Multi-omic approach identifies hypoxic tumor-associated myeloid cells that drive immunobiology of high-risk pediatric ependymoma.

Ependymoma (EPN) is a devastating childhood brain tumor. Single-cell analyses have illustrated the cellular heterogeneity of EPN tumors, identifying multiple neoplastic cell states including a mesenchymal-differentiated subpopulation which characterizes the PFA1 subtype. Here, we characterize the EPN immune environment, in the context of both tumor subtypes and tumor cell subpopulations using single-cell sequencing (scRNAseq, n = 27), deconvolution of bulk tumor gene expression (n = 299), spatial proteomics (n = 54), and single-cell cytokine release assays (n = 12). We identify eight distinct myeloid-derived subpopulations from which a group of cells, termed hypoxia myeloid cells, demonstrate features of myeloid-derived suppressor cells, including IL6/STAT3 pathway activation and wound healing ontologies. In PFA tumors, hypoxia myeloid cells colocalize with mesenchymal-differentiated cells in necrotic and perivascular niches and secrete IL-8, which we hypothesize amplifies the EPN immunosuppressive microenvironment. This myeloid cell-driven immunosuppression will need to be targeted for immunotherapy to be effective in this difficult-to-cure childhood brain tumor.

Significant increase of high-risk chromosome 1q gain and 6q loss at recurrence in posterior fossa group A ependymoma: A multicenter study.

BACKGROUND: Ependymoma (EPN) posterior fossa group A (PFA) has the highest rate of recurrence and the worst prognosis of all EPN molecular groups. At relapse, it is typically incurable even with re-resection and re-irradiation. The biology of recurrent PFA remains largely unknown; however, the increasing use of surgery at first recurrence has now provided access to clinical samples to facilitate a better understanding of this. METHODS: In this large longitudinal international multicenter study, we examined matched samples of primary and recurrent disease from PFA patients to investigate the biology of recurrence. RESULTS: DNA methylome derived copy number variants (CNVs) revealed large-scale chromosome gains and losses at recurrence in PFA. CNV changes were dominated by chromosome 1q gain and/or 6q loss, both previously identified as high-risk factors in PFA, which were present in 23% at presentation but increased to 61% at first recurrence. Multivariate survival analyses of this cohort showed that cases with 1q gain or 6q loss at first recurrence were significantly more likely to recur again. Predisposition to 1q+/6q- CNV changes at recurrence correlated with hypomethylation of heterochromatin-associated DNA at presentation. Cellular and molecular analyses revealed that 1q+/6q- PFA had significantly higher proportions of proliferative neuroepithelial undifferentiated progenitors and decreased differentiated neoplastic subpopulations. CONCLUSIONS: This study provides clinically and preclinically actionable insights into the biology of PFA recurrence. The hypomethylation predisposition signature in PFA is a potential risk-classifier for trial stratification. We show that the cellular heterogeneity of PFAs evolves largely because of genetic evolution of neoplastic cells.

Optimizing biomarkers for accurate ependymoma diagnosis, prognostication, and stratification within International Clinical Trials: A BIOMECA study.

BACKGROUND: Accurate identification of brain tumor molecular subgroups is increasingly important. We aimed to establish the most accurate and reproducible ependymoma subgroup biomarker detection techniques, across 147 cases from International Society of Pediatric Oncology (SIOP) Ependymoma II trial participants, enrolled in the pan-European "Biomarkers of Ependymoma in Children and Adolescents (BIOMECA)" study. METHODS: Across 6 European BIOMECA laboratories, we evaluated epigenetic profiling (DNA methylation array); immunohistochemistry (IHC) for nuclear p65-RELA, H3K27me3, and Tenascin-C; copy number analysis via fluorescent in situ hybridization (FISH) and MLPA (1q, CDKN2A), and MIP and DNA methylation array (genome-wide copy number evaluation); analysis of ZFTA- and YAP1-fusions by RT-PCR and sequencing, Nanostring and break-apart FISH. RESULTS: DNA Methylation profiling classified 65.3% (n = 96/147) of cases as EPN-PFA and 15% (n = 22/147) as ST-ZFTA fusion-positive. Immunohistochemical loss of H3K27me3 was a reproducible and accurate surrogate marker for EPN-PFA (sensitivity 99%-100% across 3 centers). IHC for p65-RELA, FISH, and RNA-based analyses effectively identified ZFTA- and YAP-fused supratentorial ependymomas. Detection of 1q gain using FISH exhibited only 57% inter-center concordance and low sensitivity and specificity while MIP, MLPA, and DNA methylation-based approaches demonstrated greater accuracy. CONCLUSIONS: We confirm, in a prospective trial cohort, that H3K27me3 immunohistochemistry is a robust EPN-PFA biomarker. Tenascin-C should be abandoned as a PFA marker. DNA methylation and MIP arrays are effective tools for copy number analysis of 1q gain, 6q, and CDKN2A loss while FISH is inadequate. Fusion detection was successful, but rare novel fusions need more extensive technologies. Finally, we propose test sets to guide future diagnostic approaches.

