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Supplementary table shows the information of samples invovled in this study and the information of TLDA assays.

Purpose: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.Experimental Design: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 IDH1/2, 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.Results: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 IDH 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.Conclusions:The integration of EM/PM subtyping, IDH sequencing, and low coverage WGS may improve the risk stratification and selection of treatment regimens for patients with glioma.

Figure S4 shows PM gliomas with GBM morphology carry extensive chromosome abnormalities

Background High-grade gliomas are malignant brain tumors characterized by aggressiveness and resistance to chemotherapy. Prognosis remains dismal, highlighting the need to identify novel molecular dependencies and targets. Ribosome biogenesis (RiBi), taking place in the nucleolus, represents a promising target as several cancer types rely on high RiBi rates to sustain proliferation. Publicly available transcriptomics data of glioma patients revealed a positive correlation between RiBi rates and histological grades. We, therefore, hypothesized that glioma cells could be susceptible to RiBi inhibition. Methods Transcriptomics data from glioma patients were analyzed for RiBi-related processes. BMH-21, a small molecule inhibitor of RNA pol I transcription, was tested in adult and pediatric high-grade glioma cell lines and a zebrafish transplant model. Cellular phenotypes were evaluated by transcriptomics, cell cycle analysis, and viability assays. A chemical synergy screen was performed to identify drugs potentiating BMH-21-mediated effects. Results BMH-21 reduced glioma cell viability, induced apoptosis, and impaired the growth of transplanted glioma cells in zebrafish. Combining BMH-21 with TMZ potentiated cytotoxic effects. Moreover, BMH-21 synergized with Fibroblast Growth Factor Receptor (FGFR) inhibitor (FGFRi) Erdafitinib, a top hit in the chemical synergy screen. RiBi inhibition using BMH-21, POLR1A siRNA, or Actinomycin D revealed engagement of the FGFR–FGF2 pathway. BMH-21 downregulated FGFR1 and SOX2 levels, whereas FGF2 was induced and released from the nucleolus. Conclusions This study conceptualizes the implementation of RiBi inhibition as a viable future therapeutic strategy for glioma and reveals an FGFR connection to the cellular response upon RiBi inhibition with potential translational value.

Diffuse gliomas cause significant morbidity across all age groups, despite decades of intensive research efforts. Here, we review the differences in diffuse gliomas in adults and children, as well as the World Health Organisation (WHO) 2021 classification of these tumours. We explain how DNA methylation-based classification works and list the methylation-based tumour types and subclasses for adult and paediatric diffuse gliomas. The benefits and utility of methylation-based classification in diffuse gliomas demonstrated to date are described. This entails the identification of novel tumour types/subclasses, patient stratification and targeted treatment/clinical management, and alterations in the clinical diagnosis in favour of the methylation-based over the histopathological diagnosis. Finally, we address several considerations regarding the use of DNA methylation profiling as a diagnostic tool, e.g., the threshold of the classifier, the calibrated score, tumour cell content and intratumour heterogeneity.

Background/Aim: Although fusion genes involving the proto-oncogene receptor tyrosine kinase ROS1 are rare in pediatric glioma, targeted therapies with small inhibitors are increasingly being approved for histology-agnostic fusion-positive solid tumors. Patient and Methods: Here, we present a 16-month-old boy, with a brain tumor in the third ventricle. The patient underwent complete resection but relapsed two years after diagnosis and underwent a second operation. The tumor was initially classified as a low-grade glioma (WHO grade 2); however, methylation profiling suggested the newly WHO-recognized type: infant-type hemispheric glioma. To further refine the molecular background, and search for druggable targets, whole genome (WGS) and whole transcriptome (RNA-Seq) sequencing was performed. Results: Concomitant WGS and RNA-Seq analysis revealed several segmental gains and losses resulting in complex structural rearrangements and fusion genes. Among the top-candidates was a novel TPR::ROS1 fusion, for which only the 3’ end of ROS1 was expressed in tumor tissue, indicating that wild type ROS1 is not normally expressed in the tissue of origin. Functional analysis by Western blot on protein lysates from transiently transfected HEK293 cells showed the TPR::ROS1 fusion gene to activate the MAPK-, PI3K- and JAK/STAT- pathways through increased phosphorylation of ERK, AKT, STAT and S6. The downstream pathway activation was also confirmed by immunohistochemistry on tumor tissue slides from the patient. Conclusion: We have mapped the activated oncogenic pathways of a novel ROS1-fusion gene and broadened the knowledge of the newly recognized infant-type glioma subtype. The finding facilitates suitable targeted therapies for the patient in case of relapse.

