regarding recurrent gliomas will lead to an overestimation of the results.

Incidence of acute peripheral neurotoxicity after deep regional hyperthermia of the pelvis (Article)

2008-06-01T00:00:00Z

Background: After observing rather severe acute neurotoxicity in a few patients following deep hyperthermia treatment for a pelvic tumour, we evaluated the incidence of neurotoxicity in all patients treated with deep hyperthermia of the pelvis between June 1990 and April 2004. Materials and methods: Hyperthermia treatment registrations and hospital charts of all 736 patients were reviewed. Differences between the incidence of neurotoxicity in subgroups of patients were evaluated by 2×2 exact tests. Results: Grade 2 or 3 acute neurotoxicity occurred in 2.3% of patients, grade 3 in 0.7%. The duration of symptoms was longer than 3 months in 6 patients (0.8%). Neurological examination in 5 patients showed that the most commonly involved structures are the sacral and lower lumbar nerve roots and the sacral plexus. Acute neurotoxicity occurred only after November 1999 and only in patients treated for primary cervical cancer. Comparison of applied powers and achieved temperatures in patients developing neurotoxicity did not show differences between treatment sessions which resulted in neurotoxicity and sessions not resulting in neurotoxicity. Conclusion: Acute neurotoxicity following hyperthermia for pelvic tumours is a rare complication, but can result in symptoms affecting the activities of daily life. We found no patient, tumour or treatment characteristics predictive for a risk of neurotoxicity.

Multiple sclerosis-related proteins identified in cerebrospinal fluid by advanced mass spectrometry (Article)

2008-04-01T00:00:00Z

A total of 164 cerebrospinal fluid (CSF) samples taken from neurological patients were classed into four groups according to the clinical diagnosis: multiple sclerosis (MScl, n = 44), clinically isolated syndrome of demyelination (CIS, n = 40), other inflammatory neurological disease (OIND, n = 26) and other neurological disease (OND, n = 54). After tryptic digestion, the samples were measured by MALDI-TOF MS. Spectra were analyzed using the R-project software package, in which a peak detection algorithm was developed. Subsequently, the peak lists were compared based on ranked data (non-parametric). Significant differences were observed in the comparisons of MScl vs. OND and CIS vs. OND. The comparisons of MScl vs. OIND, and CIS vs. OIND showed fewer significant differences. No significant differences were found in comparisons MScl vs. CIS and OIND vs. OND. MScl and CIS had strikingly similar profiles, probably a reflection of common pathological mechanisms. Three differentially expressed proteins in the comparison of MScl vs. OND were identified: chromogranin A, a potential marker for neurodegeneration; and two important factors in complement-mediated inflammatory reaction, clusterin and complement C3. CSF chromogranin A levels were confirmed to be significantly elevated in the MScl group using an ELISA.

A software application for comparing large numbers of high resolution MALDI-FTICR MS spectra demonstrated by searching candidate biomarkers for glioma blood vessel formation (Article)

