bluuebunny/crossref_metadata_2025 huggingface dataset:
LGG-02. Cardiac toxicity in patients receiving single-agent MEK inhibition
Authors: Cantor Evan , Shatara Margaret , Meyers Ashley , Ogle Andrea , McHugh Michele , Reiners Stephanie , Cluster Andrew , Abdelbaki Mohamed S , Navalkele Pournima , Brossier Nicole M
Published: June 2022

BACKGROUND: MEK inhibitor therapy is increasingly being utilized for the treatment of pediatric tumors, including low-grade glioma, plexiform neurofibroma and Langerhans cell histiocytosis. These drugs are well-tolerated but do have risk of toxicity, including cardiac toxicity. The purpose of this study is to better characterize MEK inhibitor-induced cardiac toxicity in pediatric patients. METHODS: Retrospective review of all patients who underwent MEK inhibitor mono-therapy for at least 3 months, 2015- 2021, age 25 years or less, at St. Louis Children’s hospital and Cardinal Glennon Children's hospital. RESULTS: We evaluated 31 patients, 19 (61%) with brain tumors and 12 (39%) without. Of the thirty-one, fifteen (48%) had NF1, 1 had Tuberous sclerosis. Cardiac toxicity consisted of asymptomatic sinus tachycardia, bradycardia, or decreased ejection fraction (EF). Thirteen patients (42%) experienced an asymptomatic decrease in left-ventricular ejection fraction (EF), Grade I-III. Time on therapy before decreased EF was 5 days to 21 months, median 2.8 months. Decreased EF developed in 5 of 13 patients receiving selumetinib and 8 of 18 receiving trametinib. Of the patients who developed decreased EF, 11 (85%) had brain tumors, 6 (46%) had NF1, and 89% had received prior systemic therapy. Out of the patients who had received no prior systemic therapy (6), 2 (33%) had decreased EF, while 11/25 (44%) of those who had received prior systemic therapy did. Drug was held temporarily for 6 patients, with dose limiting toxicity for 5 patients. Drug was discontinued for 1 patient after EF continued to decline despite dose reduction. Patients showed improvement in EF as early as 2 weeks after holding therapy. CONCLUSIONS: Cardiac toxicity in our patients was limited to asymptomatic reduction in ejection fraction, sinus bradycardia and tachycardia, reinforcing the need for appropriate monitoring via echocardiography. Prior systemic therapy was associated with decreased EF.
LGG-06. Selumetinib in pediatric patients with non-neurofibromatosis type 1-associated, non-optic pathway (OPG) and non-pilocytic recurrent/progressive low-grade glioma harboring BRAFV600E mutation or BRAF-KIAA1549 fusion: a multicenter prospective Pediatric Brain Tumor Consortium (PBTC) Phase 2 trial
Authors: Fangusaro Jason , Onar-Thomas Arzu , Poussaint Tina Young , Lensing Shelly , Wu Shengjie , Ligon Azra H , Lindeman Neal , Stewart Clinton F , Jones David T W , Pfister Stefan M , Smiley Natasha Pillay , Leach James , Packer Roger , Vezina Gilbert , Lenzen Alicia , Jaju Alok , Goldman Stewart , Doyle Laurence Austin , Smith Malcolm , Fouladi Maryam , Dunkel Ira
Published: June 2022

BACKGROUND: A greater understanding of the Ras-MAP kinase pathway in pediatric low-grade glioma (LGG) paired with the availability of selective inhibitors has enhanced the ability to target this pathway with therapeutic intent. METHODS: The PBTC conducted a multi-institutional phase II study (NCT01089101) evaluating selumetinib (AZD6244, ARRY-142886), a MEK I/II inhibitor, in children with recurrent/progressive LGG assigned to 6 strata and treated at a dose of 25 mg/m2/dose PO BID for up to two years. Here we present stratum 5 which enrolled children without NF1, non-OPG and non-pilocytic LGG harboring either a BRAFV600E mutation or BRAF-KIAA1549 fusion. RESULTS: Twenty-four of 25 children enrolled were eligible; 23 were evaluable for the primary radiologic response endpoint. Enrollment stopped early due to slow accrual and initiation of COG ACNS1931. The most common histologies were ganglioglioma (42%) and astrocytoma NOS (33%). Thirteen tumors (54%) had BRAF-KIAA1549 fusion; 11 (46%) had the BRAFV600E mutation. Five of 23 (22%) evaluable patients achieved a centrally confirmed partial response (PR), 12 (52%) had stable disease and 6 (26%) had progression with a 2-year progression-free survival of 75 + 9%. Four of 11 (36%) patients with a BRAFV600E mutation and 1/12 (8%) with a BRAF-KIAA1549 fusion achieved a PR. The 2-year PFS did not significantly differ between tumors with BRAFV600E mutation (82 + 12%) versus BRAF-KIAA1549 fusion (68 + 13%) (n=24, p=0.548). No patient remains on therapy. The most common attributable toxicities were grade 1/2 ALT/AST elevation, dry skin and leukopenia. Rare grade 3/4 toxicities included elevated CPK, rash, paronychia, fever, weight gain and sinus tachycardia. CONCLUSIONS: Despite lower than planned accrual, selumetinib met the design threshold for success in treating children with recurrent/progressive non-pilocytic, non-OPG LGG without NF1 that harbored the common BRAF aberrations. Ongoing phase 3 prospective studies will better determine the role of this agent in this population.
LGG-17. Preventing recurrence: targeting molecular mechanisms driving tumor growth rebound after MAPKi withdrawal in pediatric low-grade glioma
Authors: Kocher Daniela , Selt Florian , Valinciute Gintvile , Zaman Julia , Vonhören David , Pusch Stefan , Guiho Romain , Martinez-Barbera Juan Pedro , von Deimling Andreas , Pfister Stefan M , Jones David T W , Brummer Tilman , Witt Olaf , Milde Till , Sigaud Romain
Published: June 2022

