bluuebunny/crossref_metadata_2025 huggingface dataset:
ATRT-08. SMARCB1- and SMARCA4-deficient malignant brain tumors with complex copy number alterations andTP53 mutations may represent the first clinical manifestation of Li-Fraumeni syndrome
Authors: Hasselblatt Martin , Thomas Christian , Federico Aniello , Nemes Karolina , Johann Pascal D , Bison Brigitte , Bens Susanne , Kordes Uwe , Redlich Antje , Lessel Lienhard , Pajtler Kristian W , Mawrin Christian , Schüller Ulrich , Nolte Kay , Kramm Christof M , Hinz Felix , Sahm Felix , Giannini Caterina , Penkert Judith , Kratz Christian P , Pfister Stefan M , Siebert Reiner , Paulus Werner , Kool Marcel , Frühwald Michael C
Published: June 2022

Atypical teratoid/rhabdoid tumor (AT/RT) is a malignant central nervous system tumor predominantly affecting infants. Mutations of SMARCB1 or (rarely) SMARCA4 causing loss of nuclear SMARCB1 or SMARCA4 protein expression are characteristic features, but further recurrent genetic alterations are lacking. Most AT/RTs occur de novo, but secondary AT/RTs arising in other central nervous system tumors have been reported. Malignant gliomas, IDH-wildtype, arising in patients with Li-Fraumeni syndrome typically show somatic mutations of TP53 as well as complex copy number alterations, but little is known about loss of SMARCB1 or SMARCA4 protein expression in this context. Here we report two children, in whom malignant supratentorial brain tumors with SMARCB1-deficiency, complex copy number alterations and somatic TP53 mutations lead to the discovery of pathogenic/likely pathogenic TP53 variants in the germ line. Screening of the molecularneuropathology.org data set for cases with similar genetic and epigenetic alterations yielded another case with SMARCA4-deficiency in a young adult with Li-Fraumeni syndrome. In conclusion, SMARCB1- or SMARCA4-deficient malignant brain tumors with complex copy number alterations and somatic TP53 mutations in children and young adults may represent the first clinical manifestation of Li-Fraumeni syndrome and should prompt genetic counseling and investigation for TP53 germline status.
ATRT-10. Single-cell transcriptional profiling of ATRTs reveals heterogeneous signatures of tumor and non-malignant cell populations
Authors: Blanco-Carmona Enrique , Büllesbach Annette , Federico Aniello , Liu Ilon , Young Matthew D , Kildisuite Gerda , Behjati Sam , Vibhakar Rajeev , Donson Andrew , Foreman Nicholas , Hovestadt Volker , Shaw McKenzie , Chi Susan , Frühwald Michael , Drost Jarno , Korshunov Andrey , Hasselblatt Martin , Pfister Stefan M , Jäger Natalie , Johann Pascal , Filbin Mariella , Kool Marcel
Published: June 2022

Atypical Teratoid/Rhabdoid Tumors (ATRTs) are known for exhibiting high inter-tumor heterogeneity, even though they are almost all characterized by a common loss of SMARCB1 (or rarely SMARCA4). Three subgroups have been identified at bulk methylome and transcriptome level: ATRT-TYR, ATRT-SHH, and ATRT-MYC. To better understand the biology underlying each subgroup and potentially unveil their (different) cell(s) of origin, we performed single-cell transcriptomic analyses in 22 ATRTs using fresh frozen samples and both 10X and Smartseq technology. All data, grouped by technology, underwent quality control and normalization, regressing out the biases introduced by each sample. Tumor microenvironment (TME) and tumor bulk (TB) clusters were characterized by a combination of copy number variant analyses, enrichment in literature lists of marker genes for specific cell populations, and in-depth analysis of differentially enriched (DE) genes. Non-negative Matrix Factorization (NMF) was applied to TB to reveal major transcriptional profiles, which were grouped into meta-signatures. A total of 71 gene lists were retrieved from NMF (TB) and DE analyses (TME + TB), that gathered into 11 signature groups by Jaccard similarity, with one extra group accounting for unique signatures. Three groups targeted TME, accounting for either microglia, fibroblasts and endothelial cells, or OPCs, oligodendrocytes, astrocytes and neurons. These signatures are enriched in specific clusters across technologies. The remaining eight groups divide into two types, either enriched in clusters predominantly formed by cells of one or two ATRT subgroups or signatures enriched for a particular phenotype, such as cilial, cycling, axonogenesis or EM transition. While the first type is enriched across clusters in a gradient fashion, the second shows enrichment for selected clusters across technologies. Further analyses on the integrated dataset and additional samples are ongoing to validate and refine these 11 signature groups in ATRTs to see how this may lead to new treatment approaches.
ATRT-13. An integrative analysis of the ATRT proteome unravels novel drug targets and refines molecular subgrouping
Authors: Johann Pascal , Müller Torsten , Kalxdorf Mathias , Hasselblatt Martin , Frühwald Michael C , Pfister Stefan M , Krijgsveld Jeroen , Kool Marcel
Published: June 2022