Context Tumor progression in surgically treated patients with nonfunctioning pituitary adenomas (NFPAs) is associated with excess mortality. Reliable biomarkers allowing early identification of tumor progression are missing. Objective To explore DNA methylation patterns associated with tumor progression in NFPA patients. Methods This case-controlled exploratory trial at a university hospital studied patients who underwent surgery for NFPA that had immunohistochemical characteristics of a gonadotropinoma. Cases included patients requiring reintervention due to tumor progression (reintervention group, n = 26) and controls who had a postoperative residual tumor without tumor progression for at least 5 years (radiologically stable group, n = 17). Genome-wide methylation data from each tumor sample were analyzed using the Infinium MethylationEPIC BeadChip platform. Results The analysis showed that 605 CpG positions were significantly differently methylated (differently methylated positions, DMPs) between the patient groups (false discovery rate adjusted P value 0.2), mapping to 389 genes. The largest number of DMPs were detected in the genes NUP93 and LGALS1. The 3 hypomethylated DMPs and the 3 hypermethylated DMPs with the lowest P values were all significantly (P Conclusion In this exploratory study, DNA methylation patterns in NFPA patients were associated with postoperative tumor progression requiring reintervention. The DMPs included genes that have been previously associated with tumor development. Our study is a step toward finding epigenetic signatures to predict tumor progression in patients with NFPA.

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. Funding Agencies|Swedish government; ALF-agreement; Lions Cancer Research Fund of Western Sweden; Swedish Research Council; Swedish Cancer Society; Swedish Childhood Cancer Fund

Background The subventricular zone (SVZ) of the human brain is a site of adult stem cell proliferation and a microenvironment for neural stem cells (NSCs). It has been suggested that NSCs in the SVZ are potential cells of origin containing driver mutations of glioblastoma, but their role in the origin of diffuse lower-grade gliomas (dLGGs) is not much studied. Methods We included 188 patients ≥18 years with IDH-mutated dLGG (WHO grades 2–3) histologically diagnosed between 2007 and 2020. Tissue microarrays of tumor samples for patients between 2007 and 2016 were used for immunodetection of Nestin, SOX2, SOX9, KLF4, NANOG, CD133 cMYC, and Ki67. DNA methylation profile was used for stemness index (mDNAsi). Tumor contact with the SVZ was assessed and the distance was computed. Results Overall, 70.2% of the dLGG had SVZ contact. Tumors with SVZ contact were larger (102.4 vs 30.9 mL, P < .01), the patients were older (44.3 vs 40.4 years, P = .04) and more often had symptoms related to increased intracranial pressure (31.8% vs 7.1%, P < .01). The expression of SOX2, SOX9, Nestin, and Ki67 showed intersample variability, but no difference was found between tumors with or without SVZ contact, nor with the actual distance to the SVZ. mDNAsi was similar between groups (P = .42). Conclusions We found no statistical relationship between proximity with the SVZ and mDNAsi or expression of SOX2, SOX9, Nestin, and Ki67 in IDH-mutated dLGG. Our data suggest that the potential impact of SVZ on IDH-mutated dLGG is probably not associated with a more stemness-like tumor profile.

