GLIMMERS: glioma molecular markers exploration using long-read sequencing.

Summary: The revised WHO guidelines for classifying and grading brain tumors include several copy number variation (CNV) markers. The turnaround time for detecting CNVs and alterations throughout the entire genome is drastically reduced with the customized read incremental approach on the nanopore platform. However, this approach is challenging for non-bioinformaticians due to the need to use multiple software tools, extract CNV markers and interpret results, which creates barriers due to the time and specialized resources that are necessary. To address this problem and help clinicians classify and grade brain tumors, we developed GLIMMERS: glioma molecular markers exploration using long-read sequencing, an open-access tool that automatically analyzes nanopore-based CNV data and generates simplified reports. Availability and implementation: GLIMMERS is available at https://gitlab.com/silol_public/glimmers under the terms of the MIT license.

Clinical improvements in temporospatial gait variables after a spinal tap test in individuals with idiopathic normal pressure hydrocephalus.

Idiopathic Normal Pressure Hydrocephalus (iNPH) is a neurological condition that often presents gait disturbance in the early stages of the disease and affects other motor activities. This study investigated changes in temporospatial gait variables after cerebrospinal fluid (CSF) removal using a spinal tap test in individuals with idiopathic normal pressure hydrocephalus (iNPH), and explored if the tap test responders and non-responders could be clinically identified from temporospatial gait variables. Sixty-two individuals with iNPH were recruited from an outpatient clinic, eleven were excluded, leaving a total of 51 who were included in the analysis. Temporospatial gait variables at self-selected speed were recorded at pre- and 24-h post-tap tests which were compared using Paired t-tests, Cohen's d effect size, and percentage change. A previously defined minimal clinical important change (MCIC) for gait speed was used to determine the changes and to classify tap test responders and non-responders. A mixed model ANOVA was used to determine the within-group, between-group, and interaction effects. Comparisons of the data between pre- and post-tap tests showed significant improvements with small to medium effect sizes for left step length, right step time, stride length and time, cadence, and gait speed. Gait speed showed the largest percentage change among temporospatial gait variables. Within-group and interaction effects were found in some variables but no between-group effect was found. Tap test responders showed significant improvements in right step length and time, stride length and time, cadence, and gait speed while non-responders did not. Some individuals with iNPH showed clinically important improvements in temporospatial gait variables after the tap test, particularly in step/stride length and time, cadence, who could be classified by gait speed. However, gait-related balance variables did not change. Therefore, additional treatments should focus on improving such variables.

Exploiting nanopore sequencing for characterization and grading of IDH-mutant gliomas.

The 2021 WHO Classification of Central Nervous System Tumors recommended evaluation of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion in addition to codeletion of 1p/19q to characterize IDH-mutant gliomas. Here, we demonstrated the use of a nanopore-based copy-number variation sequencing (nCNV-seq) approach to simultaneously identify deletions of CDKN2A/B and 1p/19q. The nCNV-seq approach was initially evaluated on three distinct glioma cell lines and then applied to 19 IDH-mutant gliomas (8 astrocytomas and 11 oligodendrogliomas) from patients. The whole-arm 1p/19q codeletion was detected in all oligodendrogliomas with high concordance among nCNV-seq, FISH, DNA methylation profiling, and whole-genome sequencing. For the CDKN2A/B deletion, nCNV-seq detected the loss in both astrocytoma and oligodendroglioma, with strong correlation with the CNV profiles derived from whole-genome sequencing (Pearson correlation (r) = 0.95, P < 2.2 × 10-16 to r = 0.99, P < 2.2 × 10-16 ) and methylome profiling. Furthermore, nCNV-seq can differentiate between homozygous and hemizygous deletions of CDKN2A/B. Taken together, nCNV-seq holds promise as a new, alternative approach for a rapid and simultaneous detection of the molecular signatures of IDH-mutant gliomas without capital expenditure for a sequencer.

Inhibiting Metastasis and Improving Chemosensitivity via Chitosan-Coated Selenium Nanoparticles for Brain Cancer Therapy.