INTRODUCTION: Atypical teratoid/rhabdoid tumors (ATRT) represent frequent brain tumors in infants. In recent years, large-scale landscaping efforts on the epigenome and transcriptome of these tumors have unravelled a high degree of heterogeneity and three major molecular subgroups, termed ATRT-TYR, ATRT-SHH,ATRT-MYC, have been identified. The ATRT-proteome, in turn, still represents a largely unchartered territory. METHODS: We have performed a peptide-based screening approach to characterize the proteome of 40 ATRTs and six ATRT cell-lines. All of these samples had matching methylation data available and 28 also corresponding gene expression data. RESULTS: Unsupervised clustering recapitulated the previously described ATRT groups, revealing also a clear split of the SHH-subgroup in a supratentorial (SHH_1) and an infratentorial subgroup (SHH_2). Overall, we identified 7265 proteins, of which 1320 were differentially expressed between the groups, with an enrichment of spliceosome associated terms in SHH_1 and integrins/cell adhesions molecules in SHH_2. ATRT-MYC displayed an overrepresentation of immune cell markers and the TYR subgroup an enrichment of PI3K- as well as mTOR-signaling. Particularly, genes that have previously been described as signature genes for the ATRT-groups such as FABP7 in ATRT-SHH and OTX2 and MITF in ATRT-TYR were among the highly correlating genes that were both expressed in the proteome and the gene expression datasets. On top of this, our analysis revealed highly differentially expressed drug targets such as the tyrosine-kinase MARCKS (overexpressed in ATRT-TYR) not previously identified in ATRT transcriptome data, which warrant investigation by in vitro drug tests. CONCLUSION: Our data reveal the importance of previously described regulatory hubs in the ATRT subgroups, but additionally highlight novel drug targets that merit further exploration. Currently, drug treatment experiments in ATRT cell lines are ongoing to validate these proteins as drug targets, ultimately aiming to establish new therapeutic strategies in this deadly disease.
ATRT-20. Novel prognostic molecular signatures for improved risk-classification of Atypical Teratoid Rhabdoid Tumours
Authors: Keeling Claire , Grabovska Yura , O'Hare Patricia , Crosier Stephen , Pickles Jessica C , Finetti Martina A , Fairchild Amy R , Anderson John , Hargrave Darren , Brennan Bernadette , Jacques Thomas S , Clifford Steven C , Bailey Simon , Williamson Daniel
Published: June 2022

Malignant Rhabdoid Tumours (MRT) are aggressive paediatric malignancies seen in the central nervous system (Atypical Teratoid Rhabdoid Tumours (ATRT)), and kidney and other soft tissues (Extra-cranial Rhabdoid Tumours (ECRT)). With current therapies often proving ineffective and a lack of clear prognostic associations with consensus subgroups, we explored the possibility of using prognostic molecular signatures to further identify the biological characteristics of high risk ATRT patients. By employing a cross-validated feature selection method the methylation profiles of 121 MRT patients were analysed with clinical data to obtain meta-CpG signatures associated with prognosis for ATRT, ECRT and MRT. The relationship between these meta-CpG signatures and the consensus subgroups were further explored, along with the correlation of meta-CpGs with gene expression to establish biological significance. By selecting CpGs for their ability to predict survival this method obtained three novel prognostic methylation signatures which predict MRT outcome (ATRT-5, ECRT-14 and MRT-42). These signatures are independent of molecular subgroup and each signature was significantly associated with overall survival (OS) and event free survival (EFS) in their respective cohorts (p<0.001). Both ATRT-5 and MRT-42 maintained their significant association with OS in an independent ATRT cohort (n=64) and each meta-CPG signature is prognostically independent of other major clinical risk factors (e.g. receipt of radiotherapy and presence of metastases). Biologically, individuals with high-risk methylation signatures showed a gene expression profile suggestive of higher proliferative rates and tumours with low-risk scores in ATRT-5 and MRT-42 had an upregulated inflammatory response and increased immune infiltration. Combining these meta-CpGs with other significant clinical risk-factors produced high performing multivariate Cox-models enabling us to propose new stratification models for ATRT and MRT patients. These subgroup-independent prognostic signatures represent a distinct biology in ATRT and, if validated in prospective studies, could progress the use and efficacy of precision-based medicine in this therapeutically challenging disease.
ATRT-21. Contribution of germline mosaic alterations of SMARCB1 in rhabdoid tumor predisposition syndrome
Authors: Masliah-Planchon Julien , Maillot Laetitia , Rybak Noémie , Simaga Fatoumata , Hamza Abderaouf , Villars Marion Gauthier , Delattre Olivier , Bourdeaut Franck
Published: June 2022

