Medulloblastoma comprises four main subgroups (WNT, SHH, Group 3 and Group 4) originally defined by transcriptional profiling. In primary medulloblastoma tissues, these groups are thought to be ...distinguishable using the immunohistochemical detection of β-catenin, filamin A, GAB1 and YAP1 protein markers. To investigate the utility of these markers for in vitro studies using medulloblastoma cell lines, immunoblotting and indirect immunofluorescence were employed for the detection of β-catenin, filamin A, GAB1 and YAP1 in both DAOY and D283 Med reference cell lines and the panel of six medulloblastoma cell lines derived in our laboratory from the primary tumor tissues of known molecular subgroups. Immunohistochemical detection of these markers was performed on formalin-fixed paraffin-embedded tissue of the matching primary tumors. The results revealed substantial divergences between the primary tumor tissues and matching cell lines in the immunoreactivity pattern of medulloblastoma-subgroup-specific protein markers. Regardless of the molecular subgroup of the primary tumor, all six patient-derived medulloblastoma cell lines exhibited a uniform phenotype: immunofluorescence showed the nuclear localization of YAP1, accompanied by strong cytoplasmic positivity for β-catenin and filamin A, as well as weak positivity for GAB1. The same immunoreactivity pattern was also found in both DAOY and D283 Med reference medulloblastoma cell lines. Therefore, we can conclude that various medulloblastoma cell lines tend to exhibit the same characteristics of protein marker expression under standard in vitro conditions. Such a finding emphasizes the importance of the analyses of primary tumors in clinically oriented medulloblastoma research and the urgent need to develop in vitro models of improved clinical relevance, such as 3D cultures and organotypic slice cultures.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Nestin was originally identified as a class VI intermediate filament protein that is expressed in stem cells and progenitor cells in the mammalian CNS during development. This protein is replaced in ...the adult organism by other intermediate filament proteins; however, nestin may be re-expressed under certain pathological conditions such as ischemia, inflammation, brain injury, and neoplastic transformation. Nestin has been detected in many kinds of tumors, especially in tumors derived from the CNS. Co-expression of nestin and the CD133 surface molecule is considered to be a marker for cancer stem cells in neurogenic tumors. Our work was aimed at a detailed study of nestin expression in osteosarcomas and osteosarcoma-derived cell lines.
Using immunodetection methods, we examined nestin in tumor tissue samples from 18 patients with osteosarcomas. We also successfully established permanent cell lines from the tumor tissue of 4 patients and immunodetection of nestin and CD133 was performed on these cell lines.
Nestin-positive tumor cells were immunohistochemically detected in all of the examined osteosarcomas, but the proportion of these cells that were positively stained as well as the intensity of staining varied. Nestin-positive cells were rarely observed in 2 tumor samples, and the remaining 16 tumor samples showed various nestin expression patterns ranging from very sporadic occurrence to an overwhelming proportion of cells with strong positive staining. Three of the established osteosarcoma cell lines were demonstrated to be nestin-positive, and only one cell line showed no expression of nestin; this finding corresponds with the rare occurrence of nestin-positive cells in the respective tumor sample. Moreover, three of these osteosarcoma cell lines were undoubtedly proven to be Nes+/CD133+.
Our results represent the first evidence of nestin expression in osteosarcomas and suggest the possible occurrence of cells with a stem-like phenotype in these tumors.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2) and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and ...cyclooxygenases (COX). This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells.
Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes.
Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2) or SH-SY5Y) after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX) in combination with ATRA in both cell lines.
Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing ...child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Co-expression of CD133, cell surface glycoprotein, and nestin, an intermediate filament protein, was determined to be a marker of neural stem cells and of cancer stem cells in neurogenic tumors.
We ...examined the expression of CD133 and nestin in ten tumor tissue samples taken from patients with rhabdomyosarcomas and in five rhabdomyosarcoma cell lines. Immunohistochemistry and immunofluorescence were used to examine FFPE tumor tissue samples. Cell lines were analyzed by immunofluorescence, immunoblotting, flow cytometry, and RT-PCR. Functional assays (clonogenic in vitro assay and tumorigenic in vivo assay) were also performed using these cell lines.
CD133 and nestin were detected in all 10 tumor tissue samples and in all 5 cell lines; however, the frequency of CD133+, Nes+, and CD133+/Nes+ cells, as well as the intensity of fluorescence varied in individual samples or cell lines. The expression of CD133 and nestin was subsequently confirmed in all cell lines by immunoblotting. Furthermore, we observed an increasing expression of CD133 in relation to the cultivation. All cell lines were positive for Oct3/4 and nucleostemin; NSTS-11 cells were also able to form xenograft tumors in mice.
Our results represent the first evidence of CD133 expression in rhabdomyosarcoma tissue and in rhabdomyosarcoma cell lines. In addition, the co-expression of CD133 and nestin as well as results of the functional assays suggest a possible presence of cancer cells with a stem-like phenotype in these tumors.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Posterior fossa ependymoma comprises two distinct molecular variants termed EPN_PFA and EPN_PFB that have a distinct biology and natural history. The therapeutic value of cytoreductive surgery and ...radiation therapy for posterior fossa ependymoma after accounting for molecular subgroup is not known.