Selenium nanoparticles (SeNPs) were synthesized to overcome the limitations of selenium, such as its narrow safe range and low water solubility. SeNPs reduce the toxicity and improve the bioavailability of selenium. Chitosan-coated SeNPs (Cs-SeNPs) were developed to further stabilize SeNPs and to test their effects against glioma cells. The effects of Cs-SeNPs on cell growth were evaluated in monolayer and 3D-tumor spheroid culture. Cell migration and cell invasion were determined using a trans-well assay. The effect of Cs-SeNPs on chemotherapeutic drug 5-fluorouracil (5-FU) sensitivity of glioma cells was determined in tumor spheroids. An in vitro blood-brain barrier (BBB) model was established to test the permeability of Cs-SeNPs. SeNPs and Cs-SeNPs can reduce the cell viability of glioma cells in a dose-dependent manner. Compared with SeNPs, Cs-SeNPs more strongly inhibited 3D-tumor spheroid growth. Cs-SeNPs exhibited stronger effects in inhibiting cell migration and cell invasion than SeNPs. Improved 5-FU sensitivity was observed in Cs-SeNP-treated cells. Cellular uptake in glioma cells indicated a higher uptake rate of coumarin-6-labeled Cs-SeNPs than SeNPs. The capability of coumarin-6 associated Cs-SeNPs to pass through the BBB was confirmed. Taken together, Cs-SeNPs provide exceptional performance and are a potential alternative therapeutic strategy for future glioma treatment.

Differential cytokine profiles produced by anti-epileptic drug re-exposure of peripheral blood mononuclear cells derived from severe anti-epileptic drug patients and non-allergic controls.

Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reactions with eosinophilia and systemic symptoms (DRESS) are the most common severe cutaneous adverse drug reactions (SCARs). Anti-epileptic drugs are one of the most common drugs causing SCARs. Cytokine profiles of SCARs during culprit drug exposure have never been characterized. This study aimed to identify cytokine patterns between SCARs and non-SCARs in epilepsy patients and the patterns of DRESS and SJS/TEN. Epilepsy patients that showed allergic responses to anti-epileptic drugs that manifested as SJS/TEN or DRESS were recruited. Epilepsy patients with no drug allergy symptoms and healthy people were also recruited as control groups. Peripheral blood mononuclear cells (PBMCs) were isolated and co-cultured with assigned anti-epileptic drugs according to the lymphocyte transformation test (LTT). LTT and measurement of cytokine levels in supernatants were performed on day six of cell cultivation. This study identified different cytokine expression patterns between SCAR and non-SCAR in epilepsy patients. Significant levels of IL-10, IL-12, IL-17, and GM-CSF were detected in non-SCAR epilepsy. However, the levels of IL-2, IL-5, IL-13, and IFN-gamma were significantly higher in supernatants of PBMCs of DRESS cultivated with AEDs relative to those of SJS/TEN. These cytokine levels were positively correlated with the cell proliferation index. Production of IL-5 and IL-13 was a unique characteristic of DRESS PBMCs. This study was the first to demonstrate distinct differences in cytokine levels between SCAR and non-SCAR PBMCs in epilepsy, which could help explain the immune-pathomechanism of drug hypersensitivity in SCARs. Different patterns of cytokine production and cell proliferation between DRESS and SJS/TEN in AED hypersensitivity were also demonstrated. Production of IL-5 and IL-13 might be a promising marker to define drug hypersensitivity in DRESS.

Assessment of therapeutic effect of CD20-targeted immunoliposome in primary central nervous system lymphoma.

Primary central nervous system lymphoma (PCNSL) is a form of extranodal non-Hodgkin's B-cell lymphoma limited to the CNS. The treatment of PCNSL is ineffective partly due to the blood-brain barrier (BBB) restriction of delivery of many drugs including anti-CD20 (Rituximab; RTX) which is a standard treatment for systemic B-cell lymphomas. In this study, liposome with tween-80 surface modification was fabricated and conjugated with RTX for enhancing BBB penetration to target lymphoma cells in the CNS. Physicochemical characterizations of Lip/RTX were performed and spherical shape liposomes with narrow size distribution were demonstrated by TEM. An average diameter of Lip/RTX was 168.57 ± 1.57 nm with the percentage of RTX conjugation at 90.94. Cell internalization monitored by flow cytometry confirmed that conjugation of RTX promoted liposome entry into Raji cells expressing CD20. Antitumor activity of Lip/RTX was comparable to free RTX indicating that RTX moieties on liposome remained their therapeutic function. In addition, Lip/RTX inhibited tumor aggressiveness by limiting cell migration and invasion. Systemic administration of Lip/RTX significantly prolonged survival of mice harboring intracranial lymphoma xenografts. Taken together, Lip/RTX presents a new potential treatment for patients with PCNSL.

