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Purpose: To determine long-term outcomes of a cohort of children with germinoma treated with chemotherapy and radiation therapy without primary tumor boost even in the absence of complete response to chemotherapy. Methods: This retrospective study analyzed the outcome of patients with germinoma consecutively diagnosed and treated at a tertiary care center from January 2000 to December 2021. MRIs were reviewed by two radiologists, blinded to patient data. Tumor location at diagnosis, tumor response to chemotherapy and at completion of radiation therapy and site of relapse were assessed. Tumor response was assessed radiologically by determining the tumor size and response on diffusion-weighted imaging, in addition to biochemical, cytological parameters and neurological status. Results: Of 46 pediatric germinoma patients, 29 children (14 male; median age 12.8 years) received no primary tumor boost. Median follow-up was 63 months (range 9-187 months). Twenty-five children had localized disease and tumor location was suprasellar (n= 11), pineal (n= 10), bifocal (n= 3) and basal ganglia (n= 1) while 4 children had metastatic disease at presentation. All patients completed multi-agent chemotherapy followed by either ventricular irradiation (VI) (23.4 Gy) (n = 23), whole brain (WBI) (23.4 Gy) (n = 5) or craniospinal radiation (CSI) (23.4 Gy) (n= 1). Two children, who had localized disease at presentation and received VI after chemotherapy, relapsed 9 months and 32 months after completion of treatment respectively. No patient had a local relapse. Location of relapse was distant, outside (n= 1) and inside (n= 1) the irradiation field. Five-year PFS was 91% and overall survival (OS) was 100%. Conclusions: In this case series, excellent 5-year PFS and OS rates were achieved with chemotherapy followed by radiation therapy of 23.4 Gy delivered without primary tumor boost. No local relapse was observed despite omitting primary tumor boost in patients with localized and metastatic germinoma.

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase ( IDH) –mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1 R132H -driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

Medulloblastoma (MB) comprises a group of heterogeneous paediatric embryonal neoplasms of the hindbrain with strong links to early development of the hindbrain 1–4. Mutations that activate Sonic hedgehog signalling lead to Sonic hedgehog MB in the upper rhombic lip (RL) granule cell lineage 5–8. By contrast, mutations that activate WNT signalling lead to WNT MB in the lower RL 9,10. However, little is known about the more commonly occurring group 4 (G4) MB, which is thought to arise in the unipolar brush cell lineage 3,4. Here we demonstrate that somatic mutations that cause G4 MB converge on the core binding factor alpha (CBFA) complex and mutually exclusive alterations that affect CBFA2T2, CBFA2T3, PRDM6, UTX and OTX2. CBFA2T2 is expressed early in the progenitor cells of the cerebellar RL subventricular zone in Homo sapiens, and G4 MB transcriptionally resembles these progenitors but are stalled in developmental time. Knockdown of OTX2 in model systems relieves this differentiation blockade, which allows MB cells to spontaneously proceed along normal developmental differentiation trajectories. The specific nature of the split human RL, which is destined to generate most of the neurons in the human brain, and its high level of susceptible EOMES +KI67 + unipolar brush cell progenitor cells probably predisposes our species to the development of G4 MB.

Supplementary Figures S1-S4.

Purpose: Cancer recurrence is one of the major setbacks in oncology. Maintaining telomeres is essential for sustaining the limitless replicative potential of such cancers. Because telomerase is thought to be active in all tumor cells and normal stem cells, telomerase inhibition may be nonspecific and have detrimental effects on tissue maintenance and development by affecting normal stem cell self-renewal.Methods: We examined telomerase activity, telomere maintenance, and stem cell maturation in tumor subpopulations from freshly resected gliomas, long-term, primary, neural tumor-initiating cells (TIC) and corresponding normal stem cell lines. We then tested the efficacy of the telomerase inhibitor Imetelstat on propagation and self-renewal capacity of TIC and normal stem cells in vitro and in vivo.Results: Telomerase was undetectable in the majority of tumor cells and specific to the TIC subpopulation that possessed critically short telomeres. In contrast, normal tissue stem cells had longer telomeres and undetectable telomerase activity and were insensitive to telomerase inhibition, which results in proliferation arrest, cell maturation, and DNA damage in neural TIC. Significant survival benefit and late tumor growth arrest of neuroblastoma TIC were observed in a xenograft model (P = 0.02). Furthermore, neural TIC exhibited irreversible loss of self-renewal and stem cell capabilities even after cessation of treatment in vitro and in vivo.Conclusions: TIC exhaustion with telomerase inhibition and lack of telomerase dependency in normal stem cells add new dimensions to the telomere hypothesis and suggest that targeting TIC with telomerase inhibitors may represent a specific and safe therapeutic approach for tumors of neural origin. Clin Cancer Res; 17(1); 111–21. ©2011 AACR.

Supplementary Figure S1; Supplementary Methods; Supplementary Table S1.