Pediatric low-grade gliomas, a diverse group of WHO grade 1 and 2 glial or glioneural tumors, comprise the most common category of primary brain tumors in children. The majority of these tumors are driven by alterations in the MAPK pathway, making them in principle susceptible to MAPKi therapy. While patients often benefit from MAPKi during treatment, tumor rebound may occur once treatment is stopped, constituting a significant clinical challenge. BT-40, patient-derived cells with molecular features of pleomorphic xanthoastrocytoma (BRAFV600E, CDKN2Adel), were used to model the rebound growth in vitro, based on viable cell counts in response to treatment and withdrawal of the clinically relevant BRAFV600E specific inhibitor dabrafenib. Standard-of-care chemotherapy (vincristine and carboplatin) was used as a reference. MAPK pathway reactivation upon withdrawal was assessed by WB and qPCR analysis. Based on the observed cell-regrowth and MAPK-reactivation pattern, key-timepoints during withdrawal were identified, which are currently being further analyzed through RNAseq and phospho-/proteomics. BT-40 cells started to proliferate again two days after dabrafenib withdrawal, and earliest five days after chemotherapy withdrawal. MAPK pathway activity, based on Mek and Erk phosphorylation, reached baseline levels three hours after dabrafenib withdrawal, this was associated with 2.5-fold increased c-Fos gene expression two hours after withdrawal. The earlier cell regrowth after dabrafenib withdrawal compared to chemotherapy withdrawal matches clinical observations, making the model suitable to study the rebound. The observed MAPK overactivation suggests the growth rebound might not only be caused by a fast reactivation of the pathway but also by other mechanisms, e.g. accumulation of upstream activators due to loss of negative feedback or parallel pathways. To investigate this, key-timepoints during treatment withdrawal will be analyzed using a multi-omics approach. Based on these findings, possible rebound-driving mechanisms will be identified and further validated using BT-40 and additional PXA models in vitro and in vivo.
LGG-55. Autophagy sensitizes CNS tumors to targeted therapy by lowering their apoptotic threshold
Authors: Crespo Michele , Zahedi Shadi , Morin Andrew , Wodetzki Darya , Caino Cecilia , Levy Jean Mulcahy
Published: June 2022

Autophagy inhibition improves the effectiveness and overcomes RAF pathway inhibition (RAFi) resistance across multiple CNS tumors and molecularly distinct resistance mechanisms. Mechanistic links between autophagy and apoptotic cell death may explain this ability to improve RAFi response and reverse resistance. RAFi sensitive (MAF 794, AM38) and resistant (MAF 794R, MAF 905-3, AM38R, B76) BRAFV600E CNS tumor cell lines were analyzed at baseline, following RAFi (vemurafenib), autophagy inhibition (chloroquine or shRNAs), and combination therapy. Growth assays and caspase activation were monitored by Incucyte Zoom. qRT-PCR evaluated key pro-apoptotic BH3-only members of the BCL-2 family. Broad BH-3 profiling was completed using the Letai JC-1 Plate-Based protocol. Western blot analysis assessed protein levels. Combination pharmacologic treatment caused alterations in key pro-apoptotic BH3-only proteins including an increase in BNIP3L and PUMA. Genetically inhibiting autophagy with shRNAs for ATG5 and ATG7 (proteins required for formation of the autophagosome) produced similar results with increases in both protein and mRNA levels of BNIP3L and PUMA following RAFi treatment. This suggested autophagy-mediated regulation of BH3 proteins functions to determine cellular apoptotic threshold. Caspase activation demonstrated increased effectiveness of combined RAFi and autophagy inhibition overcoming the apoptotic threshold compared to single drug treatment. BH3 profiling demonstrated a dependence on BCL-2 to inhibit apoptosis. BH3 mimetics competitively bind to pro-survival BCL-2 family members, blocking their protective effects and pushing tumor cells towards apoptosis. Autophagy inhibition can also improve treatment response by overcoming the apoptotic threshold in RAFi resistant cells and magnifying the apoptotic response in sensitive cells. BH3 profiling reveals CNS BRAFV600E are BCL-2 dependent cells, unprimed for apoptosis, which may be good candidates for additional treatment with BH3 mimetics such as venetoclax. This presents an attractive treatment for MAPK activated CNS tumors by enhancing apoptotic cell death by targeting the MAPK pathway, autophagy and BH3.
MEDB-23. Targeting epigenetic dysregulation in medulloblastoma with poor prognosis
Authors: Badodi Sara , Pomella Nicola , Zhang Xinyu , Zabet Nicolae Radu , Basson M Albert , Marino Silvia
Published: June 2022