SIOP Ependymoma I: Final results, long-term follow-up, and molecular analysis of the trial cohort-A BIOMECA Consortium Study.

BACKGROUND: SIOP Ependymoma I was a non-randomised trial assessing event free and overall survival (EFS/OS) of non-metastatic intracranial ependymoma in children aged 3-21 years treated with a staged management strategy. A further aim was to assess the response rate (RR) of subtotally resected (STR) ependymoma to vincristine, etoposide, and cyclophosphamide (VEC). We report final results with 12-year follow-up and post hoc analyses of recently described biomarkers. METHODS: Seventy-four participants were eligible. Children with gross total resection (GTR) received radiotherapy, whilst those with STR received VEC before radiotherapy. DNA methylation, 1q, hTERT, ReLA, Tenascin-C, H3K27me3, and pAKT status were evaluated. RESULTS: Five- and ten-year EFS was 49.5% and 46.7%, OS was 69.3% and 60.5%. GTR was achieved in 33/74 (44.6%) and associated with improved EFS (P = .003, HR = 2.6, 95% confidence interval (CI) 1.4-5.1). Grade 3 tumours were associated with worse OS (P = .005, HR = 2.8, 95%CI 1.3-5.8). 1q gain and hTERT expression were associated with poorer EFS (P = .003, HR = 2.70, 95%CI 1.49-6.10 and P = .014, HR = 5.8, 95%CI 1.2-28) and H3K27me3 loss with worse OS (P = .003, HR = 4.6, 95%CI 1.5-13.2). Methylation profiles showed expected patterns. 12 participants with STR did not receive chemotherapy; a protocol violation. However, best chemotherapy RR was 65.5% (19/29, 95%CI 45.7-82.1), exceeding the prespecified 45%. CONCLUSIONS: Participants with totally resected ependymoma had the best outcomes. RR of STR to VEC exceeded the pre-specified efficacy criterion. However, cases of inaccurate stratification highlighted the need for rapid central review. 1q gain, H3K27me3 loss, and hTERT expression were all associated with poorer survival outcomes.

A retrospective analysis of recurrent pediatric ependymoma reveals extremely poor survival and ineffectiveness of current treatments across central nervous system locations and molecular subgroups.

BACKGROUND: Relapse occurs in 50% of pediatric ependymoma cases and has poor prognosis. Few studies have investigated the clinical progress of relapsed disease, and treatment lacks a standardized approach. METHODS AND MATERIALS: We analyzed 302 pediatric ependymoma cases. Tumor, demographic, and treatment variables were investigated for association with relapse risk, time to recurrence, and survival after relapse. DNA methylation profiling was performed for 135/302 cases, and predominant subgroups were EPN_PFA (n = 95) and EPN_RELA (n = 24). Chromosome 1q status was ascertained for 185/302 cases by fluorescent in-situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and DNA methylation profiles. RESULTS: Sixty-two percent of cases relapsed, with a median of two recurrences with no difference between posterior fossa and supratentorial locations (66% vs 55% relapse rate). One hundred seventeen (38%) cases relapsed within two years and five (2%) beyond 10 years. The late relapses were clinically heterogeneous. Tumor grade and treatment affected risk and time to relapse variably across subgroups. After relapse, surgery and irradiation delayed disease progression with a minimal impact on survival across the entire cohort. In the EPN_PFA and EPN_RELA groups, 1q gain was independently associated with relapse risk (subhazard ratio [SHR] 4.307, P = 0.027 and SHR 1.982, P = 0.010, respectively) while EPN_PFA had increased relapse risk compared with EPN_RELA (SHR = 0.394, P = 0.018). CONCLUSIONS: Recurrent pediatric ependymoma is an aggressive disease with poor outcomes, for which current treatments are inadequate. We report that chromosome 1q gain increases relapse risk in common molecular subgroups in children but a deeper understanding of the underlying biology at relapse and novel therapeutic approaches are urgently needed.

PI3K pathway activation provides a novel therapeutic target for pediatric ependymoma and is an independent marker of progression-free survival.

PURPOSE: Currently, there are few effective adjuvant therapies for pediatric ependymoma outside confocal radiation, and prognosis remains poor. The phosphoinositide 3-kinase (PI3K) pathway is one of the most commonly activated pathways in cancer. PI3Ks transduce signals from growth factors and cytokines, resulting in the phosphorylation and activation of AKT, which in turn induces changes in cell growth, proliferation, and apoptosis. EXPERIMENTAL DESIGN: PI3K pathway status was analyzed in ependymoma using gene expression data and immunohistochemical analysis of phosphorylated AKT (P-AKT). The effect of the PI3K pathway on cell proliferation was investigated by immunohistochemical analysis of cyclin D1 and Ki67, plus in vitro functional analysis. To identify a potential mechanism of PI3K pathway activation, PTEN protein expression and the mutation status of PI3K catalytic subunit α-isoform gene (PIK3CA) was investigated. RESULTS: Genes in the pathway displayed significantly higher expression in supratentorial than in posterior fossa and spinal ependymomas. P-AKT protein expression, indicating pathway activation, was seen in 72% of tumors (n = 169) and P-AKT expression was found to be an independent marker of a poorer progression-free survival. A significant association between PI3K pathway activation and cell proliferation was identified, suggesting that pathway activation was influencing this process. PTEN protein loss was not associated with P-AKT staining and no mutations were identified in PIK3CA. CONCLUSIONS: Our results suggest that the PI3K pathway could act as a biomarker, not only identifying patients with a worse prognosis but also those that could be treated with therapies targeted against the pathway, a strategy potentially effective in a high percentage of ependymoma patients.