CCR Translation for This Article from Neural Tumor-Initiating Cells Have Distinct Telomere Maintenance and Can be Safely Targeted for Telomerase Inhibition

Supplementary Table S2 from BRAF-KIAA1549 Fusion Predicts Better Clinical Outcome in Pediatric Low-Grade Astrocytoma

Purpose: Recent studies have revealed that the majority of pediatric low-grade astrocytomas (PLGA) harbor the BRAF-KIAA1549 (B-K) fusion gene resulting in constitutive activation of the RAS/MAPK pathway. However, the clinical significance of this genetic alteration is yet to be determined. We aimed to test the prognostic role of the B-K fusion in progression of incompletely resected PLGA.Experimental Design: We retrospectively identified 70 consecutive patients with incompletely resected “clinically relevant” PLGA. We added 76 tumors diagnosed at our institution between 1985 and 2010 as controls. We examined BRAF alterations by reverse transcriptase PCR, FISH, and single-nucleotide polymorphism array analysis and correlated that with progression-free survival (PFS).Results: Overall, 60% of tumors were B-K fusion positive. All patients with B-K fused PLGA are still alive. Five-year PFS was 61% ± 8% and 18% ± 8% for fusion positive and negative patients, respectively (P = 0.0004). B-K fusion resulted in similarly significant favorable PFS for patients who received chemotherapy. Multivariate analysis revealed that B-K fusion was the most significant favorable prognostic factor in incompletely resected PLGA and was independent of location, pathology, and age. In vitro, BRAF overexpression resulted in growth arrest associated with DNA damage (γH2AX expression). Five-year PFS was 68% ± 15% and 0% for patients with B-K fused and γH2AX-expressing PLGA versus negative tumors (P = 0.001).Conclusion: These data suggest that B-K fusion confers a less aggressive clinical phenotype on PLGA and may explain their tendency to growth arrest. Combined analysis of B-K fusion and γH2AX expression can determine prognosis and may be a powerful tool to tailor therapy for these patients. Clin Cancer Res; 17(14); 4790–8. ©2011 AACR.

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Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single nucleotide polymorphism rs55705857 (A>G), which confers a 6-fold increased risk of IDH-mutant low-grade glioma (LGG) and is amongst the highest genetic associations with cancer. By fine-mapping the locus, we reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. To functionally test rs55705857, we generated an IDH1R132H-driven LGG mouse model and show that mutating the highly conserved, orthologous mouse rs55705857 locus dramatically accelerated tumor development from 463 to 172 days and increased penetrance from 30% to 75%. Overall, our work generates new LGG models and reveals mechanisms of the heritable predisposition to lethal glioma in ∼40% of LGG-patients.

Low- and middle-income countries sustain the majority of pediatric cancer burden, with significantly poorer survival rates compared to high-income countries. Collaboration between institutions in low- and middle-income countries and high-income countries is one of the ways to improve cancer outcomes.Patient characteristics and effects of a pediatric neuro-oncology twinning program between the Hospital for Sick Children in Toronto, Canada and several hospitals in Karachi, Pakistan over 7 years are described in this article.A total of 460 patients were included in the study. The most common primary central nervous system tumors were low-grade gliomas (26.7%), followed by medulloblastomas (18%), high-grade gliomas (15%), ependymomas (11%), and craniopharyngiomas (11.7%). Changes to the proposed management plans were made in consultation with expert physicians from the Hospital for Sick Children in Toronto, Canada. On average, 24% of the discussed cases required a change in the original management plan over the course of the twinning program. However, a decreasing trend in change in management plans was observed, from 36% during the first 3.5 years to 16% in the last 3 years. This program also led to the launch of a national pediatric neuro-oncology telemedicine program in Pakistan.Multidisciplinary and collaborative efforts by experts from across the world have aided in the correct diagnosis and treatment of children with brain tumors and helped establish local treatment protocols. This experience may be a model for other low- and middle-income countries that are planning on creating similar programs.

Introduction: Moyamoya is a progressive cerebral arteriopathy affecting the arteries in the circle of Willis. Suzuki grade for moyamoya using catheter angiogram has been gold standard for the evaluation of moyamoya both in children and adults. Suzuki grade evaluates the amount of stenosis and collateral formation in the cerebral arteries. Magnetic resonance angiography (MRA) non-luminal imaging can overestimate stenosis and underestimate collaterals. With greater use of MRA for diagnosis and follow-up, it is important to understand MRA staging is truly comparable to catheter angiogram. Methods: Study neurologist and interventional radiologist reviewed both catheter angiograms and MRAs without clinical information. The modified Suzuki stage was used to evaluate the MRA. Median time from MRA to catheter angiogram was calculated. Cohen’s Kappa was used to compare modalities of Suzuki grade based on catheter angiogram to modified Suzuki grade by MRA on the same patients. Results: A total of 29 patients with moyamoya were reviewed. Median time between MRA and catheter angiogram was 2.4 months with interquartile range from 0.6-5.3 months. Unweighted Cohen’s Kappa was 0.34 (p Conclusion: Using Cohen’s kappa to compare two methods of evaluating cerebral artery stenosis in children with moyamoya, we demonstrated that there is only moderate correlation between catheter angiogram Suzuki staging compared to modified Suzuki staging with MRA. In conclusion, we suggest caution in the reliance of MRA for diagnosis and follow-up of children with moyamoya. We recommend consideration catheter angiograms at time of diagnosis and with surgical planning.