Medulloblastoma (MB) is the most common paediatric malignant brain tumour and is classified into four distinct molecular subgroups (WNT, SHH, G3 and G4), each of them further subdivided into subtypes with different prognosis and responses to therapy. Deregulation of chromatin modifier genes play an essential role in MB, particularly in the G4 subgroup. A BMI1High;CHD7Low molecular signature identifies patients with poor survival within this subgroup. We show that BMI1High;CHD7Low sustains MB growth through regulation of MAPK/ERK signalling and via a novel epigenetic regulation of inositol metabolism in both G4 MB cells and patients. These tumours display over-activation of MAPK/ERK signalling, sustaining tumour proliferation, and of AKT/mTOR pathway which leads to energetic rewiring characterised by enhanced glycolytic capacity and reduced mitochondrial function. We demonstrate that inositol administration counteracts this metabolic alteration, impairs proliferation and significantly extends survival in a pre-clinical model. Moreover, inositol synergises with cisplatin, a chemotherapy agent currently used in MB treatment, enhancing its therapeutic effect in vivo. Additionally, we identify a synergistic vulnerability of BMI1High;CHD7Low MB to a combination treatment with BMI1 and MAPK/ERK inhibitors that overcomes acquired resistance to single-drug therapy. Mechanistically, we observe a CHD7-dependent binding of BMI1 to MAPK-regulated genes underpinning the CHD7-BMI1-MAPK regulatory axis that is critical for the anti-tumour effect of the inhibitors in vitro and in a pre-clinical model. Moreover, we demonstrate that the BMI1High;CHD7Low molecular signature defines G4 MB patients with an enhanced ERK1-ERK2 phosphorylation activity. Importantly, cerebellar neural stem cells modelling the BMI1High;CHD7Low signature are not affected by BMI1 and MAPK/ERK inhibitors and do not show metabolic adaptation hence are resistant to the proposed treatments. In summary, we have identified two actionable vulnerabilities in a pre-clinical setting modelling a molecularly defined group of MB patients, paving the way for the design of signature-matched clinical trials.
MEDB-25. Do extracellular vesicles transfer a multidrug resistant phenotype via ABC transporters in medulloblastoma?
Authors: Wade Philippa , Jackson Hannah , Taylor Louisa , Hume Alistair , Kerr Ian , Coyle Beth
Published: June 2022

INTRODUCTION: Medulloblastoma is the most common malignant paediatric brain tumour accounting for 20% of all childhood tumours; approximately one-third of patients present with metastatic disease at diagnosis and the outcome for these patients remains very poor. The high frequency of recurrence and metastatic relapse in medulloblastoma supports the idea of intrinsic drug resistance within cells. This work looks at the ATP-binding cassette (ABC) transporters, known to be upregulated in several cancers, and we hypothesise that extracellular vesicles may transfer ABC transporters to surrounding cells, promoting multidrug resistance within tumours. METHODS: The Cavalli dataset, made up of 763 patient samples, was used to assess the gene expression of a number of ABC transporters across medulloblastoma subgroups. ABC transporter gene and protein expression was then further assessed in medulloblastoma cell lines, including drug-tolerant lines, using qPCR and western blot. Cell viability analysis was used to assess changes in drug response. Matched cell and EV protein samples were used for proteomic analysis. RESULTS: Patient gene expression data showed that high expression of two ABC transporters correlated with reduced patient survival in high-risk subgroups. qPCR analysis of medulloblastoma cell lines showed differential subgroup expression patterns. Additionally, qPCR analysis of drug-tolerant cell lines showed significant increases in the expression of specific drug transporters across the subgroups. RNA-seq confirmed the presence of ABC transporter mRNA in exosomes isolated from high-risk medulloblastoma cell lines. Functional studies of EV transference of ABC transporters are ongoing. CONCLUSIONS: Data to date supports the hypothesis that multidrug transporter carrying extracellular vesicles may transfer their multidrug resistant phenotype to surrounding cells in medulloblastoma, promoting drug resistance. Future work will test this hypothesis by knocking down candidate ABC transporters and assessing the effect on transference of drug resistance by extracellular vesicles.
MEDB-44. Transcriptomic resolution of subgroup-specific medulloblastoma architecture
Authors: Willard Nicholas , Riemondy Kent , Griesinger Andrea , Kaufman Michael , Venkataraman Sujatha , Foreman Nicholas , Vibhakar Rajeev , Donson Andrew
Published: June 2022

Despite a growing understanding and stratification of medulloblastoma, it remains an aggressive childhood brain tumor with high morbidity and mortality. Multimodal genomic and epigenomic analysis has permitted the classification of medulloblastoma into four subgroups with varying biology and clinical behavior: WNT, Sonic-Hedgehog (SHH), Group 3, and Group 4. In our previously published work, Single-cell RNA sequencing (scRNAseq) identified distinct tumor cell subpopulations in specific medulloblastoma groups. However, this technology is limited by its lack of architectural information. Spatial transcriptomics is a relatively new technology that permits the analysis of gene expression as it occurs within organized tissue. In our ongoing study, we utilized Visium spatial transcriptomics, integrated with scRNAseq data and immunohistochemistry, to analyze frozen samples of medulloblastomas (SHH, Group 4, and Group 3 with and without MYC amplification). In SHH in particular, we were able to identify scRNAseq populations within the geographically constricted Visium data, including SHH-C2, a population located in histologic nodules, the predominant neuronal-differentiated population SHH-C1, and progenitor populations (SHH-B1 and B2). In addition, we were able to visualize clusters not detectable by scRNAseq – a cluster lining nodules with expression of vascular endothelium marker, reticulin and M2-macrophage genes, and a novel DNA-repair cluster. In addition, Visium data permits the spatial constraint of proliferating cells, which is frequently problematic in scRNAseq, as dividing cells cluster independently. The proliferation is highest in the SHH-B2 minor progenitor population, absent in the SHH-C1 major differentiated population, and is moderate in other population including the SHH-C2 nodules. Group 3 and 4 medulloblastoma are more complex but show preliminary corroboration with scRNAseq data. In summary, Visium allows us to map subpopulations identified by scRNAseq to tumor architecture more definitively and rapidly than IHC. These novel insights advance our understanding of medulloblastoma, a critical step in improving treatment options for children with this disease.
MEDB-56. POx NPs mediated delivery of (TLR7/8) agonist resiquimod improves treatment outcomes in SHH medulloblastoma by targeting tumor associated macrophages
Authors: Hwang Duhyeong , Alicea Coral del Mar , Lim Chaemin , Park Christopher , Gershon Timothy , Sokolsky-Papkov Marina
Published: June 2022