Network-based activity induced by 4-aminopyridine in rat dorsal horn in vitro is mediated by both chemical and electrical synapses.

This study investigated the role of electrical and chemical synapses in sustaining 4-aminopyridine (4-AP)-evoked network activity recorded extracellularly from substantia gelatinosa (SG) of young rat spinal cord in vitro. Superfusion of 4-AP (50 microM) induced two types of activity, the first was observed as large amplitude field population spiking activity and the second manifested within the inter-spike interval as low amplitude rhythmic oscillations in the 4-12 Hz frequency range (mean peak of 8.0 +/- 0.1 Hz). The AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) abolished field population spiking and disrupted 4-12 Hz rhythmic oscillatory activity whereas the NMDA receptor antagonist D-AP5 (50 microM) had no significant effect on either activity component. The glycine receptor antagonist strychnine (4 microM) and the GABA(A) receptor antagonist bicuculline (10 microM) diminished and abolished, respectively, field population spiking and both antagonists reduced the power of 4-12 Hz oscillations. The non-specific gap junction blockers carbenoxolone (100 microM) and octanol (1 mM) attenuated both types of 4-AP-induced activity. By comparison, the neuronal-specific gap junction uncouplers quinine (250 microM) and mefloquine (500 nM) both disrupted 4-12 Hz oscillations but only quinine reduced the frequency of field population spiking. These data demonstrate the existence of 4-AP-sensitive neuronal networks within SG that can generate rhythmic activity, are differentially modulated by excitatory and inhibitory ionotropic neurotransmission and are at least partly reliant on neuronal and/or glial-mediated electrical connectivity. The physiological significance of these putative intrinsic SG networks and the implications in the context of processing of nociceptive inputs are discussed.

Group I mGluRs increase locomotor network excitability in Xenopus tadpoles via presynaptic inhibition of glycinergic neurotransmission.

The group I metabotropic glutamate receptor agonist (S)-3,5-dihyroxyphenylglycine (DHPG) increases the frequency of rhythmic swimming activity in Xenopus tadpoles. This study explores the possibility that group I receptor modulation occurs in part via depression of inhibitory synaptic transmission. Applications of the glycine receptor antagonist strychnine occluded the effects of DHPG, providing preliminary evidence that group I receptors affect motor network output by reducing glycinergic transmission. This evidence was supported further by intracellular and whole-cell patch-clamp recordings from presumed motorneurons. DHPG applications produced two prominent effects: (i) during swimming activity, glycinergic mid-cycle IPSPs were reduced in amplitude; and (ii) during quiescent periods, the frequency of spontaneous miniature IPSPs was also reduced. No change in membrane potential or input resistance following group I receptor activation was detected. The reduction in fast synaptic inhibition provides a plausible explanation for the increased excitability of the locomotor network, although other contributory mechanisms activated in parallel by group I receptors cannot be discounted. Aspects of this work have been published previously in abstract form [R. J. Chapman & K. T. Sillar (2003) SFN Abstracts 277.8].

Modulation of a spinal locomotor network by metabotropic glutamate receptors.

We have explored the potential involvement of the three main classes of metabotropic glutamate receptor in the modulation of a spinal locomotor network using tadpoles of the anuran amphibian Xenopus laevis. Selective activation of group I receptors in Xenopus embryos and young larvae using the general group I agonist DHPG [(S)-3,5-dihyroxyphenylglycine] significantly increased the frequency of swimming and the number of spontaneously occurring swimming episodes, as monitored by extracellular recordings from ventral roots. Group I receptor activation was without significant effect on the duration or amplitude of motor bursts, the duration of swimming episodes, or the head-to-tail delay in the propagation of swimming activity. Activation of either group II or group III receptors, however, following bath applications of the specific agonists APDC [(2R,4R)-aminopyrrolidine-2,4-dicarboxylic acid] and L-AP4 (L-2-amino-4-phosphonobutanoate), respectively, produced a net inhibitory effect on many of the parameters of fictive swimming at both developmental stages, including a reduction in swimming frequency and episode duration, along with a significant reduction in motor burst amplitude and duration in larval animals only. Applications of selective antagonists provide evidence for activation of all three groups during swimming. The group II and III antagonists EGLU (1-ethyl-2-benzimidazolinone) and MAP4 [(S)-2-amino-2-methyl-4-phosphonobutanoate], respectively, increased, while group I antagonists, CPCCOEt and MPEP, decreased swim frequency. Our findings thus provide evidence for the presence and endogenous activation of three classes of metabotropic glutamate receptor which may function as an intrinsic modulatory control system during fictive swimming in Xenopus tadpoles.