2008-03-01T00:00:00Z

Background: A Java™ application is presented, which compares large numbers (n > 100) of raw FTICR mass spectra from patients and controls. Two peptide profile matrices can be produced simultaneously, one with occurrences of peptide masses in samples and another with the intensity of common peak masses in all the measured samples, using the peak- and background intensities of the raw data. In latter way, more significantly differentially expressed peptides are found between groups than just using the presence or absence in samples of common peak masses. The software application is tested by searching angiogenesis related proteins in glioma by comparing laser capture micro dissected- and enzymatic by trypsin digested tissue sections. Results: By hierarchical clustering of the presence-absence matrix, it appears that proteins, such as hemoglobin alpha and delta subunit, fibrinogen beta and gamma chain precursor, tubulin specific chaperone A, epidermal fatty acid binding protein, neutrophil gelatinase-associated lipocalin precursor, peptidyl tRNA hydrolase 2 mitochondrial precursor, placenta specific growth hormone, and zinc finger CCHC domain containing protein 13 are significantly different expressed in glioma vessels. The up-regulated proteins in the glioma vessels with respect to the normal vessels determined by the Wilcoxon-Mann-Whitney test on the intensity matrix are vimentin, glial fibrillary acidic protein, serum albumin precursor, annexin A5, alpha cardiac and beta actin, type I cytoskeletal 10 keratin, calcium binding protein p22, and desmin. Peptide masses of calcium binding protein p22, Cdc42 effector protein 3, fibronectin precursor, and myosin-9 are exclusively present in glioma vessels. Some peptide fragments of non-muscular myosin-9 at the C-terminus are strongly up-regulated in the glioma vessels with respect to the normal vessels. Conclusion: The less rigorous than in general used commercial propriety software de-isotope algorithm results in more mono-isotopic peptide masses and consequently more proteins. Centroiding of peptide masses takes place by taking the average over more spectra in the profile matrix. Cytoskeleton proteins and proteins involved in the calcium signaling pathway seem to be most up-regulated in glioma vessels. The finding that peptides at the C-terminus of myosin-9 are up-regulated could be ascribed to splicing or fragmentation by proteases.

Analgesic Adherence Measurement in Cancer Patients: Comparison Between Electronic Monitoring and Diary (Article)

2007-12-01T00:00:00Z

Adherence to analgesics in cancer patients has scarcely been studied. In this study, the Medication Event Monitoring System (MEMS) and medication diaries were compared with respect to feasibility and adherence measurements. Forty-six outpatients with nociceptive pain caused by cancer were asked to use MEMS for their analgesics and to record their medication usage in a diary for four weeks. Seventy-nine percent of the patients used MEMS for the full four-week period; 70% did so for the diary. The majority of patients were satisfied with both MEMS and diary. Adherence data assessed by MEMS and diary were comparable. Patients used the amount of analgesics adequately (taking adherence: 87%) but took them irregularly (timing adherence: 53%). Subgroup analyses in patients using single and multiple analgesic regimens confirmed the comparable suitability of both methods. MEMS and a medication diary are equally useful for analgesic adherence measurement in cancer patients with pain.

Eye movements as a marker for cerebellar damage in paraneoplastic neurological syndromes (Article)

2007-12-01T00:00:00Z

Cerebellar disturbances can induce a variety of motor deficits, ranging from severe ataxia to mild deficits of fine motor control. Although motor disturbances appear as an important clinical feature in many neurological disorders, mild disturbances are often difficult to assess properly. Eye movement recordings using video-oculography in a group of patients with a paraneoplastic neurological disorder revealed subtle saccadic and smooth pursuit deficits when compared to controls. We conclude that an easy quantification of eye movement control may assist in the diagnosis and follow-up of mild motor disturbances in patients with neurological disorders, especially when such signs are not overt during clinical neurological examination.

The Effects of Analgesic Prescription and Patient Adherence on Pain in a Dutch Outpatient Cancer Population (Article)

2007-11-01T00:00:00Z

Insufficient awareness of cancer pain, including breakthrough pain, inadequate analgesic prescriptions, and nonadherence contribute to inadequate cancer pain management. There are insufficient data about the contribution of each of these factors. In a cross-sectional survey among 915 adult cancer outpatients, pain was assessed by the Brief Pain Inventory. Breakthrough pain was defined as a worst pain intensity rated as "7 or more" and an average pain intensity rated as "6 or less" in patients on "around-the-clock" (ATC) analgesics. The Pain Management Index (PMI) was calculated to measure the quality of treatment. Adherence was considered inadequate when below 100% of the dose prescribed. Pain was present in 27% of patients. Worst pain was rated as moderate in 26%, and as severe in 54%. Breakthrough pain was present in 45% of patients with ATC medication. The PMI indicated inadequate treatment in 65% of patients. The proportions of patients adherent to ATC analgesics varied from 59% (tramadol) to 91% (Step 3 opioids). The management of cancer pain will benefit most from improving analgesic prescriptions and patient adherence.