Cancer immunotherapy, the utilization of the patients’ own immune system to treat cancer, has emerged as a powerful new strategy in cancer treatment. Recent clinical data has demonstrated immunotherapy to be effective in a wide range of cancers, including lung, bladder, renal cell, colorectal, gastro-esophageal, and head and neck cancers5-11. However, clinical reports of immune-based treatments for medulloblastomas are scarce and preliminary. Therefore, there is a need in developing strategies to improve medulloblastoma immunotherapy. Our recent studies have confirmed that SHH medulloblastomas are enriched in Tumor Associated Macrophages (TAMs) and unlike other tumors, TAMs are associated with positive outcomes and play a positive role by impairing tumor growth. Overall, analysis of TME in medulloblastomas reveals TAMs as a potential therapeutic target. Resiquimod is a synthetic small molecule agonist of Toll-like receptors 7 and 8 (TLR7/8) that modulates innate immune cells. We have loaded resiquimod into ultra-high-capacity polyoxazoline (POx) block copolymers forming small, homogeneous nanoparticles (POx-resiquimod). Our recent study shows that loading into POx nanoparticles improves drug delivery to tumors and treatment with 3 injections of POx-res as the single-agent treatment results in a profound anti-tumor effect G-Smo mice while treatment with free drug shows no therapeutic benefit. Our data also shows that that the tumors of G-Smo mice are enriched with the mixed populations of anti-inflammatory and pro-inflammatory macrophages and the treatment with POx-resiquimod have enhanced infiltration of macrophages into the tumors, enhanced the repolarization of macrophages to M1 subtype and decreased tumor cells viability. These studies show for the first time that targeting the medulloblastoma TME with POx-resiquimod can produce a significant anti-tumor effect. Furthermore, combination of POx-resiquimod with radiation resulted in long term survivals, showing potential therapeutic combination. The expression of TLR7/8 on TAMs in patient-derived medulloblastoma samples suggests that resiquimod may produce similar anti-medulloblastoma effects in humans.
MEDB-57.GNAS inactivation as a driver for sonic hedgehog-activated medulloblastoma
Authors: Goode Erin , Montoya Liliana , Graham Eric , Pruniski Brianna , Simmons Curtis , Ngwube Alexander , Hoffman Lindsey M , Tiwari Nishant , Aldape Kenneth , Price Harper N , Paulson Vera , Mangum Ross
Published: June 2022

INTRODUCTION: Sonic hedgehog (SHH)-activated medulloblastoma is one of the four consensus molecular subgroups of medulloblastoma and is typically associated with PTCH1, SUFU, and/or SMO mutations. GNAS inactivating mutations are a less commonly recognized tumorigenic driver for SHH pathway activation.

CASE PRESENTATION: We report the case of an 11-month-old male who presented with a large posterior fossa mass, cerebellar tonsillar herniation, and obstructive hydrocephalus. Following a successful gross total resection, pathology was consistent with desmoplastic/nodular medulloblastoma. Tumor molecular profiling using a DNA-based, next-generation sequencing platform detected a pathogenic frameshift mutation in the GNAS gene (p.D189Mfs*14, NM_000516.4:c.565_568del) at a variant allele frequency of 81%, suggestive of biallelic GNAS inactivation. The same mutation was detected from a buccal swab sample, confirming germline GNAS inactivation. Whole genome methylation profiling was consistent with medulloblastoma subclass SHH B (infant). Concurrent with this brain tumor evaluation, a skin biopsy was performed of scattered subcutaneous, plate-like nodules distributed over the patient’s back and extremities. This revealed metaplastic-appearing cancellous bone within the dermis and subcutaneous tissue consistent with plate-like osteoma cutis. He was treated with high-dose chemotherapy followed by autologous stem cell rescue and remains disease-free 15 months from diagnosis.

DISCUSSION: There have been scattered case reports describing germline loss-of-function GNAS mutations acting as tumorigenic drivers of SHH medulloblastoma. However, GNAS alterations are not covered by most standard diagnostic molecular sequencing panels for medulloblastoma. Other phenotypic manifestations of germline inactivating GNAS mutations include pseudohypoparathyroidism, pseudopseudohypoparathyroidism, progressive osseous heteroplasia, and osteoma cutis.

CONCLUSION: Knowledge of the possible association between germline GNAS inactivating mutations and the development of childhood SHH-activated medulloblastoma is essential for prompt diagnosis and treatment initiation. As such, consideration should be given for inclusion of GNAS alterations in diagnostic medulloblastoma sequencing panels, especially in the setting of osteoma cutis or other endocrinopathies.
MEDB-60. Medulloblastoma with extensive nodularity mimics cerebellar development and differentiates along the granular precursor lineage
Authors: Ghasemi David R , Okonechnikov Konstantin , Tirier Stephan , Rademacher Anne , Mallm Jan-Philipp , Ernst Kati , Rippe Karsten , Korshunov Andrey , Pfister Stefan M , Pajtler Kristian W
Published: June 2022