BACKGROUND/AIM: Although fusion genes involving the proto-oncogene receptor tyrosine kinase ROS1 are rare in pediatric glioma, targeted therapies with small inhibitors are increasingly being approved for histology-agnostic fusion-positive solid tumors. PATIENT AND METHODS: Here, we present a 16-month-old boy, with a brain tumor in the third ventricle. The patient underwent complete resection but relapsed two years after diagnosis and underwent a second operation. The tumor was initially classified as a low-grade glioma (WHO grade 2); however, methylation profiling suggested the newly WHO-recognized type: infant-type hemispheric glioma. To further refine the molecular background, and search for druggable targets, whole genome (WGS) and whole transcriptome (RNA-Seq) sequencing was performed. RESULTS: Concomitant WGS and RNA-Seq analysis revealed several segmental gains and losses resulting in complex structural rearrangements and fusion genes. Among the top-candidates was a novel TPR::ROS1 fusion, for which only the 3' end of ROS1 was expressed in tumor tissue, indicating that wild type ROS1 is not normally expressed in the tissue of origin. Functional analysis by Western blot on protein lysates from transiently transfected HEK293 cells showed the TPR::ROS1 fusion gene to activate the MAPK-, PI3K- and JAK/STAT- pathways through increased phosphorylation of ERK, AKT, STAT and S6. The downstream pathway activation was also confirmed by immunohistochemistry on tumor tissue slides from the patient. CONCLUSION: We have mapped the activated oncogenic pathways of a novel ROS1-fusion gene and broadened the knowledge of the newly recognized infant-type glioma subtype. The finding facilitates suitable targeted therapies for the patient in case of relapse. CC BY-NC-ND 4.0Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.Correspondence to: Frida AbelThis work was supported by the Swedish Cancer Society (www.cancerfonden.se, grant number 2018/825 to FA and grant number 2018/652 to JAN), the Swedish Children’s Cancer Foundation (www.barncancerfonden.se, grant number PR2017-0029 to FA, PR2019-0079 to KE, and KP2019-0010 to HC), and the ALF-agreement (www.researchweb.org/is/alfgbg, ALFGBG-716231 to FA, ALFGBG-965828 to HC, and ALFGBG-719301 to JAN).

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.

Adult-type diffuse gliomas and meningiomas are the most common primary intracranial tumors of the central nervous system. DNA methylation profiling is a novel diagnostic technique increasingly used also in the clinic. Although molecular heterogeneity is well described in these tumors, DNA methylation heterogeneity is less studied. We therefore investigated the intratumor genetic and epigenetic heterogeneity in diffuse gliomas and meningiomas, with focus on potential clinical implications. We further investigated tumor purity as a source for heterogeneity in the tumors. We analyzed genome-wide DNA methylation profiles generated from 126 spatially separated tumor biopsies from 39 diffuse gliomas and meningiomas. Moreover, we evaluated five methods for measurement of tumor purity and investigated intratumor heterogeneity by assessing DNA methylation-based classification, chromosomal copy number alterations and molecular markers. Our results demonstrated homogeneous methylation-based classification of IDH-mutant gliomas and further corroborates subtype heterogeneity in glioblastoma IDH-wildtype and high-grade meningioma patients after excluding samples with low tumor purity. We detected a large number of differentially methylated CpG sites within diffuse gliomas and meningiomas, particularly in tumors of higher grades. The presence of CDKN2A/B homozygous deletion differed in one out of two patients with IDH-mutant astrocytomas, CNS WHO grade 4. We conclude that diffuse gliomas and high-grade meningiomas are characterized by intratumor heterogeneity, which should be considered in clinical diagnostics and in the assessment of methylation-based and molecular markers.

Background There is an urgent need for effective treatments against glioblastoma (GBM). In this trial, we investigated the efficacy and safety of an adoptive cell-based immunotherapy. Methods Patients with newly diagnosed GBM were recruited at 4 study sites in Sweden. The patients were randomized 1:2 to receive either radiotherapy (RT), 60 Gy/30 fractions, with concomitant and adjuvant temozolomide (TMZ) only, or RT and TMZ with the addition of Autologous Lymphoid Effector Cells Specific Against Tumor (ALECSAT) in an open-label phase II trial. The primary endpoint was investigator-assessed progression-free survival (PFS). The secondary endpoints were survival and safety of ALECSAT. Results Sixty-two patients were randomized to either standard of care (SOC) with RT and TMZ alone (n = 22) or SOC with ALECSAT (n = 40). Median age was 57 years (range 38–69), 95% of the patients were in good performance status (WHO 0–1). There was no significant difference between the study arms (SOC vs ALECSAT + SOC) in PFS (7.9 vs 7.8 months; hazard ratio [HR] 1.28; 95% confidence interval [CI] 0.70–2.36; P = .42) or in median overall survival (OS) (18.3 vs 19.2 months; HR 1.16, 95% CI 0.58–2.31; P = .67). The treatment groups were balanced in terms of serious adverse events (52.4% vs 52.5%), but adverse events ≥grade 3 were more common in the experimental arm (81.0% vs 92.5%). Conclusion Addition of ALECSAT immunotherapy to standard treatment with radiochemotherapy was well tolerated but did not improve PFS or OS for patients with newly diagnosed GBM.