Rhabdoid tumors are rare and aggressive tumors that usually arise in very young children. They are characterized by a bi-allelic inactivation of the SMARCB1 gene. Although the majority of alterations of SMARCB1 are acquired in tumors, a heterozygous germline alteration is seen in one third of patients and defines the rhabdoid tumors predisposition syndrome. Penetrance is almost complete and the vast majority of germline alterations of SMARCB1 are acquired de novo but rare familial cases with a healthy carrier have also been described. Since the advent of more sensitive molecular analysis technologies such as next-generation sequencing (NGS), the number of mosaicisms of genes involved in genetic diseases discovered from blood samples has increased considerably. The aim of our study was to explore the mosaicisms of SMARCB1 in the blood 1/ of children with rhabdoid tumors with at least one alteration of SMARCB1 previously identified in the tumor but not found in the blood with old-fashioned low-sensitivity technologies and 2/ in parents of children with heterozygous germline alteration of SMARCB1. We analyzed a custom NGS panel which covers the SMARCB1 gene with an average depth of 1.500X on blood samples of 111 children with rhabdoid tumors and 32 parents collected at the Institut Curie since 1999. The mosaicism rate found in index cases was 11.7% (13/111) and 3.1% (1/32) in parents. The variant allele frequency vary from 0.8% to 12.9%. Our results also indicate to be cautious about the possible confounding effect of circulating tumor DNA. This hitherto underestimated SMARCB1 mosaicism rate should motivate an optimization of the genetic counseling as well as the oncological monitoring of these children and thus have a significant medical impact given the catastrophic prognosis of rhabdoid tumors.
RARE-03. Vision-related quality of life in patients with childhood-onset craniopharyngioma – results of KRANIOPHARYNGEOM 2000 / 2007
Authors: Sowithayasakul Panjarat , Boekhoff Svenja , Friedrich Carsten , Müller Hermann L
Published: June 2022

BACKGROUND: Quality of life (QoL) has become a critical component of therapeutic outcomes in the survivors of childhood-onset adamantinomatous craniopharyngioma (CP) patients. Visual deficiency adversely affects daily functioning and QoL. This study aimed to report the vision-related QoL and associated factors in CP patients. PATIENTS AND METHODS: 120 CP patients recruited between 2001 and 2019 in KRANIOPHARYNGEOM 2000/2007 were included in this study. Mean age at CP diagnosis was 10 years (range: 1.3-16.8 years); age at study was 18 years (range: 6-33 years). The primary outcome measures were QoL assessed by PEDQOL, functional capacity measured by Fertigkeitenskala Münster-Heidelberg ability scale and risk factors associated with decreased QoL during 3 years follow-up in CP patients with and without visual impairment (VI). RESULTS: The most common presenting symptoms were headache (42%), VI (20%), and growth retardation (12%). After diagnosis, VI defined as visual acuity less than 20/40, was found in 87 (70%) patients. Ophthalmologic examination and PEDQOL score were evaluated at three months, one year, and 3 years after CP diagnosis. A difference in the parental assessment of CP patient social functioning within the family was found between patients with and without VI in the first year (48 vs. 39, p=0.017) and third year (43 vs. 37, p=0.011). For the PEDQOL domain autonomy, a difference in self- (51 vs. 45, p=0.029) and parental (47 vs. 42, p=0.048) assessment was observed 3 years after diagnosis. In terms of risk factors, tumor volume more than 21 mm3, incomplete tumor resection and optic atrophy at initial presentation were associated with VI during follow-up. CONCLUSIONS: VI has an impact on QoL after CP. Accordingly, early detection of VI, together with the assessment of the patient’s QoL and the provision of adequate support during follow-up plays an important role in minimizing adverse late effects after CP.
RARE-06. Expanding the clinical and molecular spectrum of pituitary blastoma
Authors: Pak-Yin Liu Anthony , Li Kay Ka-Wai , Chow Chit , Chan Shing , Leung Alex Wing-Kwan , Shing Matthew Ming-Kong , To Ka-Fai , Chan Danny Tat-Ming , Chan Godfrey Chi-Fung , Ng Ho-Keung
Published: June 2022

Pituitary blastomas (PitB) are rare DICER1-associated tumors that occur exclusively in children < 2 years of age. They are locally aggressive tumors that are driven by a combination of germline and somatic alterations involving DICER1. Here, we report two patients with pituitary neoplasms that expand the clinical and molecular spectrum of PitB. Patient 1 presented at 10 months with diabetes insipidus and was initially diagnosed with pituitary ependymoblastoma. She received debulking, chemotherapy and focal radiation with complete response achieved, but unfortunately died at the age of 8 years due to cerebral edema. Patient 2 was a survivor of infant leukemia who was treated with chemotherapy and then further chemotherapy with cranial irradiation at relapse. The patient was then diagnosed at the age of 8 years with pituitary CNS-PNET, which was treated with craniospinal irradiation and chemotherapy, and had remained in remission for 6 years. Review of histology in both cases indicates presence of neuroendocrine lobules, primitive cells, and Rathke pouch-like glandular structures, with high Ki67, ACTH and PRAME positivity compatible with PitB. Next-generation sequencing revealed presence of two DICER1 mutations (germline frameshifting and somatic missense) in Patient 1, and one somatic missense DICER1 mutation plus loss of heterozygosity in Patient 2 (no germline alteration). Surprisingly, C19MC amplification was also detected in Patient 1. Methylation profiling confirms clustering among our samples and PitB references, but not ETMR references. MicroRNA array revealed decrease in mature microRNA expression and preferential down-regulation of 5p/3p species in tumor compared to control pituitary tissue. In all, PitBs may present with clinical and molecular characteristics not conforming with the classical descriptions. It might be prudent to consider sequencing for DICER1 alteration in pediatric pituitary tumors to facilitate diagnosis of this increasingly heterogeneous rare entity.
RARE-10. Neurocutaneous melanocytosis-associated hydrocephalus: the MSK experience from 2001-2022
Authors: Rodriguez Scarlett , Sener Ugur , Elmore Kevin , Haque Sofia , Suser Stephanie , Greenfield Jeffrey , Donzelli Maria , DePass Courtnee , Pugh John , Porter Joanne , Meeker Nathan , Wells Elizabeth , Marghoob Ashfaq , Khakoo Yasmin
Published: June 2022