Background: Moyamoya is a progressive arteriopathy condition characterized by steno-occlusion of the arteries of the circle of Willis. MRI performed with hypercapnic challenge can image blood-oxygen-level-dependent cerebrovascular reactivity (BOLD-CVR) thereby assessing cerebrovascular reserve. MRI studies of children Methods: Children with moyamoya underwent two BOLD-CVR imaging in the same session under GA (GA-CVR). Differences in CVR estimates and intraclass correlation coefficient (ICC) between repeated scans were examined to determine repeatability across grey and white matter tissue and vascular territories. Bland-Altman plots were used to visulaize the overall variation between the scans. The associations with age, moyamoya types, and stroke presentation were also examined. Qualitative scoring by visual inspection was also conducted by trained neurologists. Results: Thirty-two paired GA-CVR studies (sixty-four scans in total) were analyzed (mean age: 7.07 (2.74 -17.95) years, 13 females). Forty-one percent (41%) were under 7 and 77%, under 10. No significant differences between repeated scans were found for any of the CVR estimates, when summarized by tissue and vascular territory. Of the paired studies, repeatability (ICC) for the whole-brain CVR estimates was excellent (≥0.74) in 14 (43.8%), good (≤0.59, >0.74) in 7 (21.9%), fair (≤0.41, >0.59) in 6 (18.8%) and poor ( Conclusion: Our study support the clinical use of GA-CVR across all ages and disease conditions. The GA-CVR provides a feasible, repeatable, and reliably interpretable tool for the assessment of cerebrovascular reserve of very young and behaviourally challenged children with moyamoya.

The rate limiting enzyme of the polyamine pathway, SAT1, is significantly upregulated in non-cycling GSCs. However, higher SAT1 expression predicts poorer survival in GBM suggesting a role in quiescence and recurrence post-treatment. Treating GSCs with N1N11-Diethylnorspermine (DENSpm) recapitulates SAT1 upregulation and triggers a chemoprotective, long-term reversible growth arrest, while not affecting expression of stem, differentiation, nor senescence-associated markers. Remarkably, DENSpm strongly induces double-stranded RNA in GSCs. Interestingly, S-Adenosyl methionine (SAM) is an essential aminopropyl donor for polyamine biosynthesis. We found a significant correlation between DNA methylation, SAM, and polyamines across GSCs suggesting that SAM depletion may cause epigenetic and post-transcriptional alterations leading to re-activation of endogenous retroviral elements (ERVs) and viral mimicry-driven quiescence. However, while we confirmed DENSpm-induced SAM depletion, we did not detect DNA, histone, nor RNA demethylation. Noteworthy, ERVs are the most accessible transposable elements in GSCs indicative of a poised state. Furthermore, the correlation between expression of ERVs and interferon stimulated genes reveals a spectrum of viral mimicry amongst GSCs. Using a larger cohort of GSCs and hfNSCs, we identified three DENSpm response subgroups. A resistant subgroup comprised of cells able to proliferate through treatment, a reversible one whereby cells become quiescent and readily re-enter cell cycle post-treatment, and an irreversible consisting of cells that enter a deep quiescent state which they cannot escape. Importantly, we found that DENSpm response correlates with the cell's basal inflammatory state and the activity of viral defense pathways. These results indicate that a viral mimicry-driven quiescence may be specific to slower-growing neurodevelopmental GSCs, while more proliferative, inflammatory-poised GSCs are impervious to these additional cues. We propose that polyamine metabolism, glioma stem cell hierarchy and tumour inflammatory context play a central role in regulating quiescence and have important clinical implications in the initial response to treatment, aggressiveness, and ultimately recurrence of GBM.

Glioblastoma is a highly malignant tumour driven by a subset of self-renewing cells termed glioblastoma stem cells. This self-renewal phenotype is largely epigenetically driven, however, the exact epigenetic mechanisms underpinning it are poorly understood. We found that the histone variant macroH2A2 is repressed in a subset of glioblastoma tumours, and that this repression is associated with poorer survival. We interrogate the function of macroH2A2 using in vitro and in vivo patient-derived models. We show that macroH2A2 antagonizes self-renewal and expression of NPC and OPC-like markers, and rewires accessible chromatin by maintaining accessibility at a subset of enhancer elements. We identify small molecules that can upregulate macroH2A2 expression, and find that treatment with a small molecule reduces self-renewal, induces interferon sensitive genes, and a viral mimicry response, effects which are abolished by macroH2A2 knockdown. In summary, we identify macroH2A2 as a repressor of self-renewal in glioblastoma and suggest it may be a novel biomarker and potential marker of therapeutic susceptibility.