BACKGROUND: The heterogeneous group of tumors, primitive neuro-ectodermal tumors of the central nervous system (CNS-PNETs), is a group of rare childhood embryonal tumors associated with a poor prognosis. In recent years, molecular analyzes have shown that CNS-PNETs consist of high-grade gliomas (HGG), ependymomas, different embryonal entities like atypical teratoid /rhabdoid tumors (AT/RT), CNS neuroblastoma FOXR2 and embryonal tumor with multi-layered rosettes (ETMR). Each of these tumor types is unusual and long-term clinical follow-up data are sparse. METHODS: We retrospectively re-evaluated all children (0-18 years old) diagnosed with a CNS-PNET in Sweden during 1984-2015. In total, 88 supratentorial CNS-PNETs were identified in the Swedish Childhood Cancer Registry and from these formalin-fixed paraffin-embedded tumor material was available for 69 patients. All tumors were reviewed histopathologically by an experienced neuropathologist and were analyzed using genome-wide DNA methylation profiling and classified by the MNP brain tumor classifier. RESULTS: The largest entities, after re-evaluation, were HGG (30%), CNS NB-FOXR2 (12%), AT/RT (10%) and ETMR (8%). Some tumors were difficult to classify and will be further evaluated molecularly. Some examples: Best treatment results were seen for patients with CNS-NB FOXR2 (5-year PFS: 100%) where all patients had received craniospinal radiotherapy (CSI). Patients with ETMR were all very young and survival data show early progression and poor survival (5-year OS 34%). CONCLUSIONS: Although the patient material is relatively small, it is population-based with long follow-up times. Our findings are in line with other studies and shows that CSI is important for cure for CNS-NB FOXR2 and that intensive multi-modal therapies needs to be evaluated in up-front studies for these rare embryonal tumors.

BackgroundDNA methylation profiling has facilitated and improved the classification of a wide variety of tumors of the central nervous system. In this study, we investigated the potential utility of DNA methylation profiling to achieve molecular diagnosis in adult primary diffuse lower-grade glioma (dLGG) according to WHO 2016 classification system. We also evaluated whether methylation profiling could provide improved molecular characterization and identify prognostic differences beyond the classical histological WHO grade together withIDHmutation status and 1p/19q codeletion status. All patients diagnosed with dLGG in the period 2007–2016 from the Västra Götaland region in Sweden were assessed for inclusion in the study.ResultsA total of 166 dLGG cases were subjected for genome-wide DNA methylation analysis. Of these, 126 (76%) were assigned a defined diagnostic methylation class with a class prediction score ≥ 0.84 and subclass score ≥ 0.50. The assigned methylation classes were highly associated with theirIDHmutation status and 1p/19q codeletion status.IDH-wildtype gliomas were further divided into subgroups with distinct molecular features.ConclusionThe stratification of the patients by methylation profiling was as effective as the integrated WHO 2016 molecular reclassification at predicting the clinical outcome of the patients. Our study shows that DNA methylation profiling is a reliable and robust approach for the classification of dLGG into molecular defined subgroups, providing accurate detection of molecular markers according to WHO 2016 classification.