OBJECTIVE: We hypothesize that patients with neurocutaneous melanocytosis-associated melanoma and ventriculoperitoneal shunts are at risk of developing intraperitoneal spread of melanoma. BACKGROUND: Neurocutaneous melanocytosis, a rare condition characterized by excessive proliferation and deposition of melanocytes in the leptomeninges and brain parenchyma, typically occurs in children with large congenital melanocytic nevi and multiple smaller congenital nevi. These patients are at heightened risk for developing NRAS+ melanomas in the central nervous system, which in turn may lead to symptomatic hydrocephalus requiring cerebrospinal fluid diversion for symptom relief. METHODS: Retrospective single-institution study of patients with histologically or radiographically confirmed NCM evaluated at Memorial Sloan Kettering Cancer Center (MSKCC) from 2001-2022. RESULTS: Of the 47 patients with a diagnosis of NCM, 44 patients had symptomatic neurological complications. Eleven patients developed hydrocephalus, 10 had CNS melanoma, and required ventriculoperitoneal shunt placement. Nine of the 10 patients ultimately died of their disease. Three patients were diagnosed with intraperitoneal melanoma, though data are unavailable for the remaining eight. CONCLUSIONS: All (n=11) patients with NCM-associated CNS melanoma required VP shunts for symptomatic relief. Ten of these patients died within 4.3 years of VP shunt placement, with a range of 1 month to 13.5 years prior to succumbing to their disease. While the intraperitoneal pathology remains unknown for 7 of the cases, 3 had confirmed intraperitoneal melanoma, suggesting that VP shunts provided the conduit to CNS melanoma seeding of the peritoneum. Obtaining baseline abdominal imaging studies prior to VP shunt placement may be helpful in the follow-up of these patients.
RARE-11. 60 years single centre experience of craniopharyngioma management
Authors: Rodrigues Beryl , Adamski Jenny , English Martin , Curry Helen , Solanki Guirish , Peet Andrew , Apps John
Published: June 2022

Adamantinomatous craniopharyngiomas are challenging intracranial tumours associated with significant morbidity. Management includes surgery and radiotherapy, with a shift towards more conservative surgery in recent years, aimed at preserving hypothalamic function. The West Midlands Regional Children’s Tumour Registry collects detailed clinical, pathological and follow up information on patients treated within the region from 1957. 52 cases (26 male, 26 female) of craniopharyngioma treated at Birmingham Children’s Hospital 1957-2018, were identified, with further clinical details obtained from patient records, where available. Visual symptoms were the commonest presenting feature (63%), followed by headache (48%), vomiting (31%), neurological symptoms (31%) and features of endocrine disorders (21%) with a median symptom duration of 6 months (range <1-24). Initial management was with gross total resection (GTR) in 14 patients, subtotal resection in 22 patients and subtotal resection with adjuvant radiotherapy in seven patients. Two patients received radiotherapy without resection, and five patients underwent cystic drainage procedures alone. Two patients initially underwent shunt insertion alone, but received radiotherapy at progression. 30 (58%) patients underwent relapse/progression, with a median time to progression of 1.2 years (range 0.2-6.3). 15 had further surgery. Radiotherapy was used in 14/15 patients who had not previously received radiotherapy, with the other undergoing a GTR. To date 10 patients have died, nine from tumour related reasons and one from pulmonary embolism. Where data was available at follow up, all patients had at least one endocrinopathy, with 38/45 patients having diabetes insipidus. Hypothalamic obesity was identified in 14/36 (39%) patients with sufficient records, with this more common in those undergoing GTR (7/9 (78%)) compared to other surgical procedures (7/27)(26%)(p<0.05). Three patients have developed neurovascular complications and three fatty liver disease. This experience is consistent with the literature and supports the increasing usage of hypothalamic sparing surgical management.
RARE-13. Clinical management and functional and survival outcomes in pediatric craniopharyngioma, a patient and family perspective
Authors: Marshall Emily , Cheng Sylvia , Crowley Julia , McCormack Shana , Rood Brian , Hankinson Todd , DeCuypere Michael , Lam Sandy , Goldman Stewart , Boekhoff Svenja , Muller Hermann L , Velasco Ryan , Storm Phillip B , Resnick Adam , Prados Michael , Mueller Sabine , Kline Cassie , Malbari Fatema
Published: June 2022