OBJECTIVE Gliomas in adolescents and young adults (AYA) are commonly treated with a standard chemo-radiation approach. Molecular alterations have not been comprehensively described to date. METHODS We compiled a multi-institutional cohort of patients diagnosed with glioma between 15-39.9 years over 20 years. Molecular analysis, therapeutic data and outcome was collected. For specific alterations, analysis included patients aged 0-39.9 years. RESULTS A total of 1900 patients with 876 AYA gliomas were included. Ongoing analysis reveals genetic alterations in 95% of available tumours. IDH p.R132H was found in 49% of tumours, while non-canonical IDH mutations were found in 7%. Paediatric-type mutations were found in 33% of AYA tumours with IDH-WT GBM accounting for the remaining 11%. The most common paediatric alterations in AYAs included BRAF p.V600E (11%) and FGFR alterations (7%) while BRAF fusions, H3 p.K27M and H3.3 p.G34R were rarely observed (4%, 4% and 1% respectively). BRAF fused tumours with non-canonical binding partners were enriched in AYAs. Analysis of BRAF-V600E gliomas between ages 0-40 revealed increased tendency for malignant tumours in patients > 20 years suggesting malignant transformation possibly due to higher rate of secondary hits. This resulted in worse overall-survival for AYA patients with BRAF-V600E glioma when compared to children under 20 years (p=0.0032). Ten-year OS of 100%, 90% and 95% was seen for BRAF fused, BRAF-V600E and FGFR-altered AYA low grade glioma respectively, compared to 14% and 25% for BRAF-V600E and FGFR-altered high grade glioma. In contrast, continuous decline was observed in the IDH-mutant gliomas with 10-year OS of 50% which declined to 29% at 15 years. CONCLUSIONS Gliomas in AYA often have non-canonical alterations that may evade standard molecular analysis. They are enriched for paediatric-type alterations with distinct molecularly-based outcomes. These tumours may respond to targeted inhibitors and would benefit from comprehensive diagnostic and therapeutic approaches.

Glioblastoma (GBM) is an incurable disease of adults and children and the deadliest form of central nervous system (CNS) malignancy. Despite major advances in our understanding of GBM biology in recent years, the prognosis for patients who develop this disease has not improved. If we hope to find new treatments for GBM that are safe and effective, we desperately need to reform our thinking. The brain’s chemical milieu is rich with neurotransmitters, and our lab’s screen of 680 neuroactive compounds on patient-derived glioblastoma stem cells (GSCs) in vitro strongly implicates neurotransmitter pathways as critical regulators of the GSC niche. More specifically, disrupting dopamine signaling by inhibiting its receptor D4 on GSCs causes substantial GSC apoptosis in vitro and attenuates GBM growth in vivo (Dolma et al., Cancer Cell, 2016). Dopamine (DA) is a catecholamine neurotransmitter that is essential for reward learning and movement and has numerous roles in cognition. Consequentially, dysregulation of DA signaling is associated with a diversity of brain diseases ranging from drug addiction to schizophrenia to Parkinson’s. Our research aims to determine how DA signaling affects normal neural stem cell (NSC) and tumorigenic GSC populations, as we hypothesize that GSCs arising/residing in DA projection zones exploit dopaminergic (DAergic) activity for GBM growth. Toward these aims, we have developed in vivo model systems and harnessed them to study NSC and GSC niches in the context of either controlled activation or depletion of DAergic neurons. These manipulations of the brain's DAergic neurons are achieved using optogenetic stimulation, genetic depletion, and neurotoxin-mediated ablation in mice. Ultimately, unraveling the dopaminergic influence on GBM may contribute to a redeployment of existing treatments—that modulate DA signaling and are already approved to treat CNS disorders—to patients with this deadly brain cancer.

OBJECTIVE The objective of this study was to describe the management of hydrocephalus in a cohort of pediatric patients with germinoma. METHODS The authors conducted a retrospective chart review of patients with germinoma and symptomatic hydrocephalus treated at the Hospital for Sick Children between 2002 and 2020. Descriptive data included tumor location, CSF diversion procedure (external ventricular drain [EVD], endoscopic third ventriculostomy [ETV], ventriculoperitoneal [VP] shunt) and outcomes. The frontooccipital horn ratio (FOR) method was used to determine the presence of ventriculomegaly. RESULTS Of 39 patients with germinoma, 22 (73% male) had symptomatic hydrocephalus at diagnosis (11 pineal, 4 suprasellar, and 7 bifocal). Management of hydrocephalus included EVD (n = 5, 22.7%), ETV (n = 5, 22.7%), and combination ETV and EVD (n = 7, 31.8%), whereas 5 patients (22.7%) did not undergo surgical intervention. The median FOR at diagnosis was 0.42 (range 0.38–0.58), which correlated with moderate to severe ventriculomegaly. Carboplatin and etoposide–based chemotherapy induced fast tumor shrinkage, avoiding CSF diversion (n = 5) and resolving hydrocephalus with a transient EVD (n = 5). The median duration until EVD removal was 7 days (range 2–10 days). Two of 12 patients with EVD ultimately required a VP shunt. Kaplan-Meier overall survival was 100% and progression-free survival was 96.4% at a median follow-up of 10.4 years. CONCLUSIONS Timely initiation of chemotherapy is imperative to rapidly reduce tumor bulk in children with germinoma and limits the need for VP shunt insertions. In children in whom CSF diversion is required, hydrocephalus may be successfully managed with a temporary EVD ± ETV.