Background Radiation is an important therapeutic tool. However, radiotherapy has the potential to promote co-evolution of genetic and epigenetic changes that can drive tumour heterogeneity, formation of radioresistant cells and tumour relapse. There is a clinical need for a better understanding of DNA methylation alterations that may follow radiotherapy to be able to prevent the development of radiation-resistant cells. Methods We examined radiation-induced changes in DNA methylation profiles of paediatric glioma stem cells (GSCs) in vitro. Five GSC cultures were irradiated in vitro with repeated doses of 2 or 4 Gy. Radiation was given in 3 or 15 fractions. DNA methylation profiling using Illumina DNA methylation arrays was performed at 14 days post-radiation. The cellular characteristics were studied in parallel. Results Few fractions of radiation did not result in significant accumulation of DNA methylation alterations. However, extended dose fractionations changed DNA methylation profiles and induced thousands of differentially methylated positions, specifically in enhancer regions, sites involved in alternative splicing and in repetitive regions. Radiation induced dose-dependent morphological and proliferative alterations of the cells as a consequence of the radiation exposure. Conclusions DNA methylation alterations of sites with regulatory functions in proliferation and differentiation were identified, which may reflect cellular response to radiation stress through epigenetic reprogramming and differentiation cues.

Purpose: 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. Experimental Design: 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 IDH1/2, 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. Results: 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 IDH 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. Conclusions: The integration of EM/PM subtyping, IDH sequencing, and low coverage WGS may improve the risk stratification and selection of treatment regimens for patients with glioma.

This chapter discusses analysis and interpretation of large-scale Illumina DNA methylation microarray data, used in the context of cancer studies. We outline commonly used normalization procedures and list issues to consider regarding data preprocessing. Focusing on software packages for R, we describe methods for finding features in the methylation data that are of importance for generating and testing hypotheses in cancer research, like differentially methylated positions or regions and global methylation trends.

This chapter discusses analysis and interpretation of large-scale Illumina DNA methylation microarray data, used in the context of cancer studies. We outline commonly used normalization procedures and list issues to consider regarding data preprocessing. Focusing on software packages for R, we describe methods for finding features in the methylation data that are of importance for generating and testing hypotheses in cancer research, like differentially methylated positions or regions and global methylation trends.

Background: More than half of all surgically treated patients with non-functioning pituitary adenomas (NFPA) experience tumor progression during their follow-up, with some of these adenomas exhibiting locally aggressive behavior. Tumor progression is strongly associated with excess morbidity and mortality. Tumor control is, therefore, of vital importance for the patient outcome. There is no reliable method to predict tumor behavior after the initial surgical treatment. The aim of this study was therefore to study associations between DNA methylation patterns and tumor progression in patients with NFPA. Methods: In a case-controlled trial, 43 patients operated for NFPA with immunohistochemical characteristics of gonadotroph subtype were selected. Cases included patients who needed reintervention due to tumor progression (Reintervention group, n = 26), and controls had residual tumor without tumor progression during at least five years of follow-up (Radiologically stable group, n = 17). Genome-wide methylation analysis of the tumor tissue from each patient was performed on the Infinium MethylationEPIC BeadChip platform. Results: Principal component analysis (PCA) showed that clinical course was a strong contributor to the difference in DNA methylation profiles between the two groups. Also, unsupervised hierarchical clustering of the top 5,000 most variable CpG sites revealed three methylation profiles corresponding to, (1) the Reintervention group, (2) the Radiologically stable group, and (3) a mix of the two groups. Although the overall average methylation levels in the two groups did not differ (P = 0.16), 142,521 Differentially Methylated Positions (DMPs) were identified. Of these, 650 showed a difference in beta value methylation larger than 0.2. Interestingly, among the genes harboring the most significantly DMPs was GABRA1, and among the genes containing the largest number of DMPs were GATA2, NUP93, and LGALS1. In addition, the three most hypomethylated and the three most hypermethylated DMPs were all significantly (P