Four independent nonoverlapping retrospective cohorts of posterior fossa ependymomas (n = 820) were profiled using genome-wide methylation arrays. Risk stratification models were designed based on known clinical and newly described molecular biomarkers identified by multivariable Cox proportional hazards analyses.
Molecular subgroup is a powerful independent predictor of outcome even when accounting for age or treatment regimen. Incompletely resected EPN_PFA ependymomas have a dismal prognosis, with a 5-year progression-free survival ranging from 26.1% to 56.8% across all four cohorts. Although first-line (adjuvant) radiation is clearly beneficial for completely resected EPN_PFA, a substantial proportion of patients with EPN_PFB can be cured with surgery alone, and patients with relapsed EPN_PFB can often be treated successfully with delayed external-beam irradiation.
The most impactful biomarker for posterior fossa ependymoma is molecular subgroup affiliation, independent of other demographic or treatment variables. However, both EPN_PFA and EPN_PFB still benefit from increased extent of resection, with the survival rates being particularly poor for subtotally resected EPN_PFA, even with adjuvant radiation therapy. Patients with EPN_PFB who undergo gross total resection are at lower risk for relapse and should be considered for inclusion in a randomized clinical trial of observation alone with radiation reserved for those who experience recurrence.
BACKGROUNDCo-expression of CD133, cell surface glycoprotein, and nestin, an intermediate filament protein, was determined to be a marker of neural stem cells and of cancer stem cells in neurogenic ...tumors. METHODSWe examined the expression of CD133 and nestin in ten tumor tissue samples taken from patients with rhabdomyosarcomas and in five rhabdomyosarcoma cell lines. Immunohistochemistry and immunofluorescence were used to examine FFPE tumor tissue samples. Cell lines were analyzed by immunofluorescence, immunoblotting, flow cytometry, and RT-PCR. Functional assays (clonogenic in vitro assay and tumorigenic in vivo assay) were also performed using these cell lines. RESULTSCD133 and nestin were detected in all 10 tumor tissue samples and in all 5 cell lines; however, the frequency of CD133+, Nes+, and CD133+/Nes+ cells, as well as the intensity of fluorescence varied in individual samples or cell lines. The expression of CD133 and nestin was subsequently confirmed in all cell lines by immunoblotting. Furthermore, we observed an increasing expression of CD133 in relation to the cultivation. All cell lines were positive for Oct3/4 and nucleostemin; NSTS-11 cells were also able to form xenograft tumors in mice. CONCLUSIONOur results represent the first evidence of CD133 expression in rhabdomyosarcoma tissue and in rhabdomyosarcoma cell lines. In addition, the co-expression of CD133 and nestin as well as results of the functional assays suggest a possible presence of cancer cells with a stem-like phenotype in these tumors.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Medulloblastoma comprises four distinct molecular subgroups: WNT, SHH, Group 3, and Group 4. Current medulloblastoma protocols stratify patients based on clinical features: patient age, metastatic ...stage, extent of resection, and histologic variant. Stark prognostic and genetic differences among the four subgroups suggest that subgroup-specific molecular biomarkers could improve patient prognostication.
Molecular biomarkers were identified from a discovery set of 673 medulloblastomas from 43 cities around the world. Combined risk stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariable Cox proportional hazards analyses. Identified biomarkers were tested using fluorescent in situ hybridization (FISH) on a nonoverlapping medulloblastoma tissue microarray (n = 453), with subsequent validation of the risk stratification models.
Subgroup information improves the predictive accuracy of a multivariable survival model compared with clinical biomarkers alone. Most previously published cytogenetic biomarkers are only prognostic within a single medulloblastoma subgroup. Profiling six FISH biomarkers (GLI2, MYC, chromosome 11 chr11, chr14, 17p, and 17q) on formalin-fixed paraffin-embedded tissues, we can reliably and reproducibly identify very low-risk and very high-risk patients within SHH, Group 3, and Group 4 medulloblastomas.
Combining subgroup and cytogenetic biomarkers with established clinical biomarkers substantially improves patient prognostication, even in the context of heterogeneous clinical therapies. The prognostic significance of most molecular biomarkers is restricted to a specific subgroup. We have identified a small panel of cytogenetic biomarkers that reliably identifies very high-risk and very low-risk groups of patients, making it an excellent tool for selecting patients for therapy intensification and therapy de-escalation in future clinical trials.
Tuberous sclerosis complex is a neurocutaneous syndrome that results from a germline mutation in TSC1 or TSC2 genes. The pathogenic activation of mTORC1 leads to the development of subependymal giant ...cell astrocytomas in patients with tuberous sclerosis complex. Blocking of the dysregulated pathway with mTOR inhibitors has the potential to reduce the volume of this low-grade brain tumor. This article reviews the current knowledge on the pharmacological treatment of subependymal giant cell astrocytomas. A longterm followup and early therapeutic intervention should lead to mortality and morbidity reduction and quality of life improvement in patients with tuberous sclerosis complex associated tumors.
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