BACKGROUND: Medulloblastoma with extensive nodularity (MBEN) represents a rare type of cerebellar tumors of infancy comprising two histologically distinct components that differ in cell differentiation and mitotic activity. Whereas some children suffering from MBEN experience disease recurrence, MBEN can also spontaneously differentiate and discontinue to grow. The underlying mechanisms of this variable biological behavior may offer insight into how embryonal tumors develop. METHODS: Fresh frozen and FFPE tumor tissue from nine MBEN-patients was subjected to multi-omics characterization including bulk sequencing, microdissection followed by RNA sequencing, single nucleus RNA-sequencing using the 10X Genomics- and SMART Seq. V2-protocols and spatial transcriptomics via RNAscope. RESULTS: All cases were molecularly classified as Sonic Hedgehog (SHH)-MB, and harbored somatic mutations within the SHH-pathway. After quality control, a total of ~30.000 cells were subjected to downstream analysis. Several non-malignant cell types, such as glial cells, were identified. In accordance with previous studies, we found only sparse immune infiltration. Unsupervised clustering identified cell clusters that differed in differentiation state and represented a continuum from embryonal-like cells with SHH-upregulation over intermediate cell states, to neuronal-like, postmitotic cells. Mapping to a single nucleus sequencing atlas of cerebellar development indicated that tumor cells reflected various stages of normally developing cerebellar granular precursors. Interestingly, one cluster of malignant cells with tumor-specific copy number alterations showed both transcriptomic features of astrocytes and embryonal cells. Using spatial transcriptomics, we were able to correlate different clusters of MBEN cells with distinct histologic MBEN compartments, with astrocyte-like tumor cells being located in the internodular compartment and in close proximity to mitotically active cancer cells. CONCLUSION: MBEN is formed by a continuum of malignant cell differentiation along the granular precursor lineage, with a subset of cells developing into cells that may represent tumor astrocytes. This differentiation process is reflected in the bicompartmental structure of MBEN.
MEDB-66. Investigating intra-tumoral heterogeneity of extrachromosomal DNA in SHH medulloblastoma
Authors: Chapman Owen , Sridhar Sunita , Wechsler-Reya Robert J , Chavez Lukas , Mesirov Jill P
Published: June 2022

Extrachromosomal circular DNA (ecDNA) is an important driver of aggressive cancers, including medulloblastoma (MB), the most common malignant pediatric brain tumor. Our study’s aim is to better understand how ecDNA containing cells can potentiate malignant growth. EcDNA’s role in the development of treatment resistance and association with poor outcomes is hypothesized to arise from its contribution to intra-tumoral heterogeneity and its potential to promote oncogene dependency switching. To analyze the intra-tumoral distribution of ecDNA, we have now simultaneously analyzed the accessible chromatin and gene expression in single cells of a SHH medulloblastoma (MB) patient using multiome single-cell ATAC-seq and gene expression (10X Genomics). Whole genome sequencing (WGS) of this tumor previously revealed a heterozygous somatic TP53 mutation and two distinct ecDNAs: a 3.2Mbp amplicon comprising 3 regions of chr1 and another 4.5Mbp amplicon comprising 23 segments originating from chr7 and chr17. We then used multimodal analysis to describe the tumor cell types, gene expression, variant signatures and estimate ecDNA copy number in the medulloblastoma tumor sample. We identified 12 distinct clusters in the human tumor, 5 of which were determined to be normal non-tumor [OSC1] cells, as identified by specific cell type markers, and 7 of which were determined to be tumor cells. Enrichment of ecDNA was restricted to only one of these tumor clusters. In addition, we also performed the same multiome single-cell analyses in an orthotopic xenograft mouse model derived from this SHH MB patient tumor. In the PDX, 17 clusters were identified, all of which were determined to be tumor cells and enriched for ecDNA. Our preliminary results indicate that tumor cells with ecDNA in the human tumor (particularly the ecDNA enriched cluster) almost exclusively account for [OSC2] the cells in the corresponding PDX, emphasizing the aggressiveness of ecDNA containing cells.
MEDB-76. Evaluating the B7-H3 checkpoint in Medulloblastoma
Authors: Moziak Kirsten , Picarda Elodie , Perez Natalia Muñoz , Galbo Phillip , Zheng Deyou , Zang Xingxing , Martin Allison
Published: June 2022

BACKGROUND: There is currently no curative therapy for recurrent/refractory MB. Novel approaches to MB include immunotherapy, such as targeting the immune checkpoint molecule B7-H3. B7-H3 is implicated in tumor metastasis and is highly expressed in MB. This study explores the effects of genetically knocking down B7-H3 in a murine model of recurrent/refractory medulloblastoma. METHODS: Murine MB cells were transduced with a CRISPR/Cas9 lentivirus to create a B7-H3 knockout. Knockout population was sorted twice via FACS by the AECOM flow cytometry CORE and confirmed by western blot and flow cytometry. Three healthy clones were used in subsequent studies, and compared to the wild type and the scramble control. IncuCyte live imaging technology was used to evaluate spheroid growth. Matrigel Boyden chambers were used to evaluate migration. Bulk RNA-seq was performed by the Yale University Core. RESULTS: B7-H3 knockout was successful in the murine MB model. Morphological differences were noted in the B7-H3 knockout cells. Spheroid formation assays show one of the clones with statistically slower growth kinetics compared to controls. Migration results are pending. RNA seq revealed similar clustering amongst knockouts, separate from controls with an enrichment in genes of morphologic development, WNT signaling and amoeboid migration. CONCLUSIONS: The morphologic changes in the B7-H3 knockouts suggest a potential growth differential. Although in vitro growth assays have shown mixed results regarding the effect of knocking out B7-H3 in spheroid formation, B7-H3 has been more directly implicated in migration and immune signaling. If migration is impaired, this will suggest that B7-H3 enhances malignant and metastatic potential in MB. Functional in vivo immune studies in syngeneic mice will investigate immune mediated effects of B7-H3 knockout in this tumor. If our studies support a role for B7-H3 in the development of MB, it may have important clinical implications, particularly for relapsed patients.
MEDB-80. CDK8 promotes stemness of MYC-driven medulloblastoma
Authors: Wang Dong , Veo Bethany , Pierce Angela , Venkataraman Sujatha , Vibhakar Rajeev
Published: June 2022