Background: Primary brain tumors are the most common solid tumors in children. Increasing evidence demonstrates diverse intratumoral immune signatures, which are tentatively reflected in peripheral blood. Procedure: Twenty cytokines were analyzed in preoperative plasma samples from five healthy children and 45 children with brain tumors, using a multiplex platform (MesoScale Discovery V-PLEX®). Tumor types included medulloblastoma (MB), ependymoma, sarcoma, high-grade glioma, pilocytic astrocytoma, and other low-grade gliomas. Results: A panel of four cytokines [VEGFA, interleukin (IL)-7, IL-17A, and tumor necrosis factor (TNF)-β] delineated two distinct patient groups, identified as VEGFAhighIL-7highIL-17AlowTNF-βlow (Group A) and VEGFAlowIL-7lowIL-17AhighTNF-βhigh (Group B). Healthy controls and the vast majority of patients with MB were found within Group A, whereas patients with other tumor types were equally distributed between the two groups. Unrelated to A/B affiliation, we detected trends toward increased IL-10 and decreased IL-12/23 and TNF-α in several tumor types. Finally, a small number of patients displayed evidence of enhanced systemic immune activation, including elevated levels of interferon-γ, granulocyte monocyte colony-stimulating factor, IL-6, IL-12/23, and TNF-α. Following tumor resection, cytokine levels in a MB patient approached the levels of healthy controls. Conclusions: We identify common features and individual differences in the systemic immune profiles of children with brain tumors. Overall, patients with MB displayed a uniform cytokine profile, whereas other tumor diagnoses did not predict systemic immunological status in single patients. Future characterization and monitoring of systemic immune responses in children with brain tumors will have important implications for the development and implementation of immunotherapy. (Less)

OBJECTIVES Pancreatic ductal adenocarcinomas (PDACs) are found in more than 85% of patients with pancreatic cancer and with 5-year survival of less than 10%. Effective treatment may be radical surgery, which is hampered by rapid relapse. Therefore, our aim was to compare DNA sequence alterations in patients with short and long survival to evaluate if confirmed DNA alterations predict short postoperative survival. METHODS DNA was extracted from tumor tissue from 59 PDAC patients, analyzed for KRAS mutations, and hybridized to 180 K CGH + SNP microarrays and 450 K methylation arrays. Analyses were based on postoperative survival where less than 12 months was considered to be short survival and more than 18 months was considered long survival. RESULTS Ninety-three percent of the patients had KRAS mutations in tumor DNA. Great heterogeneity of whole genome DNA sequence alterations were observed among chromosomes within the patient materials. Specific DNA sequence alterations did not directly predict postoperative survival, although short survivors had significantly more and larger DNA amplifications (P < 0.006). Amplifications on chromosome 11 and 21 and deletions on chromosome 2 predicted short postoperative survival (P < 0.03). DNA methylation was not related to survival. CONCLUSIONS Highly variable genetic differences among DNA regions in PDAC tumors were demonstrated. Postoperative short survival was related to tumor sequence DNA alterations on chromosome 2, 11, and 21.

Glioblastoma multiform (GBM) is the most common brain tumor characterized by a dismal prognosis. GBM cancer stem cells (gCSC) or tumor-initiating cells are the cell population within the tumor-driving therapy resistance and recurrence. While temozolomide (TMZ), an alkylating agent, constitutes the first-line chemotherapeutic significantly improving survival in GBM patients, resistance against this compound commonly leads to GBM recurrence and treatment failure. Although the roles of protein-coding transcripts, proteins and microRNA in gCSC, and therapy resistance have been comprehensively investigated, very little is known about the role of long noncoding RNAs (lncRNAs) in this context. Using nonoverlapping, independent RNA sequencing and gene expression profiling datasets, we reveal that TP73-AS1 constitutes a clinically relevant lncRNA in GBM. Specifically, we demonstrate significant overexpression of TP73-AS1 in primary GBM samples, which is particularly increased in the gCSC. More importantly, we demonstrate that TP73-AS1 comprises a prognostic biomarker in glioma and in GBM with high expression identifying patients with particularly poor prognosis. Using CRISPRi to downregulate our candidate lncRNA in gCSC, we demonstrate that TP73-AS1 promotes TMZ resistance in gCSC and is linked to regulation of the expression of metabolism- related genes and ALDH1A1, a protein known to be expressed in cancer stem cell markers and protects gCSC from TMZ treatment. Taken together, our results reveal that high TP73-AS1 predicts poor prognosis in primary GBM cohorts and that this lncRNA promotes tumor aggressiveness and TMZ resistance in gCSC.

