Chapter 4: Cross-Cultural Communication Eubanks, Robin L.; McFarland, Marilyn R.; Mixer, Sandra J. ...
Journal of transcultural nursing,
10/2010, Letnik:
21, Številka:
4_suppl
Journal Article
We have established a robust and reliable cell line-based glioma stem cell xenograft mouse model, genetically and epigenetically similar to its originating patient tumours. The survival of patients ...and injected mice correlate significantly, demonstrating that the transplanted mice mimic the clinical course of the patient.
Abstract
The leading cause of cancer-related mortality among children is brain tumour, and glioblastoma multiforme (GBM) has the worst prognosis. New treatments are urgently needed, but with few cases and clinical trials in children, pre-clinical models such as patient-derived tumour xenografts (PDTX) are important. To generate these, tumour tissue is transplanted into mice, but this yields highly variable results and requires serial passaging in mice, which is time-consuming and expensive. We therefore aimed to establish a cell line-based orthotopic mouse model representative of the patient tumour. Glioma stem cell (GSC) lines derived from paediatric GBM were orthotopically transplanted into immunodeficient mice. Overall survival data were collected and histological analysis of the resulting neoplasias was performed. Genome-wide DNA methylation arrays were used for methylation and copy-number alterations (CNA) profiling. All GSC lines initiated tumours on transplantation and the survival of the mice correlated well with the survival of the patients. Xenograft tumours presented histological hallmarks of GBM, and were also classified as GBM by methylation profiling. Each xenograft tumour clustered together with its respective injected GSC line and patient tumour based on the methylation data. We have established a robust and reproducible cell line-based xenograft paediatric GBM model. The xenograft tumours accurately reflected the patient tumours and mirrored the clinical course of the patient. This model can therefore be used to assess patient response in pre-clinical studies.
Thioflavin-T binds to and detects amyloid fibrils via fluorescence enhancement. Using a combination of linear dichroism and fluorescence spectroscopies, we report that the relation between the ...emission intensity and binding of thioflavin-T to insulin fibrils is nonlinear and discuss this in relation to its use in kinetic assays. We demonstrate, from fluorescence lifetime recordings, that the nonlinearity is due to thioflavin-T being sensitive to self-quenching. In addition, thioflavin-T can induce fibril compaction but not alter fibril structure. Our work underscores the photophysical complexity of thioflavin-T and the necessity of calibrating the linear range of its emission response for quantitative in vitro studies.
High-grade gliomas (HGGs) are very aggressive brain tumors with a cancer stem cell component. Cells, including cancer stem cells, release vesicles called exosomes which contain small non-coding RNAs ...such as microRNAs (miRNAs). These are thought to play an important role in cell-cell communication. However, we have limited knowledge of the types of exosomal miRNAs released by pediatric HGG stem cells; a prerequisite for exploring their potential roles in HGG biology. Here we isolated exosomes released by pediatric glioma stem cells (GSCs) and compared their repertoire of miRNAs to genetically normal neural stem cells (NSCs) exosomes, as well as their respective cellular miRNA content. Whereas cellular miRNAs are similar, we find that the exosomal miRNA profiles differ between normal and tumor cells, and identify several differentially expressed miRNAs. Of particular interest is miR-1290 and miR-1246, which have previously been linked to 'stemness' and invasion in other cancers. We demonstrate that GSC-secreted exosomes influence the gene expression of receiving NSCs, particularly targeting genes with a role in cell fate and tumorigenesis. Thus, our study shows that GSCs and NSCs have similar cellular miRNA profiles, yet differ significantly in the repertoire of exosomal miRNAs and these could influence malignant features of HGG.
Abstract
AIMS: Paediatric brain tumours are rare and establishing a precise diagnosis can be challenging. Analysis of DNA methylation profiles has been shown to be a reliable method to classify ...central nervous system (CNS) tumours with high accuracy. We aimed to prospectively analyse CNS tumours diagnosed in Sweden, to assess the clinical impact of adding DNA methylation-based classification to standard paediatric brain tumour diagnostics in an unselected cohort. Methods: All CNS tumours diagnosed in children (0-18 years) during 2017-2020 were eligible for inclusion provided sufficient tumour material was available. Tumours were analysed using genome-wide DNA methylation profiling and classified by the MNP brain tumour classifier. The initial histopathological diagnosis was compared to the DNA methylation-based classification. For incongruent results, a blinded re-evaluation was performed by an experienced neuropathologist. Results: 240 tumours with a histopathology-based diagnosis were profiled. A high-confidence methylation score of 0.84 or more was reached in 78% of the cases. In 69%, the histopathological diagnosis was confirmed and for some of these also refined, 6% were incongruent and the re-evaluation favoured the methylation-based classification. In the remaining 3% of cases, the methylation class was non-contributory or could not be predicted. The change in diagnosis would have had a direct impact on the clinical management in 5% of all patients.Conclusions: Integrating DNA methylation-based tumour classification into routine clinical analysis improves diagnostics and molecular information important for treatment decisions. The results from methylation profiling should be interpreted in the context of clinical and histopathological information.