Cyclin-dependent kinase 8 (CDK8) belongs to the transcription-related cyclin dependent protein kinase family. CDK8 and cyclin C associate with the mediator complex to regulate gene transcription. Although CDK8 has been shown to be implicated in the malignancy of several types of cancer, its functional role and mechanism in medulloblastoma remains largely unknown. Here, we demonstrate how CDK8 plays an essential role in maintaining stemness and tumorigenicity in medulloblastoma stem cell. CDK8 inhibition suppresses stem cell associated signaling in medulloblastoma cells and inhibits tumor cell self-renewal. Additionally, CDK8 is amplified in MYC-driven medulloblastoma, is positively correlated with c-MYC expression in human medulloblastoma specimens and associates with poor survival in patients. Using cut&run assay, we found CDK8 associates with MED1 to activate transcription of MYC target genes. CDK8 attributes to MYC-driven transcriptional programs mediating DNA repair. Pharmaceutic inhibitors and genetic depletion result in cessation of tumor growth in xenograft mouse models and increase in apoptosis and DNA damage. Collectively, our studies establish the selective inhibition of CDK8 inhibition as a viable therapeutic strategy in MYC-driven medulloblastoma.
MEDB-88. BAF60C/SMARCD3-mediated novel neurodevelopmental epigenomic program promotes metastatic dissemination in medulloblastoma
Authors: Zou Han , Poore Bradley , Brown Emily , Hu Zhongliang , Li Xuejun , Pollack Ian , Friedlander Robert , Hainer Sarah , Taylor Michael , Hu Baoli
Published: June 2022

Normal brain development relies on precise genetic and epigenetic spatiotemporal regulation of gene expression. How dysregulation of neurodevelopment relates to medulloblastoma, the most common pediatric brain tumor, remains elusive. Here, we uncovered a novel neurodevelopmental epigenomic program that regulates Purkinje cell migration in developing cerebellum is hijacked to induce tumor metastatic dissemination in medulloblastoma. Integrating publicly available datasets with our in-house data, unsupervised analyses revealed that BAF60C/SMARCD3, a subunit of SWI/SNF chromatin remodeling complex, promotes tumor cell migration in vitro and metastasis in vivo. Based on analyzing the single-cell RNAseq data of cerebellum developmental trajectory in mice and humans, aligning with the medulloblastoma patients’ datasets, we found that BAF60C/SMARCD3 regulated DAB1-mediated Reelin signaling is involved in Purkinje cell positioning during cerebellum development and medulloblastoma metastasis by orchestrating the cis-regulatory elements (CREs) at the DAB1 gene locus. Analysis of spatiotemporal gene expression and chromatin architecture in the human and mouse cerebellum demonstrated that transcription activity of the BAF60C/SMARCD3-DAB1 circuit is downregulated in a mature state of cerebellar development, however, is upregulated in metastatic medulloblastoma. We further identified that a core set of transcription factors, enhancer of zeste homolog 2 (EZH2) and nuclear factor I X (NFIX), bi-directionally control BAF60C/SMARCD3 transcriptional regulation by coordinating with the CREs at the BAF60C/SMARCD3 gene locus to form a chromatin hub during developing cerebellar development and medulloblastoma metastatic dissemination. Highly expressed BAF60C/SMARCD3 activates the Reelin/DAB1 signaling pathway downstream Src kinase, which was validated in the pair-wised primary and metastatic tumors from medulloblastoma patients. Preclinical medulloblastoma mouse models revealed that inhibiting Src activity reduces tumor cell migration and metastatic dissemination at a lower and safe dose. Together, these data deepen our understanding of how the developmental program influences disease progression and provide an opportunity for the development of therapeutics for this devastating brain cancer in children.
NFB-17. "Optic Pathway findings in children with Neurofibromatosis type-1 (NF-1)
Authors: Roka Kleoniki , Kokkinou Eleftheria , Gavra Maria , Tsina Efthymia , Mparka Konstantina , Zarafonitis Georgios , Kosma Konstantina , Makrythanasis Periklis , Tzetis Maria , Chasiotou Maria , Kanaka-Gantenbein Christina , Pons Roser , Kattamis Antonis
Published: June 2022

BACKGROUND/OBJECTIVES:Optic-pathway-glioma(OPG) represents the most common central-nervous tumor in children with neurofibromatosis type-1(NF1), occurring at an incidence of 15-20%. It is estimated that 1/3 of NF1 patients(pts) with OPG will need treatment. DESIGN-METHODS:We performed a retrospective-review of all NF1-pts examined in the First Hellenic Multidisciplinary-Clinic – Center of Expertise for Neurocutaneous-disorders. Gender, age, MRI-radiological and ophthalmological findings, presence of OPG, management and outcome were analyzed. RESULTS:Since the establishment of the Clinic in 2016, 198pts with clinical diagnosis of NF1 based on NIH1988-criteria were evaluated and of them, 165(73 females, median age:5.5y, range:0.3-17.1y), who had imaging studies were included in this analysis. Eighty three pts(50.3%) had NF1-positive genetic-testing and 45NF1-family-history(27.3%). Imaging-findings from optic pathway were found in 55/165pts(28females). Percentage of pts with findings were 51.7% for <3y, 45.4% for 3-5y, 34.7% for 5-10y and 8.5% for >10y, respectively. The median age of their first brain-MRI imaging was 2.82y. Upon 1stMRI-imaging, 70.9% presented thickness of the optic nerves(ON)(25,4%bilateral, 20% optic chiasm,18.1% right ON, 10.0% left ON), 14.5%ON-tortuosity, 38.1%OPG(43,5% in the optic-chiasm) and 34.5% contrast enhancement. Of notice, 14pts presented an OPG after a median follow-up time of 1.79y. According to LGG2004-protocol indications for treatment, only 15/55pts had to be treated(27,2%, 5pts with family history, 33.3% between 5-10y). Severe vision-loss with need for immediate start of treatment upon 1stMRI imaging was found in 4pts, of whom 75% had family-history and first evaluation after the 5th year of age. Of notice, only 2pts<3y had to receive treatment, one with family-history and one with symptoms(diencephalic syndrome). CONCLUSIONS: Pts with NF1 should be followed by a multidisciplinary-team. Management should be individualized and imaging-studies can be limited to patients at high-risk. Positive family-history may be a negative prognostic factor for OP lesions.
QOL-02. Paediatric medical traumatic stress in children with cancer and their parents: Differences in levels of posttraumatic stress symptoms
Authors: Klašnja Sandra , Hausmeister Ivana Kreft , Kavčič Marko , Masten Robert
Published: June 2022