INTRODUCTION: A hallmark of glioblastoma (GBM) is the cellular and molecular heterogeneity both inter- and intra-tumourally. This adds to the complexity of GBM regarding risk for sampling bias and tumour escape of targeted therapy. Heterogeneity of gene expression within GBM is well documented, but little is known regarding the epigenetic heterogeneity. This could have implications for diagnostics, as DNA methylation can be used for tumour classification, subtyping and determination of the clinically used biomarker MGMT promoter methylation status. We therefore aimed to profile the intra-tumour epigenetic heterogeneity in GBM. MATERIALS AND METHODS: Three biopsies per tumour from spatially separated regions were collected from 10 adult GBM patients. Only samples that were highly fluorescent using Gliolan (5-ALA) were collected and the location was documented with neuronavigation. We then performed genome-wide DNA methylation analysis to determine the methylation level of ~850 000 CpG sites and compared inter and intra-tumour variation. The tumour content of the biopsies was assessed based on histology by a specialist in clinical neuropathology. RESULTS: Hierarchical clustering of the CpG sites with the most variable methylation clustered the biopsies from each patient respectively together except for the biopsies from one patient. A large number of differentially methylated probes (DMP) existed between the intra-tumour biopsies and some were located in GBM-related genes. All samples were classified as GBM by methylation profiling, but the strength of the classification and the subtype differed within tumours. We also found intra-tumour heterogeneity in the methylation status of the MGMT promoter in one of the patients. CONCLUSIONS: Intra-tumour biopsies did not cluster together for all patients and displayed many DMP demonstrating that intra-tumour DNA methylation heterogeneity exists in GBM. The tumour type (GBM) is classified homogenously within the tumour for all patients, while the strength of the classification and the subtype differs. The observed intra-tumour heterogeneity in DNA methylation needs to be taken into consideration for methylation-based biomarkers and future stratification of GBM subtypes.

BACKGROUND: The leading cause of cancer-related mortality among children is brain tumours and glioblastoma multiforme (GBM) has the worst prognosis. Epigenetic deregulation is believed to be a driver as these patients have few mutations compared to adults. New treatments are urgently needed but few pre-clinical in vivo models exist. One approach of generating these is to transplant tumour tissue into mice, but it yields highly variable results and requires serial passaging in mice, which is time-consuming, expensive and ethically questionable. We therefore aimed to establish a cell line-based orthotopic mouse model representative of the patient tumour. METHODS: Patient-derived cancer stem cell (CSC) lines from paediatric GBM were orthotopically transplanted into immunodeficient mice. The xenograft tumours were evaluated by histology for glioma features. Genome-wide DNA methylation analysis was performed on the CSC, xenograft and patient tumours. RESULTS: All CSC lines initiated tumours and the survival of the mice correlated with the survival of their respective patient. The xenograft tumours had key glioma features and were classified as GBM by histology and methylation profiling. The methylation profile of the xenograft tumour clustered together with the patient tumour (

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., 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.

Glioblastoma (GBM) is the most aggressive primary brain tumor with a median survival of less than 15 months, emphasizing the need for better treatments. Immunotherapy as a treatment for improving or aiding the patient’s own immune defense to target the tumor has been suggested for GBM. A randomized clinical trial of adoptive cell transfer using ALECSAT (Autologous Lymphoid Effector Cells Specific Against Tumor Cells) is currently ongoing in Sweden. Here we performed a paired pre-clinical study to investigate the composition and in vitro effect of ALECSAT and identify determinants for the effect using autologous GBM-derived cancer stem cells (CSC), immunocytochemistry and flow cytometry. We show a clear dose-response relationship of ALECSAT on CSC, suggesting that the number of infused cells is of importance. In addition, the in vitro effect of ALECSAT on CSC correlated significantly to the blood count of T helper (Th) cells in the patient indicating a potential benefit of collecting cells for ALECSAT preparation at an even earlier stage when patients generally have a better blood count. The factors identified in this study will be important to consider in the design of future immunotherapy trials to achieve prolonged survival.