Anti-Yo-associated paraneoplastic cerebellar degeneration in a man with adenocarcinoma of the gastroesophageal junction (Article)

2007-10-01T00:00:00Z

Anti-Yo-associated paraneoplastic cerebellar degeneration is a cancer-related syndrome affecting the nervous system. This syndrome occurs almost exclusively in middle-aged women with gynecological cancers and it is rarerly found in patients with other types of cancer or in males. In this report we describe a male patient adenocarcinoma of the gastroesophageal junction and PCD with anti-Yo antibodies. To our knowledge, this is only the third report of PCD with positive anti-Yo antibodies in an esophageal tumor and the first report in a tumor of the gastroesophageal junction. Copyright

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

2007-07-01T00:00:00Z

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 negative SCLC patients without PNS and 54 healthy controls. Peripheral blood mononuclear cells (PBMC) were stimulated with HuD protein-spanning peptide pools (15-mers) and individual HuD-derived peptides (9-mers) and analysed by cytokine flow cytometry and interferon-γ ELISPOT-assays. Additionally, HuD-based Class I HLA multimers were used to visualize HuD-specific CD8+T cells. Results: No HuD-specific CD8+T cells could be detected in the blood of Hu-PNS patients or controls. Conclusions: Our results do not support a role for HuD-specific CD8+T cells in Hu-PNS. Further studies should focus on the detection of circulating HuD-specific CD4+T cells and examine the antigen specificity of T cells in affected tissues.

Increased levels of circulating endothelial progenitor cells and circulating endothelial nitric oxide synthase in patients with gliomas (Article)

2007-07-01T00:00:00Z

Objective: Gliomas are among the highest vascularized tumors. We hypothesized that patients with gliomas have increased levels of circulating endothelial progenitor cells (EPCs) and circulating endothelial nitric oxide synthase (eNOS). Methods: The fraction of EPCs was quantified by fluorescence-activated cell sorter analysis using anti-CD34, -CD133 and -KDR (kinase insert domain receptor) monoclonal antibodies in unselected peripheral blood samples of 32 patients with gliomas. Control groups included 47 patients with other central nervous system tumors or diseases, 10 patients with recent ischemic strokes, and 19 healthy blood donors. The circulating eNOS concentration of plasma was measured by a colorimetric assay in the same samples. In addition, CD34+CD105+KDR+and CD34+CD146+KDR-cell fractions were measured. Results: The percentage of CD34+CD133+KDR+EPCs in the blood of glioma patients is significantly greater than that in the blood of patients with other central nervous system tumors or diseases (p = 0.003), stroke patients (p = 0.005), or healthy donors (p = 0.013). The plasma eNOS concentration is also significantly greater in glioma patients compared with each of the control groups (p < 0.001 for all groupwise comparisons). No significant differences in the levels of the EPCs or eNOS between any of the control groups were demonstrated. In the glioma patients, the level of eNOS correlated with the fraction of CD34+CD105+KDR+cells (r = 0.748; p = 0.008). Interpretation: The data are suggestive of increased mobilization of EPCs contributing to neoplastic vasculogenesis in glioma. The increased levels of EPCs and eNOS in the peripheral blood of glioma patients trigger further investigations as to their value as independent parameters for use in clinical practice.