Paediatric medical traumatic stress (PMTS) is a set of children’s and their parents’ psychological and physiological responses to pain, injury, serious illnesses, and other experiences with the medical environment. Paediatric cancer patients have the highest prevalence of PMTS as the illness involves a set of stressors that trigger many negative psychological reactions. Posttraumatic stress symptoms (PTSS) are one of the most common psychopathologies among cancer patients. We examined the incidence of PMTS in children with cancer and their parents due to coping with a serious illness and treatment complications. We analysed the following risk factors for PTSS: selected groups of individuals, medical interventions, complications, and treatment modalities. The study involved 183 parents of 133 children and 63 children and adolescents who were treated between 2009 and 2019 at Clinical Department of Paediatric Haematology and Oncology of Paediatric Clinic in Ljubljana. We collected the data using The Intensity of treatment rating scale 2.0 [IRT-2], PTSD checklist for Children/Parent [PCL-C/PR], The PTSD Checklist for DSM-5 [PCL-5] and The Child PTSD Symptoms Scale for DSM-5 [CPSS-5]. PMTS is frequently present in both, children and their parents, regardless of the cancer type, treatment duration, treatment outcome, and child’s age. Mothers, patients with relapse, patients who were diagnosed after age 5, patients with more intensive treatment, and parents of the latter are at higher risk for PMTS occurrence. Additionally, we found a decreasing trend of traumatic responses after five or more years post cancer diagnosis and that parents are more traumatized than children. Our findings will contribute to the systematic prevention of PMTS and medical trauma and to endeavour to use trauma-informed care.
QOL-12. Relaxation technique of imagery based story telling reduces manifestation of anxiety in children and adolescents undergoing brain tumor surgery
Authors: Goßler Christina , Schweitzer Tilmann , Krauß Jürgen , Andres Oliver , Rueckriegel Stefan Mark
Published: June 2022

Children and adolescents undergoing surgical resection of brain tumors are prone to marked psychologic burden. Especially fear of surgery and its consequences like pain or neurologic sequelae is an important issue. Techniques of relaxation might reduce the intensity of the experienced anxiety and therefore might improve quality of life. In this study, we aimed at determining the effect of a standardized imagery story telling on experienced anxiety as quantified by the questionaire KAT III and cardiac frequency (CF) before and after intervention at two time points (before and after surgery). 12 patients (age: 6-17 years) undergoing brain tumor resection were included in the study. KAT III-scores and CF were determined and compared before and after interventions using a dependend t-test. Mean KAT III-score before first intervention was 0.23 (SD: 0.23), while it was 0.15 (SD: 0.21) after (p = 0.11). Mean KAT III-score before second intervention was 0.11 (SD: 0.13), after: 0.05 (SD: 0.9), p = 0.07. Mean CF before first intervention was 77.1 (SD: 10.3), after: 68,36 (SD: 6.8), p = 0.003. Mean CF before second intervention was 71.67 (SD: 9.57), after: 65 (SD: 8.72), p = 0.003. CF was significantly lower post-interventionally after the first and the second intervention. KAT III-score showed a trend to be lower post-interventionally after the second intervention. Hence, our study points at an efficacy of the deployed relaxation technique of imagery story telling in children and adolescents undergoing brain tumor surgery, although it was limited by a small patient number. Further studies with larger patient numbers and a comparison of randomized intervention vs. non-intervention groups are warranted.
QOL-21. Language development and outcomes in pediatric brain tumor
Authors: Janke Kelly , Paltin Iris , Schofield Hannah , Sy Megan , Kearns Zoe
Published: June 2022

RATIONALE: Pediatric brain tumor (PBT) survivors are at risk for speech (e.g., articulation, prosody, fluency) and language (e.g., vocabulary, grammar, narratives, pragmatics) difficulties, though group outcomes vary with study design and individual risk factors (Hodges et al 2020). This study characterizes language functioning for clinically referred PBT survivors diagnosed in early childhood, a time of rapid language development and vulnerability. METHODS: Participants were 98 PBT patients (56 supratentorial) diagnosed by age 6 (M = 43.8 months, SD 24.4) who received tumor-directed intervention (surgery, chemotherapy, and/or radiation). Age at neuropsychological evaluation (2019 – 2022) was 2-19 years (M = 9.1, SD 4.1). Patients were 56% male, 73.5% White, and fluent English speakers. Verbal IQ, naming, working memory, fluency, comprehension, syntactic and phonological processing, and parent-reported functional communication outcomes were assessed. Rates of weak performance (1 SD<Mean) were compared to the expected base rate of 16%. RESULTS: Group means significantly diverged from age-expected performance in all domains except fluency. IQ correlated with most language measures, age at diagnosis (r .280, p .010), and time from diagnosis (r -.276, p .012). Weakness was identified on at least 1 verbal subtests for 71.4% of the full sample and most (57%) patients with IQ>84. Patients with NF1 (n=15) or treatment-related hearing concerns had lower reported functional communication. Naming difficulty was associated with supratentorial tumors; there were no other significant differences by tumor type or location. While functional communication correlated with several measures, weaknesses on performance-based and parent-report measures only corresponded for 44.6% of the sample. CONCLUSIONS: Language weaknesses are prevalent following early childhood PBT diagnosis. IQ and functional communication measures are useful indicators of risk, but more detailed and higher-level language measures are needed, particularly for cognitively intact patients. Additional implications and research needs will be discussed.
QOL-31. A psychosocial support program for young adult childhood cancer survivors in Austria: a qualitative evaluation study
Authors: Pletschko Thomas , Krottendorfer Kerstin , Schlifelner Juliana , Schwarzinger Agathe , Fohn-Erhold Verena , Weiler-Wichtl Liesa , Kienesberger Anita , Leiss Ulrike
Published: June 2022