Background: Hypermutant gliomas in children are caused by inherited or therapy related replication repair deficiency (RRD), the latter typically following treatment with the alkylating agent temozolomide (TMZ). Recently, immune checkpoint inhibitors (ICIs) revealed clinical responses and prolonged survival in 30% of pediatric-inherited hypermutant gliomas but none of the treatment-related adult hypermutant gliomas. We hypothesized that high-throughput screening of hypermutant and RRD patient derived glioma cell lines from both glioma types (PDGCL) would reveal specific molecular vulnerabilities immediately translatable as targeted therapies to be used in combination with ICIs. Methods: We screened two pediatric hypermutant and RRD PDGCLs (1806, 1260); one pediatric non-hypermutant non-RRD PDGCL (477); an adult non-hypermutant non-RRD PDGCL (189EW); matched recurrent post-TMZ treatment hypermutant RRD PDGCL (248z, 248xy) from patient 189 against a library of 3336 bioactive compounds, including 1500 FDA approved drugs supplemented with 16 additional compounds (BET, PLK1, EZH2, PARP1 inhibitors) at 100nM.Each plate screened contained negative (0.2% DMSO) and positive controls (50nM bortezomib). Cell viability was calculated at 96hr by ATP measurement. Raw values from negative and positive control wells were aggregated and used to calculate Z’-factors for each assay plate, as a measure of assay performance and data quality, with a Z’-factor >0.5 representing high quality data. Only drugs scoring Normalized Percentage of Inhibition (NPI) >50% and z-score>3 were considered potential hits for follow-up evaluation, focusing on those enriched in the pediatric and adult hypermutant PDGCLs. Results: We identified 45 drugs active in all three of the pediatric PDGCLs and 23 common hits in the adult cell lines. Broad target classes were identified such as microtubule associated inhibitors, heat shock protein inhibitors for PDGCLs and proteasome inhibitors for adult cell lines. Unique targets were also identified. Importantly, the inherited and treatment related mismatch repair deficient glioma cells (MMR1260,248z, 248xy) and the hypermutant pediatric cell line (1806) exhibited significant response to PI3K/mTOR inhibitors. Ultrahypermutant RRD cell line and the treatment-related hypermutant cell lines (1806 248z 248xy ) displayed sensitivity to topoisomerase I inhibitors. Preliminary in vitro data confirm sensitivity to topotecan of hypermutant pediatric and adult cell lines. Conclusion: We have identified both mechanism specific and common intrinsic vulnerabilities in hypermutant PDGCLs that can be tested as adjuvant therapies for ICI. Functional validation of the lead candidates is ongoing and will be reported. Citation Format: Laura Scolaro, Nuno Miguel Nunes, Michelle Kushida, David C. Schultz, Sara Cherry, Kanupriya Whig, Peter Dirks, Uri Tabori, John M. Maris. Identification of intrinsic molecular vulnerabilities in inherited and treatment-related hypermutant patient-derived glioma cell line models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB188.

OBJECTIVE: Gliomas in adolescents and young adults (AYA) are commonly treated with a standard chemo-radiation approach based on data from adults. The clinical impact of paediatric-type alterations in these tumours is unknown. METHODS: We compiled a multi-institutional cohort of patients diagnosed with glioma between 15-39.9 years over 20 years. Complete molecular analysis, therapeutic data and outcome was collected. For specific alterations, analysis included patients aged 0-39.9 years. RESULTS: A total of 1900 patients with 876 AYA gliomas were included. Ongoing analysis reveals genetic alterations in 95% of available tumours. IDH-mutant tumours account for only 53%, while paediatric-type mutations were found in 35% of AYA tumours with IDH-WT GBM accounting for the remaining 12%. The most common paediatric alterations in AYAs included BRAF p.V600E (11%) and FGFR alterations (6%) while BRAF fusions, H3 p.K27M and H3.3 p.G34R were rarely observed (4%, 4% and 1% respectively). BRAF fused tumours with non-canonical binding partners were enriched in AYAs. Analysis of BRAF-V600E gliomas between ages 0-40 revealed increased tendency for malignant tumours in patients >20 years suggesting malignant transformation possibly due to higher rate of secondary hits including TP53, CDKN2A and ATRX mutations. This resulted in worse overall-survival for AYA patients with BRAF-V600E glioma when compared to children under 20 years (p=0.0032). Ten-year OS of 100%, 90% and 95% was seen for BRAF fused, BRAF-V600E and FGFR-altered AYA low grade glioma respectively, compared to 14% and 25% for BRAF-V600E and FGFR-altered high grade glioma. In contrast, continuous decline was observed in the IDH-mutant gliomas with 10-year OS of 50% which declined to 29% at 15 years. CONCLUSIONS: Gliomas in AYA are enriched for paediatric-type alterations with distinct molecularly-based outcomes. As these tumours carry different outcomes than childhood glioma and may respond to targeted inhibitors, AYA gliomas would benefit from comprehensive diagnostic and therapeutic approaches.