Brain tumors are the leading cause of cancer-related death in children but high-grade gliomas in children and adolescents have remained a relatively under-investigated disease despite this. A better ...understanding of the cellular and molecular pathogenesis of the diseases is required in order to improve the outcome for these children. In vitro-cultured primary tumor cells from patients are indispensable tools for this purpose by enabling functional analyses and development of new therapies. However, relevant well-characterized in vitro cultures from pediatric gliomas cultured under serum-free conditions have been lacking. We have therefore established patient-derived in vitro cultures and performed thorough characterization of the cells using large-scale analyses of DNA methylation, copy-number alterations and investigated their stability during prolonged time in culture. We show that the cells were stable during prolonged culture in serum-free stem cell media without apparent alterations in morphology or growth rate. The cells were proliferative, positive for stem cell markers, able to respond to differentiation cues and initiated tumors in zebrafish and mice suggesting that the cells are cancer stem cells or progenitor cells. The cells accurately mirrored the tumor they were derived from in terms of methylation pattern, copy number alterations and DNA mutations. These unique primary in vitro cultures can thus be used as a relevant and robust model system for functional studies on pediatric brain tumors.
Aims
Paediatric brain tumours are rare, and establishing a precise diagnosis can be challenging. Analysis of DNA methylation profiles has been shown to be a reliable method to classify central ...nervous system (CNS) tumours with high accuracy. We aimed to prospectively analyse CNS tumours diagnosed in Sweden, to assess the clinical impact of adding DNA methylation‐based classification to standard paediatric brain tumour diagnostics in an unselected cohort.
Methods
All CNS tumours diagnosed in children (0–18 years) during 2017–2020 were eligible for inclusion provided sufficient tumour material was available. Tumours were analysed using genome‐wide DNA methylation profiling and classified by the MNP brain tumour classifier. The initial histopathological diagnosis was compared with the DNA methylation‐based classification. For incongruent results, a blinded re‐evaluation was performed by an experienced neuropathologist.
Results
Two hundred forty tumours with a histopathology‐based diagnosis were profiled. A high‐confidence methylation score of 0.84 or more was reached in 78% of the cases. In 69%, the histopathological diagnosis was confirmed, and for some of these also refined, 6% were incongruent, and the re‐evaluation favoured the methylation‐based classification. In the remaining 3% of cases, the methylation class was non‐contributory.
The change in diagnosis would have had a direct impact on the clinical management in 5% of all patients.
Conclusions
Integrating DNA methylation‐based tumour classification into routine clinical analysis improves diagnostics and provides molecular information that is important for treatment decisions. The results from methylation profiling should be interpreted in the context of clinical and histopathological information.
In this population‐based study, a successful diagnostic molecular classification using DNA methylation was achieved in 78% of the cohort. Six per cent of the initial diagnoses were on re‐evaluation changed by an experienced neuropathologist favouring the methylation‐based diagnosis. The change in diagnosis would have influenced the management of the patients in 5% of the cohort.
We aimed to develop a diagnostic platform to capture the transcriptomic resemblance of individual adult diffuse gliomas of WHO grades II to IV to neural development and the genomic signature ...associated with glioma progression.
Based on the EM/PM classification scheme, we designed a RT-PCR-based TaqMan low-density array (TLDA) containing 44 classifier and 4 reference genes. Samples of a training dataset (GSE48865), characterized by RNA-sequencing, were utilized to optimize the TLDA design and to develop a support vector machine (SVM)-based prediction model. Complemented with Sanger sequencing for
/
and low coverage whole genome sequencing (WGS), the TLDA and SVM prediction model were tested in a validation (31 gliomas) and a test (121 gliomas) dataset.
Independent of morphologically defined subtypes and grades, gliomas can be individually assigned into the EM and PM glioma subtypes with the respective areas under ROC curves at 0.86 and 0.85 in the validation dataset. The EM gliomas showed a medium overall survival (OS) of 15.6 months, whereas the medium OS for PM gliomas was not reached (HR = 3.55; 95% confidence interval, 1.96-6.45). The EM and PM gliomas showed distinct patterns of genomic alterations, with
mutation and 1p19q codeletion in the PM gliomas and gain of chromosome 7/loss of chromosome 10 in the EM gliomas. Extensive chromosomal abnormalities marked the progression of PM gliomas.
The integration of EM/PM subtyping,
sequencing, and low coverage WGS may improve the risk stratification and selection of treatment regimens for patients with glioma.
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