Identification of glioma neovascularization-related proteins by using MALDI-FTMS and Nano-LC fractionation to microdissected tumor vessels (Article)

2007-07-01T00:00:00Z

The identification of angiogenesis-related proteins is important for the development of new antiangiogenic therapies, and such proteins are potential new biomarkers for gliomas. The aim of this study was to identify proteins that are exclusively present in glioma neovasculature and not in the vasculature of normal brain. We combined advanced proteomics techniques to compare the expression profiles of microdissected blood vessels from glioma with blood vessels of normal control brain samples. We measured the enzymatic generated peptide profiles from these microdissected samples by MALDI-FTMS. Subsequently, the samples were fractionated by nano-LC prior to MALDI-TOF/TOF. This combined approach enabled us to identify four proteins that appeared to be exclusively expressed in the glioma blood vessels. Two of these proteins, fibronectin and colligin 2, were validated on tissue sections using specific antibodies. We found that both proteins are present in active angiogenesis in glioma, other neoplasms, and reactive conditions in which neoangiogenesis takes place. This work proves that gel-free mass spectrometric techniques can be used on relatively small numbers of cells generated by microdissection procedures to successfully identify differentially expressed proteins.

Identification of differentially regulated splice variants and novel exons in glial brain tumors using exon expression arrays (Article)

2007-06-15T00:00:00Z

Aberrant splice variants are involved in the initiation and/or progression of glial brain tumors. We therefore set out to identify splice variants that are differentially expressed between histologic subgroups of gliomas. Splice variants were identified using a novel platform that profiles the expression of virtually all known and predicted exons present in the human genome. Exon-level expression profiling was done on 26 glioblas tomas, 22 oligodendrogliomas, and 6 control brain samples. Our results show that Human Exon arrays can identify subgroups of gliomas based on their histologic appearance and genetic aberrations. We next used our expression data to identify differentially expressed splice variants. In two independent approaches, we identified 49 and up to 459 exons that are differentially spliced between glioblastomas and oligodendrogliomas, a subset of which (47% and 33%) were confirmed by reverse transcription-PCR (RT-PCR). In addition, exon level expression profiling also identified >700 novel exons. Expression of ∼67% of these candidate novel exons was confirmed by RT-PCR. Our results indicate that exon level expression profiling can be used to molecularly classify brain tumor subgroups, can identify differentially regulated splice variants, and can identify novel exons. The splice variants identified by exon level expression profiling may help to detect the genetic changes that cause or maintain gliomas and may serve as novel treatment targets.

Identification of novel biomarkers in pediatric primitive neuroectodermal tumors and ependymomas by proteome-wide analysis (Article)

2007-06-01T00:00:00Z

The aim of this study was to identify aberrantly expressed proteins in pediatric primitive neuroectodermal tumors (PNETs) and ependymomas. Tumor tissue of 29 PNET and 12 ependymoma patients was subjected to 2-dimensional difference gel electrophoresis. Gel analysis resulted in 79 protein spots being differentially expressed between PNETs and ependymomas (p < 0.01, fold change difference in expression >2). Three proteins, stathmin, annexin A1, and calcyphosine, were chosen for validation by immunohistochemistry. Stathmin was expressed 2.6-fold higher in PNETs than in ependymomas, and annexin A1 and calcyphosine were expressed 2.5- and 37.6-fold higher, respectively, in ependymomas. All PNETs showed strong staining for stathmin, and all ependymomas were strongly positive for annexin A1, whereas control tissues were negative. Calcyphosine immunoreactivity was observed in 59% of the ependymomas and was most profound in ependymoma tissue showing epithelial differentiation. mRNA expression levels of stathmin, annexin A1, and calcyphosine significantly correlated (Rs = 0.65 [p < 0.0001], Rs = 0.50 [p = 0.001], and Rs = 0.72 [p < 0.0001], respectively) with protein expression levels. In conclusion, using a proteome-wide approach, stathmin, annexin A1, and calcyphosine were successfully identified as tumor-specific proteins in pediatric PNETs and ependymomas. Ongoing studies are focused on characterizing the role of these proteins as tumor markers and potential drug targets in pediatric brain tumors.