BACKGROUND: Response-based radiation therapy has been the approach for germinoma after chemotherapy. However, the presence of residual lesions at the end of chemotherapy did not demonstrate a negative impact on progression-free survival (PFS). Similarly, resection of residual tumors after chemotherapy did not show a survival benefit. AIM: Our study objective was to determine long-term outcomes of a cohort who received chemotherapy and radiation therapy without primary tumor boost even in the absence of complete response to chemotherapy. METHOD: This retrospective study analyzed the outcome of germinoma patients diagnosed and treated at a tertiary care center from January 2006 to December 2021. RESULTS: Twenty-nine children (14 male; median age 12.8 years) were identified. Median follow-up was 63 months (range 9-187 months). Twenty children had localized disease and tumor location was suprasellar (n= 9), pineal (n= 10), and bifocal (n= 1) while 9 children had metastatic disease at presentation. All patients completed multi-agent chemotherapy followed by either whole ventricular (WVI) (23.4 Gy) (n = 23), whole brain (WBI) (23.4 Gy) (n = 5) or craniospinal radiation (CSI) (23.4 Gy) (n= 1). Two children, who had localized disease at presentation and received WVI after chemotherapy, relapsed 9 months and 32 months after completion of treatment respectively. None of them had local relapses. Location of relapse was distant, outside (n= 1) and inside (n= 1) the radiation field. Five-year PFS was 93% and overall survival (OS) was 100%. CONCLUSION: In this limited experience, excellent 5-year PFS and OS rates were achieved with chemotherapy followed by radiation therapy delivered without primary tumor boost. This study also demonstrated the absence of local relapse despite omitting primary tumor boost in patients with localized and metastatic germinoma.

Cancers arising from germline DNA mismatch repair deficiency or polymerase proofreading deficiency (MMRD and PPD) in children harbour the highest mutational and microsatellite insertion–deletion (MS-indel) burden in humans. MMRD and PPD cancers are commonly lethal due to the inherent resistance to chemo-irradiation. Although immune checkpoint inhibitors (ICIs) have failed to benefit children in previous studies, we hypothesized that hypermutation caused by MMRD and PPD will improve outcomes following ICI treatment in these patients. Using an international consortium registry study, we report on the ICI treatment of 45 progressive or recurrent tumors from 38 patients. Durable objective responses were observed in most patients, culminating in a 3 year survival of 41.4%. High mutation burden predicted response for ultra-hypermutant cancers (>100 mutations per Mb) enriched for combined MMRD + PPD, while MS-indels predicted response in MMRD tumors with lower mutation burden (10–100 mutations per Mb). Furthermore, both mechanisms were associated with increased immune infiltration even in ‘immunologically cold’ tumors such as gliomas, contributing to the favorable response. Pseudo-progression (flare) was common and was associated with immune activation in the tumor microenvironment and systemically. Furthermore, patients with flare who continued ICI treatment achieved durable responses. This study demonstrates improved survival for patients with tumors not previously known to respond to ICI treatment, including central nervous system and synchronous cancers, and identifies the dual roles of mutation burden and MS-indels in predicting sustained response to immunotherapy., Hypermutation and microsatellite burden determine responses and long-term survival following PD-1 blockade in children and young adults with refractory cancers resulting from germline DNA replication repair deficiency.

Het beheersen van aardappelopslag in landbouwgewassen is van groot belang voor de bodemgezondheid en verspreiding van aardappelziekten. De relatief hoge kosten voor handmatige bestrijding maken inzet van high tech oplossingen interessant. In dit project is een Deep Learning herkenningsalgoritme voor aardappelplanten in een tweetal akkerbouwgewassen ontwikkeld. Tevens is het algoritme getest met een prototype van een spotsprayer, een apparaat dat kleine hoeveelheden middel precies op gedetecteerde planten kan spuiten. Dit rapport beschrijft de behaalde resultaten en de verbeterstappen die nodig zijn om de gewenste kwaliteit in de praktijk te realiseren.

This paper studies the effect dwelling mixed-traffic arterial Bus Rapid Transit (BRT-“Lite”) buses have on general traffic conditions and intersection capacity. Queue length and flow rate d...