Oligosarcomas, IDH-mutant are distinct and aggressive.

Oligodendrogliomas are defined at the molecular level by the presence of an IDH mutation and codeletion of chromosomal arms 1p and 19q. In the past, case reports and small studies described gliomas with sarcomatous features arising from oligodendrogliomas, so called oligosarcomas. Here, we report a series of 24 IDH-mutant oligosarcomas from 23 patients forming a distinct methylation class. The tumors were recurrences from prior oligodendrogliomas or developed de novo. Precursor tumors of 12 oligosarcomas were histologically and molecularly indistinguishable from conventional oligodendrogliomas. Oligosarcoma tumor cells were embedded in a dense network of reticulin fibers, frequently showing p53 accumulation, positivity for SMA and CALD1, loss of OLIG2 and gain of H3K27 trimethylation (H3K27me3) as compared to primary lesions. In 5 oligosarcomas no 1p/19q codeletion was detectable, although it was present in the primary lesions. Copy number neutral LOH was determined as underlying mechanism. Oligosarcomas harbored an increased chromosomal copy number variation load with frequent CDKN2A/B deletions. Proteomic profiling demonstrated oligosarcomas to be highly distinct from conventional CNS WHO grade 3 oligodendrogliomas with consistent evidence for a smooth muscle differentiation. Expression of several tumor suppressors was reduced with NF1 being lost frequently. In contrast, oncogenic YAP1 was aberrantly overexpressed in oligosarcomas. Panel sequencing revealed mutations in NF1 and TP53 along with IDH1/2 and TERT promoter mutations. Survival of patients was significantly poorer for oligosarcomas as first recurrence than for grade 3 oligodendrogliomas as first recurrence. These results establish oligosarcomas as a distinct group of IDH-mutant gliomas differing from conventional oligodendrogliomas on the histologic, epigenetic, proteomic, molecular and clinical level. The diagnosis can be based on the combined presence of (a) sarcomatous histology, (b) IDH-mutation and (c) TERT promoter mutation and/or 1p/19q codeletion, or, in unresolved cases, on its characteristic DNA methylation profile.

Simplified approach for pathological diagnosis of diffuse gliomas in adult patients.

The most recent WHO classification (2016) for gliomas introduced integrated diagnoses requiring both phenotypic and genotypic data. This approach presents difficulties for countries with limited resources for laboratory testing. The present study describes a series of 118 adult Thai patients with diffuse gliomas, classified by the WHO 2016 classification. The purpose was to demonstrate how a diagnosis can still be achieved using a simplified approach that combines clinical, morphological, immunohistochemical, and fewer molecular assays than typically performed. This algorithm starts with tumor location (midline vs. non-midline) with diffuse midline glioma identified by H3 K27M immunostaining. All other tumors are placed into one of 6 categories, based on morphologic features rather than specific diagnoses. Molecular testing is limited to IDH1/IDH2 mutations, plus co-deletion of 1p/19q for cases with oligodendroglial features and TERT promoter mutation for cases without such features. Additional testing for co-deletion of 1p/19q, TERT promoter mutation and BRAF mutations are only used in selected cases to refine diagnosis and prognosis. With this approach, we were able to reach the integrated diagnosis in 117/118 cases, saving 50 % of the costs of a more inclusive testing panel. The demographic data and tumor subtypes were found to be similar to series from other regions of the world. To the best of our knowledge, this is to the first reported series of diffuse gliomas in South-East Asia categorized by the WHO 2016 classification system.

Active targeting liposome-PLGA composite for cisplatin delivery against cervical cancer.