Craniopharyngiomas are rare, histologically benign, sellar/parasellar tumors with significant tumor and therapy related morbidity and impairment in quality of life (QOL). We report survey results from patients/families affected by childhood-onset craniopharyngioma to identify opportunities for improvement in management. An anonymous REDCap survey was distributed via social media and clinic visits to patients/families of craniopharyngioma survivors. Survey questions investigated perspectives on clinical management and functional and survival outcomes at initial diagnosis and recurrence. A total of 159 patients/families completed the survey, 40% (n=64) reported craniopharyngioma recurrence. For primary craniopharyngioma, maximal safe resection was the most frequent treatment reported (n=84), followed by partial resection (n=40), radiation (n=8), biopsy (n=5), and chemotherapy (n=3). Most patients (n=120) decided on a treatment plan within one week, 63 (40%) decided in one day. For recurrent craniopharyngioma, maximal safe resection and radiation were the most frequent interventions (n=33 each), followed by partial resection (n=13), chemotherapy (n=4) and biopsy (n=2). Multiple treatment options and/or participation in a clinical trial were offered to similar numbers of patients across primary and recurrent diagnoses (~21% for each). Most recurrent craniopharyngioma patients decided on management within one week (n=43). Long term effects related to tumor and treatment were identified as the primary concern in all respondents. The most common deficits for all patients were neuro-endocrine followed by vision and neurocognition problems. Neuro-endocrine complications were self-reported as the biggest impact on QOL. Families reported that they would prefer treatment options with the potential for improved QOL, even if these options also carried an increased risk of recurrence. Craniopharyngioma continues to be predominantly treated with surgery and radiation initially and with recurrence. Survivors have multiple comorbidities, with an interest in targeted therapies that preserve QOL. Novel therapies to prevent co-morbidities and provide long term benefits are necessary and upcoming.
RARE-21Sox2 plays an important role in choroid plexus tumor development
Authors: Faltings Lukas , Sarowar Tamanna , Virga James , Singh Navleen , Kwa Brightlyn , Zhao Haotian
Published: June 2022

Choroid plexus (CP) tumors are rare primary brain neoplasms found most commonly in children and are thought to arise from CP epithelial cells. Sox2 is a transcription factor that not only plays a role in development in the ventricular zone, CP, and roof plate, but also contributes to cancer stemness, tumorigenesis, and drug resistance. Gene expression studies demonstrate aberrant Sox2 expression in human CP tumors, suggesting a role in tumor development. A subset of CP tumors exhibit abnormal NOTCH pathway activity. Using animal models, we previously show that sustained NOTCH activity leads to CP tumors. Immunofluorescence, RT-qPCR, and RNA scope assays have revealed increased Sox2 levels in NOTCH-driven CP tumors compared to wild type CP in mice. To investigate the role of Sox2 in CP tumors, we eliminated Sox2 expression in NOTCH-driven CP tumors. Loss of Sox2 almost completely blocked NOTCH-driven CP tumor growth in these mice, supporting a role for Sox2 in these tumors. Ciliation regulation is one proposed functional pathway for tumorigenesis in CP tumors. Using immunofluorescence assays for cilia (ARL13b) and aquaporin transport protein 1 (AQP1) in combination with super resolution microscopy, we observe a stark contrast between wild type CP epithelial cells which are multiciliated and homogeneously express AQP1, indicative of normal epithelial differentiation, compared to NOTCH-driven CP tumors consisting of mono-ciliated cells with loss of AQP1 expression. In Sox2-deficient NOTCH-driven CP tumors, we observe tumor cells remain mono-ciliated and AQP1-negative, indicating that Sox2 loss does not affect the ciliation machinery. Together this warrants further study into the mechanisms of Sox2 functions in CP tumors. By unraveling the role of Sox2 in CP tumors, we may better understand their origin and biology to ultimately design improved treatment options.
RARE-24. The use of novelin vitro models to study adamantinomatous craniopharyngioma disease biology and drug response
Authors: Trudeau Tammy , Prince Eric , Chatain Oscar , Chee Keanu , Jackson Eric , Limbrick David , Naftel Robert , Feldstein Neil , Grant Gerald , Ginn Kevin , Niazi Toba , Smith Amy , Kilburn Lindsay , Chern Joshua , Drapeau Annie , Lam Sandi , Johnston James , Dudley Roy , Staulcup Susan , Hankinson Todd
Published: June 2022