Background Intracranial pyogenic complications of sinusitis in children are relatively uncommon but can lead to serious sequelae. The objective of this study was to characterize the clinical, epidemiologic and microbiologic characteristics of children with such complications over a 20-year period (2000- 2019). Methods Single-center retrospective chart review. Cases were identified based on International Classification of Diseases (ICD)-10 diagnostic codes (intracranial abscess or granuloma, extradural and subdural abscess, Pott’s puffy tumor, acute or chronic sinusitis) and by reviewing all microbiological samples of intracranial pus, tissue or fluid. Results 108 cases of clinically and/or radiologically diagnosed sinusitis were included after review of 1591 charts. The majority were adolescents (median age 12, IQR 9-14); 72 were male (67%). The most common presenting symptoms were fever (84%), headache (87%) and symptoms of upper respiratory tract infection (57%). Median symptom duration was 10 days (IQR 5-21) and 55 cases (51%) received oral antibiotics prior to admission. The most frequent complications were epidural empyema (n=50, 46%), subdural empyema (n=46, 43%) and Pott’s puffy tumor (n= 31, 29%). 50% (n=54) underwent neurosurgery, of which 20% (n=11) required multiple craniectomies. 38% (n=41) underwent otolaryngological surgery. Microbiological data from sterile specimens demonstrated single organisms in 36 cases (59%) and polymicrobial growth in 25 cases (41%). The most frequently identified pathogens were Streptococcus anginosus (n=40, 66%) followed by Fusobacterium species (n=10, 16%) and Prevotella species (n=10,16%). Most cases were treated with combination antibiotic therapy (n=68, 63%) and 14% (n=15) with a carbapenem. The median duration of intravenous antibiotic therapy was 51 days (IQR 42-80). One child died and 23% (n=25) suffered neurological sequelae (median follow-up 344 days). 48 cases (44%) occurred between 2014-2019. Conclusion Intracranial complications of sinusitis continue to cause significant morbidity in children. The predominant causative pathogen was Streptococcus anginosus. Polymicrobial infections are common, confirming the need for prolonged broad-spectrum antibiotic treatment. Disclosures Sharon Cushing, MD, MSc, FRCSC, Cochlear Corporation (Research Grant or Support)Cochlear Corporation (Speaker’s Bureau)Interacoustics (Speaker’s Bureau)Plural publishing (Other Financial or Material Support, Royalties: editor: manual of pediatric balance disorders)

DNA replication-associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10(-13)). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in

Norrin is an atypical WNT ligand that binds the Frizzled-4 (FZD4) and Low-density lipoprotein receptor-related protein (LRP5/6) receptor complex to activate canonical WNT/ β-catenin signaling. Norrin/FZD4 signaling is involved in the regulation of vasculature in several tissues including the retina and for blood-brain barrier function. The role of Norrin in cancer is not very well characterized. Here, we show that NDP, the gene encoding for Norrin, is expressed in a wide range of cancer types, with a particular enrichment in glioblastoma (GBM) and lower grade glioma (LGG). Kaplan-Meier survival analysis of publicly available datasets revealed a significant correlation between NDP expression and survival in GBM, LGG and neuroblastoma. To investigate the function of NDP in GBM, we performed a set of NDP and FZD4 gain and loss of function experiments in patient-derived GBM stem cell (GNS) lines. Recently Achaete-scute homolog 1 (ASCL1) expression was shown to stratify GNS lines into two cohorts with different tumorigenic, proliferation and differentiation dynamics. Surprisingly, we found that NDP manipulation resulted in opposite effects in ASCL1hi versus ASCL1lo lines. NDP inhibited proliferation and sphere formation in ASCL1lo lines through the WNT/ β-catenin pathway, while it stimulated proliferation and sphere formation in ASCL1hi lines independently of the WNT/β-catenin pathway. The NDP/FZD4 effects on growth in both GNS subtypes were recapitulated in xenografts, confirming effects of this pathway on tumor progression in vivo. To gain insight into the cellular and molecular effects of NDP/FZD4 on GNS growth we performed immunocytochemistry (ICC) and RNA-Seq analyses in GNS cells with NDP knockdown. In parallel with our in vitro and in vivo observations, we found that NDP/FZD4 signaling affects GNS proliferation index and cell cycle kinetics and the expression of cell cycle regulatory genes in both GNS subtypes, but in opposite ways. RNA-Seq analysis identified cell cycle regulatory genes and gene sets that are commonly affected by NDP in both GNS types. Interestingly, the analysis also identified several genes and genesets unique to each GNS type, consistent with the divergence of NDP functions between GNS subtypes. Collectively, our results uncover novel functions of NDP in regulating GBM stem cell progression, and that NDP functions in GBM stem cells stratify with ASCL1 expression. Additionally, our results highlight the significance of precision medicine in targeting tumor subtypes based on the molecular signature. Citation Format: Ahmed A. Elsehemy, Hayden Selvadurai, Arturo Ortin-Martinez, Nenad Pokrajac, Yasin Mamatjan, Katherine Rowland, Kenneth Aldape, Peter Dirks, Valerie Wallace. Novel biological roles of the atypical WNT ligand, Norrin, on glioblastoma stem cells segregate with ASCL1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4671.