Cisplatin (Cis) is a widely used chemotherapeutic drug for cancer treatment. However, toxicities and drug resistance limit the use of cisplatin. This study was aimed to improve cisplatin delivery using a targeting strategy to reduce the toxicity. In the present study, combinations of poly lactic-co-glycolic acids (PLGA) and liposomes were used as carriers for cisplatin delivery. In addition, to target the nanoparticle towards tumor cells, the liposome was conjugated with Avastin®, an anti-VEGF antibody. Cisplatin was loaded into PLGA using the double emulsion solvent evaporation method and further encapsulated in an Avastin® conjugated liposome (define herein as L-PLGA-Cis-Avastin®). Their physicochemical properties, including particle size, ζ-potential, encapsulation efficiency and drug release profiles were characterized. In addition, a study of the efficiency of tumor targeted drug delivery was conducted with cervical tumor bearing mice via intravenous injection. The therapeutic effect was examined in a 3D spheroid of SiHa cell line and SiHa cells bearing mice. The L-PLGA-Cis-Avastin® prompted a significant effect on cell viability and triggered cytotoxicity of SiHa cells. A cell internalization study confirmed that the L-PLGA-Cis-Avastin® had greater binding specificity to SiHa cells than those of L-PLGA-Cis or free drug, resulting in enhanced cellular uptake. Tumor targeting specificity was finally confirmed in xenograft tumors. Taken together, this nanoparticle could serve as a promising specific targeted drug for cervical cancer treatment.

Effect of spinal tap test on the performance of sit-to-stand, walking, and turning in patients with idiopathic normal pressure hydrocephalus.

The aim of the study was to investigate the effect of the spinal tap test on sit-to-stand (STS), walking, and turning and to determine the relationship among the outcome measures of STS, walking, and turning in patients with iNPH. Twenty-seven patients with clinical symptoms of iNPH were objectively examined for STS, walking, and turning by the Force Distribution Measurement (FDM) platform connected with a video camera. Assessments were performed at before and 24 hours after spinal tap. Motor abilities were assessed by the STS time, time of walking over 3 meters, and time and number of steps when turning over 180 degrees. Significant improvements were found in the STS time (p = 0.046), walking time (p = 0.048), and turning step (p = 0.001). In addition, turning time was improved but not statistically significant (p = 0.064). Significant relationships were found among all outcome measures (p < 0.001). The relationship among these outcome measures indicated that the individuals had similar ability levels to perform different activities. This may serve as a new choice of outcome measures to evaluate the effect of intervention in different severity levels of patients with iNPH.

Brainstem dysfunction heralding disseminated cryptococcosis.

Cryptococcal meningoencephalitis is one of the most common central nervous system infections affecting immunocompromised patients. However, brainstem involvement is extremely rare and may represent a diagnostic challenge to clinicians. We report a non-HIV infected, chronically immunosuppressed, patient with fatal disseminated cryptococcosis presented with subcutaneous masses at both thighs and progressive brainstem dysfunction. Magnetic resonance imaging demonstrated multiple brainstem infarcts likely derived from small vessel vasculopathy. Anti-fungal treatment led to partial neurologic improvement but the patient succumbed to a fatal sepsis from hospital-acquired pneumonia.

Targeting Netrin-1 in glioblastoma stem-like cells inhibits growth, invasion, and angiogenesis.

Glioblastoma (GBM) is an aggressive malignant brain tumor that still lacks effective therapy. Glioblastoma stem cells (GBM-SCs) were identified to contribute to aggressive phenotypes and poor clinical outcomes for GBM. Netrin-1, an axon guidance molecule, has been found in several tumors in adults. However, the role of Netrin-1 in GBM-SCs remains largely unknown. In this study, CD133-positive U251 GBM cells were used as a putative GBM-SC population to identify the functions of Netrin-1. Using lentiviral transduction, Netrin-1 miR RNAi vectors were transduced into CD133-positive U251 cells. We demonstrated that cell proliferation and survival were decreased following targeted deletion of Netrin-1. Cell invasion was dramatically diminished in Netrin-1 knockdown GBM-SCs. Moreover, Netrin-1 knockdown GBM-SCs exhibited less proangiogenic activity. In conclusion, Netrin-1 may represent a therapeutic target in glioblastoma.

High Carbonic Anhydrase-9 Expression Identifies a Subset of 1p/19q Co-Deletion and Favorable Prognosis in Oligodendroglioma.