The impact of impurities in synthetic peptides on the outcome of T-cell stimulation assays (Article)

2007-04-04T00:00:00Z

Protein-spanning peptide pools have proven valuable as a screening tool for detecting T-lymphocyte responses against a wide range of proteins. We have used this approach in our search for T cells reactive to the onconeural protein HuD. We found positive responses in only 3 of 127 individuals; however, these were highly unusual in that the same class I HLA alleles and peptides were involved. These T-cell responses were not confirmed when peptides re-synthesized by the same manufacturer with similar and with higher purity levels were used. Our observations indicated that these T-cell responses were not directed against the designed HuD peptides. Here, we report on (i) comparisons of the peptide batches analyzed by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) that did -and did not - elicit T-cell responses and (ii) a detailed analysis of the various by-products of peptides, irrespective of T-cell assay outcome. We found numerous differences between the peptide batches, such as omissions of amino acids in the primary structure of the peptides. Furthermore, some batches revealed strong interactions with calcium ions or contained sulfated peptides. Our data reveal that different batches from the same peptide may contain artefacts that influence the outcome of HLA-restricted T-cell response assays. Copyright

MALDI-TOF mass spectrometry analysis of cerebrospinal fluid tryptic peptide profiles to diagnose leptomeningeal metastases in patients with breast cancer. (Article)

2005-09-01T00:00:00Z

Leptomeningeal metastasis (LM) is a devastating complication that occurs in 5% of patients with breast cancer. Early diagnosis and initiation of treatment are essential to prevent neurological deterioration. However, early diagnosis of LM remains challenging because 25% of cerebrospinal fluid (CSF) samples produce false-negative results at first cytological examination. We developed a new, MS-based method to investigate the protein expression patterns present in the CSF from patients with breast cancer with and without LM. CSF samples from 106 patients with active breast cancer (54 with LM and 52 without LM) and 45 control subjects were digested with trypsin. The resulting peptides were measured by MALDI-TOF MS. Then, the mass spectra were analyzed and compared between patient groups using newly developed bioinformatics tools. A total of 895 possible peak positions was detected, and 164 of these peaks discriminated between the patient groups (Kruskal-Wallis, p<0.01). The discriminatory masses were clustered, and a classifier was built to distinguish patients with breast cancer with and without LM. After bootstrap validation, the classifier had a maximum accuracy of 77% with a sensitivity of 79% and a specificity of 76%. Direct MALDI-TOF analysis of tryptic digests of CSF gives reproducible peptide profiles that can assist in diagnosing LM in patients with breast cancer. The same method can be used to develop diagnostic assays for other neurological disorders.

Increased incidence of neurological complications in patients receiving an allogenic bone marrow transplantation from alternative donors (Article)

2000-01-01T00:00:00Z

OBJECTIVE: To compare the frequency and type of neurological complications after bone marrow transplantation (BMT) with an HLA identical unrelated donor or a mismatched related donor (alternative donors) to the neurological complications after matched sibling BMT for standard and high risk leukaemia or myelodysplastic syndromes. METHODS: Retrospective analysis of consecutively treated patients with (a) BMT from alternative donors (n=39), (b) treated with matched sibling BMT for standard risk leukaemia, myelodysplastic syndromes, or aplastic anaemia (n=53), and (c) treated with matched sibling BMT for high risk leukaemia, myelodysplastic syndromes, or aplastic anaemia (n=49). RESULTS: A total of 72 neurological complications were found. Most of these occurred within the first 6 months after transplant. Thirty six patients developed a severe neurological complication: 17 Alternative donor patients (44%) by contrast with six standard risk patients (11%) and 13 high risk patients (27%; p<0.005). The most frequent complication was a metabolic encephalopathy occurring in 18% of patients. Most of the encephalopathies were caused by either the transplant procedure, cyclosporin, systemic infections, microangiopathic thrombopathy, or by complications induced by graft versus host disease. Infections of the CNS developed in 9% of patients, cerebrovascular lesions in 3%. CONCLUSIONS: Severe neurological complications are more frequent after BMT from alternative donors. This is mainly due to increased treatment related morbidity and to more profound immunosuppression after BMT from alternative donors.