Background: Moyamoya disease is a progressive steno-occlusive arteriopathy that causes recurrent ischaemic events and neurological decline. Cerebrovascular reactivity (CVR) is an indicator of tissue level perfusion impairment and stroke risk. Quantitative BOLD MRI using carbon dioxide as a vasoactive stimulus has been validated in adults and region of CVR abnormality shown to be concordant with angiographic region of abnormality. However the evidence in paediatric literature remains scarce and mainly refers to the use of targeted-controlled delivery of CO2 which has limited utility in the paediatric population. Objective: To examine whether hypercapnic challenge BOLD CVR using endogenous CO2 in the awake (breath-hold [BH]) and sleep (general anaesthetic [GA]) state in children is reliable and repeatable. We also sought to explore whether regional abnormalities of CVR using these techniques were concordant with angiographic regions of abnormality. Method: Consecutive children with angiographic confirmation of MM had BH or GA CVR studies. All repeat studies - conducted on the same day in the same MRI session - were assessed for reliability and repeatability of qualitative measures of CVR. Results: Thirty seven children (16 male; median age MM diagnosis 8.61 years, range 0.6 - 16.7; median age at CVR 10.7 years, range 1.08-17.7) had CVR studies. Children who had a CVR study under GA were significantly younger at diagnosis of MM (mean age 7.4 years, range .67-16.58) compared to those studied using BH (mean age 10.47 years, range .83-15.58). CVR region of abnormality was concordant with region of angiographic abnormality. Twenty nine had repeat studies (14 GA, 15 BH). Intraclass correlation was fair (0.783, 95% confidence interval .534-.899) to excellent (.910, 95% confidence interval .577-.908) and agreement between repeat measures good. Conclusion: Qualitative measures of CVR using general anaesthetic and breath-hold techniques are reliable, repeatable and interpretable for use in clinical practice. However standardization of protocols would allow more reliable application of these tools for assessment of ischaemic risk in childhood cerebrovascular disease.

Objective During seizures, intracranial EEG electrodes can record ictal muscle movements. Our purpose was to differentiate the high-frequency oscillations (HFOs) of extracranial muscle contractions from those of intracranial epileptogenic discharges. Methods Using intracranial video-EEG (IVEEG), we recorded seizures in a 17-year-old boy with left mesial–temporal lobe epilepsy. We used multiple band frequency analysis (MBFA) to differentiate extracranial HFOs of craniofacial muscle activities from intracranial HFOs recorded ictally and interictally. Results During 11 seizures, IVEEG showed low-amplitude fast waves (∼60 Hz) starting at the left mesial–temporal electrodes. Ictal facial grimacing projected low-amplitude (∼20 μV) fast waves (∼160 Hz) on inferior lateral–temporal electrodes. Interictal chewing projected medium-amplitude (∼100 μV) fast waves (∼140 Hz) correlating to mouth movements. MBFA topographic power spectrograms revealed a sustained, consistent ictal fast-frequency band from electrodes in the seizure-onset zone and randomly scattered HFOs without a specific frequency band from ictal and interictal extracranial muscle contractions. Conclusions MBFA power spectrograms differentiated randomly scattered muscle HFOs without a specific frequency band at electrodes close to temporal muscles from ictal epileptic HFOs with a sustained, fast-frequency band in the seizure-onset zone. Significance The pattern and distribution of frequency power spectrograms of extracranial HFOs differ from those of intracranial HFOs.

The determinants of outcomes for adult survivors of pediatric low-grade glioma (PLGG) are largely unknown.This study collected population-based follow-up information for all PLGG patients diagnosed in Ontario, Canada from 1985 to 2012 (n = 1202) and determined factors affecting survival. The impact of upfront radiation treatment on overall survival (OS) was determined for a cohort of Ontario patients and an independent reference cohort from the Surveillance, Epidemiology, and End Results database.At a median follow-up of 12.73 years (range, 0.02-33 years), only 93 deaths (7.7%) were recorded, and the 20-year OS rate was 90.1% ± 1.1%. Children with neurofibromatosis type 1 had excellent survival and no tumor-related deaths during adulthood. Adverse risk factors included pleomorphic xanthoastrocytoma (P .001) and a thalamic location (P .001). For patients with unresectable tumors surviving more than 5 years after the diagnosis, upfront radiotherapy was associated with an approximately 3-fold increased risk of overall late deaths (hazard ratio [HR], 3.3; 95% confidence interval [CI], 1.6-6.6; P = .001) and an approximately 4-fold increased risk of tumor-related deaths (HR, 4.4; 95% CI, 1.3-14.6; P = .013). In a multivariate analysis, radiotherapy was the most significant factor associated with late all-cause deaths (HR, 3.0; 95% CI, 1.3-7.0; P = .012) and tumor-related deaths (HR, 4.4; 95% CI, 1.3-14.6; P = 0.014). A similar association between radiotherapy and late deaths was observed in the independent reference cohort (P .001). In contrast to early deaths, late mortality was associated not with PLGG progression but rather with tumor transformation and non-oncological causes.The course of PLGG is associated with excellent long-term survival, but this is hampered by increased delayed mortality in patients receiving upfront radiotherapy. These observations should be considered when treatment options are being weighed for these patients.