OBJECTIVE: To investigate the relationship between 3 hypoxic markers, carbonic anhydrase-9 (CA-9), hypoxia-inducible factor (HIF)-1α, and HIF-2α and the traditional genetic markers, deletions of chromosomes 1p and 19q and Isocitrate dehydrogenase 1 (IDH1) R132H mutation in oligodendrogliomas. METHODS: Thirty-one oligodendrogliomas (27 World Health Organization Grade [WHO] II and 4 WHO Grade III) were processed into tissue microarray. Fluorescence in situ hybridization was exploited to detect chromosome deletion, whereas immunohistochemistry was performed to assess IDH1R132H mutation, CA-9, HIF-1α, and HIF-2α expression. RESULTS: The frequencies of 1p/19q co-deletion and IDH1 R132H mutation were 68% and 71%, respectively. High expression of CA-9 was observed in 42% and was associated with longer survival (P = 0.04) in WHO Grade II oligodendroglioma. High CA-9 expression also identified 62% of 1p/19q-codeleted oligodendroglioma (P = 0.001). In addition, all tumors with high CA-9 expression displayed 1p/19q-codeletion. HIF-1α and HIF-2α provided no additional prognostic value for survival. CONCLUSIONS: High expression of CA-9, a marker for hypoxia and acidosis, is associated with favorable prognosis in oligodendroglioma. In addition, it may serve as a simple screening test for 1p/19q co-deletion if validated in larger cohorts.

Diagnostic performance of advanced MRI in differentiating high-grade from low-grade gliomas in a setting of routine service.

OBJECTIVE: To evaluate the usefulness of advanced MRI techniques in differentiating high-grade (HGG) from low-grade gliomas (LGG). MATERIAL AND METHOD: Sixty-four patients with suspected gliomas were prospectively evaluated by conventional and advanced MRI studies including MR spectroscopy (MRS), diffusion tensor imagining (DTI), and dynamic susceptibility contrast (DSC) MRI. The parametric measurements of metabolic profile, cerebral blood volume, flow (CBV, CBF), apparent diffusion coefficient (ADC), fractional anisotropy, and their ratios by internal normalization were analyzed to differentiate LGG from HGG. Histopathologic findings were used as the gold standard. RESULTS: Forty-three cases with pathologically-proven gliomas were included The best discriminating features between HGG and LGG were CBV and CBF of the solid tumoral region (p < 0.05) whereas the minADC/corpus callosum ratio for DTI and the ratio of Cho/Cr for MRS of the solid tumoral region provided the best diagnostic performance (p < 0.05). With a predetermined threshold for each parametric measurement, the combination of all advanced MRI modalities was associated with the best accuracy whereas the combination of DSC MRI and MRS provided the highest specificity. When all parametric measurements were positive, the probability of HGG was 0.889. CONCLUSION: Comprehensive advanced MRI studies provided better diagnostic performance than using conventional MRI alone in the evaluation of gliomas.

Diagnostic performance of perfusion MRI in differentiating low-grade and high-grade gliomas: advanced MRI in glioma, A Siriraj project.

BACKGROUND: To determine the usefulness of the perfusion MRI technique at Siriraj Hospital for differentiating between high- and low-grade gliomas by using pathological results as the gold standard. MATERIAL AND METHOD: The authors prospectively investigated 64 consecutive patients who were suspected as cerebral glioma from prior conventional imaging. Cerebral perfusion study was achieved during the first pass of a bolus of gadolinium-based contrast agent. All post-processing MRI images were interpreted by two board-certified neuroradiologists (more than 10-year-experience), one radiology resident and one well-trained technician, who separately performed and blinded from the pathological results. RESULTS: Forty-four patients diagnosed as glioma were included in this study. There were 26 cases of high-grade and 18 cases of low-grade gliomas. The cerebral blood volume and flow and its ratios had a strong association with the grade of glioma. The areas under the ROC curve for CB K CBVratio (rCBV), CBF and CBF ratio (rCBF) are 0.778, 0.769, 0.769, and 0.772, respectively. On the basis of equal misclassification rates, a cutoff value of 6.15 for CBV (sensitivity, 81.5%; specificity, 64.7%), a cutoff value of 2.38 for the rCBV (sensitivity, 88.9%; specificity, 64.7%), a cutoff value of 0.66 for CBF (sensitivity 81.5%; specificity 70.6%), and a cutoff value of 2.6 for the rCBF (sensitivity, 85.2%; specificity, 70.60%) best discriminated the high and low-grade gliomas. CONCLUSION: Preoperative radiologic grading of gliomas based on conventional MR imaging is sometimes unreliable. The cerebral perfusion measurements can significantly improve the sensitivity and predictive values of radiologic glioma grading. The rCBV measurement is the best parameter for tumor grading due to the highest sensitivity.