The elucidation of mechanisms involved in resistance to therapies is essential to improve the survival of patients with malignant gliomas. A major feature possessed by glioma cells that may aid their ability to survive therapy and reconstitute tumors is the capacity for self-renewal. We show here that glioma stem cells (GSCs) express low levels of MKP1, a dual-specificity phosphatase, which acts as a negative inhibitor of JNK, ERK1/2, and p38 MAPK, while induction of high levels of MKP1 expression are associated with differentiation of GSC. Notably, we find that high levels of MKP1 correlate with a subset of glioblastoma patients with better prognosis and overall increased survival. Gain of expression studies demonstrated that elevated MKP1 impairs self-renewal and induces differentiation of GSCs while reducing tumorigenesis in vivo. Moreover, we identified that MKP1 is epigenetically regulated and that it mediates the anti-tumor activity of histone deacetylase inhibitors (HDACIs) alone or in combination with temozolomide. In summary, this study identifies MKP1 as a key modulator of the interplay between GSC self-renewal and differentiation and provides evidence that the activation of MKP1, through epigenetic regulation, might be a novel therapeutic strategy to overcome therapy resistance in glioblastoma., O.A. (BFI_2011_195), L.G.-R. (PRE_2013_1_760), L.M.-C. (PRE_2014_1_92), and J.A.-I. (PRE_2016_1_0375) were each recipient of a predoctoral fellowship from the Department of Education, University and Research of the Basque Government, and P.A. from the Spanish Association Against Cancer (AECC Gipuzkoa). This work was supported by grants from the Instituto de Salud Carlos III and FEDER Funds (PI13/02277, PI14/01495, PI15/00186, CP16/00039, PI16/01580, DTS16/084), European Union (Marie Curie CIG 2012/712404, REFBIO13/BIOD/009, and 011), and AICR/WCR [13–1270].

The CD24 glycoprotein is a mediator of neuronal proliferation, differentiation and immune suppression in the normal CNS, and a proposed cancer biomarker in multiple peripheral tumor types. We performed a comparative analysis of CD24 gene expression in a large cohort of pediatric and adult brain tumors (n = 813), and further characterized protein expression in tissue sections (n = 39), primary brain tumor cultures (n = 12) and a novel orthotopic group 3 medulloblastoma xenograft model. Increased CD24 gene expression was demonstrated in ependymomas, medulloblastomas, anaplastic astrocytomas and glioblastomas, although medulloblastomas displayed higher expression than all other tumor entities. Preferential expression of CD24 in medulloblastomas was confirmed at protein level by immunostaining and computerized image analysis of cryosections. Morphologies and immunophenotyping of CD24(+) cells in tissue sections tentatively suggested disparate functions in different tumor subsets. Notably, protein staining of medulloblastoma cells was associated with prominent cytoplasmic and membranous granules, enabling rapid and robust identification of medulloblastoma cells in clinical tissue samples, as well as in experimental model systems. In conclusion, our results implicate CD24 as a clinically and experimentally useful medulloblastoma immunomarker. Although our results encourage further functional studies of CD24 as a potential molecular target in subsets of brain tumors, the promiscuous expression of CD24 in vivo highlights the importance of specificity in the future design of such targeted treatment.

Hepatocytes derived from human pluripotent stem cells (hPSC-HEP) have the potential to replace presently used hepatocyte sources applied in liver disease treatment and models of drug discovery and development. Established hepatocyte differentiation protocols are effective and generate hepatocytes, which recapitulate some key features of their in vivo counterparts. However, generating mature hPSC-HEP remains a challenge. In this study, we applied transcriptomics to investigate the progress of in vitro hepatic differentiation of hPSCs at the developmental stages, definitive endoderm, hepatoblasts, early hPSC-HEP, and mature hPSC-HEP, to identify functional targets that enhance efficient hepatocyte differentiation. Using functional annotation, pathway and protein interaction network analyses, we observed the grouping of differentially expressed genes in specific clusters representing typical developmental stages of hepatic differentiation. In addition, we identified hub proteins and modules that were involved in the cell cycle process at early differentiation stages. We also identified hub proteins that differed in expression levels between hPSC-HEP and the liver tissue controls. Moreover, we identified a module of genes that were expressed at higher levels in the liver tissue samples than in the hPSC-HEP. Considering that hub proteins and modules generally are essential and have important roles in the protein-protein interactions, further investigation of these genes and their regulators may contribute to a better understanding of the differentiation process. This may suggest novel target pathways and molecules for improvement of hPSC-HEP functionality, having the potential to finally bring this technology to a wider use.