BACKGROUND: Challenges around the design and investigation of cell culture models of adamantinomatous craniopharyngioma (ACP) have arisen from the cellular heterogeneity of these tumors, with populations that harbor disparate requirements in culture. Novel approaches to in vitro modeling of ACP are needed. METHODS: Intraoperatively collected tumor specimens were mechanically digested and plated under conditions tailored to the cell population of interest. ACP tumor-derived fibroblasts and epithelial cells were isolated using serum-containing and keratinocyte-specific media respectively. ACP-derived epithelial cells were immortalized via SV40 virus transfection and puromycin treatment for stable cell-line generation. Cell line validation included immunofluorescence with markers appropriate for the cell population of interest. RNA sequencing of cell lines was compared to ACP transcriptome reference data. Cell typing was conducted using short tandem repeat sequencing. RESULTS: ACP fibroblasts and ACP epithelial cells maintained spindle-like and cobblestone morphologies respectively, even after 4 passages. Immunofluorescence staining confirmed high levels of Vimentin expression in ACP-derived fibroblasts, and panCK and B-catenin in ACP-derived epithelial cells. Point mutation in exon 3 of the CTNNB1 gene was identified in ACP-derived epithelial cells. CONCLUSION: Initial limits related to cell line development in ACP may be addressed through the isolation and culture-specific ACP cell populations. This experience demonstrates the maintenance of validated markers of the cell populations of interest ex vivo. While preliminary, such cell lines offer promise as tools for the identification and study of potential therapeutic vulnerabilities in ACP.
RARE-26. Evaluating the clinical utility of DNA methylation profiling for choroid plexus tumors
Authors: Macrae Cassie , Gampel Bradley , Yeo Kee Kiat , Lidov Hart , Chi Susan , Wright Karen , Fehnel Katie , Baird Lissa , Aldape Ken , Alexandrescu Sanda , Clymer Jessica
Published: June 2022

INTRODUCTION: Choroid plexus tumors (CPT) are rare, potentially aggressive CNS tumors with defined histologic criteria for grading. In recent years, several patients within our practice have demonstrated discordance between histological diagnosis and clinical behavior. DNA methylation profiling has emerged as a potential diagnostic adjunct for aiding clinical planning and treatment approach. In this study, we sought to retrospectively evaluate the clinical utility of DNA methylation profiling within our cohort of patients with CPT. METHODS: We performed a retrospective chart review of all patients with choroid plexus tumors treated at Dana-Farber / Boston’s Children’s Cancer and Blood Disorder Center between 1990-2021, evaluating the histology, treatment approach, and clinical outcome. Available tissue samples were sent to the National Institute of Health for DNA methylation profiling. RESULTS: Seventeen patients with CPT were identified. Median age at diagnosis was 1.8 years (range: 0.4-27.7). Histologic diagnosis included choroid plexus papilloma (CPP; n=4), atypical choroid plexus papilloma (aCPP; n=5), and choroid plexus carcinoma (CPC, n=8). DNA methylation in an initial subset placed these tumors with the pediatric type A (n=5), pediatric type B (n=6), and adult (n=1) subgroups. For one patient, methylation profiling returned as unclassifiable (possibly representing an alternative diagnosis). Discrepancies with the histologic grade were noted in several cases: one patient diagnosed with CPP grouped with pediatric type B CPT on methylation analysis, had rapid recurrence, and a diagnosis of CPC was made on a re-resection specimen; another patient with aCPP with concerning features was classified as pediatric type A by methylation, and is without evidence of disease after initial complete resection. Survival outcomes based on histologic diagnosis and molecular subgroups are compared and reported. CONCLUSION: DNA methylation profiling is a useful tool for the diagnosis of CPT and may have the potential to guide clinical planning and management.
RARE-31. Radiation induced malignancy associated with pediatric craniopharyngioma: a single institution experience
Authors: Chien Franklin , Schniederjan Matthew , Castellino R C , Janss Anna , Esiashvili Natia , Eaton Bree , Indelicato Daniel , Mazewski Claire
Published: June 2022

BACKGROUND: Therapy for pediatric craniopharyngioma, a rare suprasellar tumor, includes surgical resection with consideration for intracranial radiation. Radiation is associated with increased risk of secondary malignancies. Between 2000 and 2021, 81 pediatric patients with craniopharyngioma were treated at our institution; 3 of 54 (5.6%) who received radiation therapy(RT) developed secondary malignancy within the treatment field. CASE DESCRIPTIONS: In all 3 cases, initial imaging demonstrated cystic/solid suprasellar mass and underwent resection; pathology revealed calcifications and wet keratin consistent with craniopharyngioma. None had known cancer predispositions. The first patient (male), presented at 4-years-old with headaches. He underwent subtotal resection (STR) with cyst fenestration(w/CF) and received 55.8Gy photon 3D-Conformal RT. Six-years later, the tumor progressed (edge of RT field). Patient underwent a second STRw/CF and fractionated RT(50.4Gy). Both pathologies were consistent with(c/w) papillary craniopharyngioma. Eight-years from first RT, progression occurred again within the RTfield; pathology revealed an (adeno)squamous carcinoma. The second patient, a 5-year-old female, presented with vision loss, underwent partial resection and received 54Gy focal proton therapy for adamantinonatous craniopharyngioma. Almost 5-years later, an unresectable right basal ganglia/globus pallidus mass was noted in the 30-54Gy field. Pathology was c/w anaplastic astrocytoma(AA). The third, a 9-year-old female was treated with 54Gy photon radiation and 7 years later had evidence of increasing mass. Pathology revealed high-grade-diffuse-glioma(HGDG). Molecular analysis of AA/HGDG both revealed PDGFRA amplification and CDKN2A/B homozygous loss. DISCUSSION/CONCLUSION: Malignant CNS tumors are reported following radiotherapy for a variety of primary CNS lesions. While radiation is a valuable therapy in achieving long-term disease control of pediatric craniopharyngiomas, it is important to understand the risk of developing secondary malignant neoplasms. Our report adds to the body of literature describing secondary malignancies post radiation therapy for the treatment of pediatric craniopharyngioma.
DIPG-01. Targeting p300: A master regulator of childhood diffuse intrinsic pontine glioma (DIPG)
Authors: Chowdhury Pinki , Lober Robert
Published: June 2022