Value of diffusion tensor imaging in differentiating high-grade from low-grade gliomas.

OBJECTIVE: To determine the usefulness of diffusion tensor imaging (DTI) in differentiating high-grade glioma (HGG) from low-grade glioma (LGG). MATERIAL AND METHOD: Patients with cerebral gliomas underwent conventional MRI and DTI before surgery. All proven pathologies were classified into two groups, i.e. LGG and HGG. The authors measured fractional anisotropy (FA) and apparent diffusion coefficient (ADC) values in region of interest (ROI) including solid tumoral region, necrotic region, peritumoral edema, contralateral normal appearing white matter (NAWM) and normal corpus callosum as well as calculated ADC ratios. Pairwise comparisons were performed by using the t-test. The ROC curves of imaging parameters were employed to determine the best parameter for differentiating the two entities. RESULTS: Forty-three patients with cerebral gliomas, 17 with LGG and 26 with HGG, no statistical significant difference between LGG and HGG using mean FA values in each ROI. The ADC and minimal ADC values of solid tumoral region and peritumoral edema, the ADC and minimal ADC ratios of solid tumoral region are statistical significant to differentiate HGG from LGG, p < 0.05. The ratio ADC solid tumoral region to normal corpus callosum had highest predictive accuracy to differentiate the two entities with AUC of 0.74. CONCLUSION: The ADC value, minimal ADC value, and ADC ratios of solid tumoral region appeared to be useful for differentiating HGG from LGG.

Siriraj Acute Stroke Unit: 10 years experience.

BACKGROUND: Stroke is the second most common cause of death and leading cause of adult disability worldwide. The recent publication guidelines suggest that there are treatment strategies for optimizing the management of acute stroke patients including thrombolytic therapy, antiplatelet drugs and the establishment of a stroke unit. In Thailand, the first stroke unit has been established since May 1997 and was named Siriraj Acute Stroke Unit (SASU). MATERIAL AND METHOD: The authors retrospectively analyzed the data of stroke patients who were admitted in the SASU from May 1997 to May 2007, as well as hyper acute stroke (within 3 hours after onset). The statistical analysis was performed by using SPSS 11.0. RESULTS: There were 2,109 patients admitted to the SASU during 10 year-period. The mean age of all patients was 65.35 years (range 14-94 years, median 69.2 years). Stroke subtypes were classified as infarction (including transient ischemic attack) in 1799 patients (86.7%) and hemorrhage in 310 patients (13.26%). The most common stroke mechanism was small vessel disease (38.97%). The mainly ischemic stroke distribution was middle cerebral artery territory (77.14%). Risk factors of stroke were as follow: hypertension (61.79%), diabetes mellitus (35.47%), hyperlipidaemia (46.58%), smoking (21.02%) and prior stroke or coronary heart disease (23.74%). Mortality rate was 3.4% mainly due septicemia (26 patients). The mean total hospital stay of stroke patients at the SASU was 13.81 days (ranging from 1-120 days). There were thirty hyper-acute ischemic stroke patients who received intravenous thrombolytic therapy (between August 2005 and May 2007) for which the mean age was 70.7 years (ranges 48-88 years, median 74.5 years). Mean initial National Institutes of Health Stroke Scale (NIHSS) was 14.27. Intracranial hemorrhage after intravenous thrombolytic therapy was found in 8 patients which include 3 patients with symptomatic hemorrhage. CONCLUSION: The standard of care in SASU is not inferior to other stroke unit worldwide. Admission of acute stroke patient to the SASU provides a better chance of survival as well as a shorter length of hospital stay.