PURPOSE: Many adolescents and young adult (AYAs) childhood cancer survivors face disease- or therapy-related late-effects, which limit their participation in various areas of daily life. AYAs are often left alone in our health care system and many worry about their ability to cope with long-term sequelae and some are even lost to follow up. Therefore, in the present study a targeted aftercare program was developed and evaluated with the goal of facilitating three important “life skills”: (1) self-perception, (2) social interaction and conflict management as well as (3) self-conscious communication of support needs. METHODS: A total of n=13 participants (19.2-30.2 years, mean age: 22.8 years) completed a three-day aftercare seminar, at the end of which each participant wrote a reflection letter (“letter to my future self”), elaborating on observed effects of the seminar, applicability of the given information in daily life and the direct impact of the seminar on their individual circumstances. The reflection letters were analyzed using qualitative content analysis. RESULTS: All target life skills were mentioned in the reflection letters. The participants reported individual benefits from the program especially with respect to self-perception and self-confidence, giving and taking feedback, and acceptance of personal strengths and weaknesses. Moreover, the feeling of “not being alone” was associated with the survivors’ experience of emotional and social support. CONCLUSIONS: This evaluation highlights the potential of a one weekend aftercare seminar to address important life skills that are known to positively influence health behavior in AYAs. The detailed description of the seminar can serve as a basis for making this kind of aftercare accessible for other people in similar circumstances.
SURG-08. 5-Aminolevulinic acid (5-ALA)-guided resection of pediatric brain tumors
Authors: Schwake Michael , Bruns Ann-Katrn , Müther Michael , Stummer Walter
Published: June 2022

BACKGROUND: 5-Aminolevulinic acid (5-ALA)-guided resection of gliomas in adults enables better differentiation between tumor and normal brain, allowing a higher degree of resection, and improved patient outcomes. In recent years, several reports have emerged regarding the use of 5-ALA in other brain tumor entities, including pediatric brains tumors. Since gross total resection (GTR) of many brain tumors in children is crucial, the role of 5-ALA-guided resection requires elucidation. METHODS: A systematic literature review of EMBASE and MEDLINE/PubMed databases revealed 20 eligible publications encompassing 186 5-ALA-guided operations on pediatric brain tumors. To reduce bias, publications were revised independently by two authors. RESULTS: 5-ALA-guided resection enabled the surgeons to identify the tumor more easily and was considered helpful mainly in cases of glioblastoma (GBM, 21/27, 78%), anaplastic ependymoma WHO grade III (10/14, 71%), and anaplastic astrocytoma (4/6, 67%). In contrast, cases of pilocytic astrocytomas (PAs) and medulloblastomas 5-ALA-guided surgery did not show consistent fluorescent signals and 5-ALA was considered helpful only in 12% and 22% of cases, respectively. Accumulation of fluorescent porphyrins seems to depend on WHO tumor grading. In case fluorescence signal was considered helpful, it was associated to a greater degree of resection. One study showed an association between visible fluoresce signal and concentration of protopophyrin IX (PPIX) concentration. A threshold of 4μg/ml was required in order to visualize the fluorescence signal. The rate of adverse events related to 5-ALA was negligible, especially new postoperative sequelae. CONCLUSION: 5-ALA could play a role in resection of malignant, contrast enhancing, supratentorial pediatric brain tumors. At present, we are conducting a prospective phase I-II multicenter clinical trial to evaluate side effects and feasibility of 5-ALA guided surgery
OTHR-07. A new framework for missing value tolerant data integration
Authors: Voß Hannah , Schlumbohm Simon , Wurlitzer Marcus , Dottermusch Matthias , Neumann Philipp , Barwikowski Philip , Schlüter Hartmut , Krisp Christoph , Neumann Julia
Published: June 2022

Dataset integration is common practice to overcome limitations, e.g., in statistically underpowered omics datasets. This is of particular importance when analyzing rare tumor entities. However, combining datasets leads to the introduction of biases, so called 'batch effects', which are due to differences in quantification techniques, laboratory equipment or used tissue type. A common problem is the missing quantification for features like gene transcripts or proteins within a dataset. These missing values can appear at random in a given dataset and also get introduced by combination of multiple datasets. Currently, strategies beyond common normalization for batch effect reduction are either missing entirely or are unable to handle absence of data points and therefore rely on error-prone data imputation. We introduce a framework that enables batch effect adjustments for combined datasets while avoiding data loss by appropriately handling missing values without imputation. The underlying idea is based on a matrix dissection approach, adjusting common information from the integrated dataset under guarantee of sufficient data presence. The strategy is implemented within the R environment and linked with popular software stacks that are built on top of R. Successful data adjustment is exemplarily shown for proteomic data generated by different quantification approaches and LC-MS/MS instrumentation setups.