Diffuse intrinsic pontine gliomas (DIPG) are heterogeneous, highly aggressive, accounting for ~10% of all childhood central nervous system tumors that are poorly understood. The heterogeneity of the cell populations could contribute to major variability in tumor growth and chemoresistance followed by inadequate treatment responses. Approximately 80% of DIPG patients harbor mutations on lysine-27 of histone-3 (H3K27M), which forms heterotypic nucleosomes with normal, acetylated-H3K27 (H3K27ac), and colocalizes with bromodomain proteins to sites of active transcription. p300 (or EP300) bromodomain has recently been exploited as a potential therapeutic target in other cancer, with a small molecule inhibitor showing antiproliferative activity. Bromodomain inhibitors (BrDi) have been suggested as a potential therapeutic target for DIPG too which showed antiproliferative activity in vitro. We therefore hypothesize that p300 bromodomain inhibition is a potential therapeutic strategy that must be urgently explored in DIPG. p300 is a multi-domain enhancer protein, expressed in 80% of gliomas, responsible for H3K27ac mark and localizes to sites of H3K27ac enrichment, where it serves as an essential binding partner of histones to various transcription factors. p300 interacts with BMI-1, a polycomb repressive complex 1 (PRC1) group of transcription factor, through an intermediate protein NANOG, which promotes cell proliferation in head-neck squamous cell carcinoma. However, immunohistochemical study demonstrated that there is no NANOG expression found in DIPG patient samples. Therefore, we propose that p300 and BMI-1 interact with each other directly or through an intermediate molecule, essential for tumor growth and thus can be considered as a possible therapeutic target for DIPG. The overall outcome of this research will reveal the unexplored mechanisms of DIPG cell proliferation to overcome chemoresistance in sick children and develop drug/combination against this deadly malignancy, which can be used for future clinical trials.
DIPG-05. How do potassium channels contribute to the growth and invasion of highly-aggressive brain cancers?
Authors: Boyle Yasmin , Fletcher Emily , Johns Terrance
Published: June 2022

Glioblastoma (GBM) is a lethal form of high-grade glioma with a dismal median survival time of just 15 months. Characterised by its’ highly diffuse and intrinsic growth pattern, GBM invades the healthy brain at an alarming rate. While diffuse midline glioma (DMG) is a much rarer disease, it is even more lethal, with a 5-year survival rate of just 2%. Though GBM is largely diagnosed in adults, DMG primarily affects young children aged 5-9 years and accounts for 10% of all childhood CNS cancers. Thus, while rare, DMG is highly aggressive and currently has no effective treatments to extend survival times beyond the median of 9 months. There is a pressing need for the development of novel and improved targeted therapies for each of these devastating diseases. Ion channels have long been implicated in the progression of numerous cancer types, due to their integral roles in proliferation, cell cycle transition, apoptosis, migration, and cellular plasticity. Voltage-gated potassium channels (VGKCs), in particular, have strong links to the key processes of proliferation, migration and invasion in GBM tumours. Given that the majority of GBM-related deaths are attributed to secondary tumours and metastasis, targeting proteins that are integral to these processes could result in reduced recurrence. Preliminary evidence suggests a potential role for the VGKC subtypes Kv5.1, Kv7.2, and the Kv4 subfamily in GBM and DMG, due to observed upregulation of these genes in both patient-derived cell lines and tumour samples. These particular VGKC subtypes are highly novel with regards to these cancers, while their significant upregulation suggests they may be associated with tumour progression. Thus, we aim to further explore the relationship between ion channel function and tumourigenesis in GBM and DMG, with a specific focus on VGKC subtypes and their potential therapeutic value.
DIPG-06. Uncovering the FACTs in Diffuse Midline Glioma (DMG)
Authors: Holliday Holly , Ehteda Anahid , Khan Aaminah , Krug Brian , Rouaen Jourdin R C , Jayatilleke Nisitha , Mayoh Chelsea , Vittorio Orazio , Jabado Nada , Tsoli Maria , Ziegler David S
Published: June 2022

Effective treatments are urgently needed for the incurable paediatric brainstem tumour Diffuse Midline Glioma (DMG). Most DMGs contain Histone H3 mutations (H3K27M), which produce extensive epigenetic dysregulation by inhibiting EZH2-mediated trimethylation of H3K27 (H3K27me3). Global depletion of the repressive H3K27me3 modification results in aberrantly open chromatin and is central to DMG tumorigenesis. Thus, targeting the epigenome is a promising avenue of treatment for DMG. We found that targeting the histone chaperone complex Facilitates Chromatin Transcription (FACT) with the curaxin drug CBL0137 to have potent pre-clinical efficacy against DMG, leading to a paediatric Phase I/II clinical trial for CBL0137 which includes a DMG/DIPG cohort (NCT04870944). In this project we aim to elucidate CBL0137’s molecular mechanism in DMG. FACT is critical for maintaining chromatin homeostasis during DNA replication, transcription, and repair. We therefore hypothesised that FACT maintains the aberrant epigenetic landscape resulting from H3K27M. Consistently, we found CBL0137 to be more cytotoxic against H3K27M-mutant, compared to H3-WT or isogenic DMG cells lacking the mutation. Furthermore, FACT directly interacts with H3K27M, and FACT inhibition increases both EZH2 catalytic activity and global H3K27me3 levels. We are now using ChIP-seq to discover the genome-wide distribution of FACT and H3K27M, and will interrogate the consequence of CBL0137 treatment on the epigenome and transcriptome. Preliminary results suggest that FACT is located at certain genes co-occupied by H3K27M, the active histone mark H3K27ac, and the BET protein BRD4. This implies that FACT is involved in maintaining open chromatin and perhaps transcription at these regions. Combining CBL0137 with other epigenetic drugs, such as BET inhibitors, could therefore represent a rational therapeutic opportunity for DMG. This work will ultimately inform mechanism-based targeted therapies to combine with CBL0137 to improve its efficacy and uncover valuable new insights into DMG epigenetics and pathobiology.
DIPG-09. Diffuse Midline Glioma-Adaptive Combinatory Trial (DMG-ACT): A biology-driven platform trial in pediatric and young adult patients with diffuse midline glioma
Authors: Franson Andrea , Kline Cassie , Molinaro Annette , Zhang Yalan , Hitchner Kelly , Koschmann Carl , Nazarian Javad , Mueller Sabine
Published: June 2022

BACKGROUND: Despite advances in our understanding of the biology of diffuse midline gliomas (DMGs), little progress has been made in improving outcomes. Therapy development is limited by lack of preclinical data across multiple model systems and laboratories; limited knowledge about blood-brain barrier penetrance; and lack of multi-agent therapies. We aim to address these issues through DMG-ACT, where we translate robust preclinical data using ONC201 as the therapy backbone in a multi-arm, combination strategy within an innovative trial design. DESIGN: DMG-ACT is an open-label, multi-institutional trial of combination therapy for patients with DMG between 2 and 39 years of age. The trial utilizes a novel Bayesian drug combination platform design with adaptive shrinkage (ComPAS). ComPAS allows ongoing assessment of therapy efficacy with borrowing of data across different arms and the ability to eliminate ineffective drug combinations and add new promising combinations throughout the trial. ONC201 is the backbone therapy in each arm and given in combination with other agents that show additive or synergistic benefit in preclinical testing. Patients enter into one of three cohorts: newly diagnosed (Cohort 1), post-radiation (Cohort 2), and relapsed/progressive (Cohort 3). The cohorts offer a target validation option to assess intratumoral pharmacokinetics and pharmacodynamics of drug given prior to tumor biopsy. Cohort 1 and 3 offer radiation or re-irradiation with concomitant single agent therapy followed by maintenance combination therapy. The primary efficacy endpoints are median progression-free survival at 6 months (Cohort 1 and 2) and overall survival at 7 months (Cohort 3). Exploratory endpoints include intratumoral drug concentration, toxicity profile of combination therapy during radiation therapy, toxicity profile and efficacy of combination therapy, CSF and ctDNA analysis, and health related quality of life, cognitive, and patient/proxy-reported outcome measures. This trial was successfully launched in November 2021 with updates to be presented at the meeting.
DIPG-10. A Phase I trial of panobinostat following radiation therapy in children with diffuse intrinsic pontine glioma (DIPG) or H3K27M-mutated thalamic diffuse midline glioma (DMG): Report from the Pediatric Brain Tumor Consortium (PBTC-047)
Authors: Monje Michelle , Cooney Tabitha , Glod John , Huang Jie , Baxter Patricia , Vinitsky Anna , Kilburn Lindsay , Robison Nathan J , Peer Cody J , Figg William D , Fouladi Maryam , Fangusaro Jason , Onar-Thomas Arzu , Dunkel Ira J , Warren Katherine E
Published: June 2022

INTRODUCTION: Panobinostat is an oral HDAC inhibitor with pre-clinical activity against DIPG. The phase I study in children with progressive DIPG (stratum 1) defined the maximum-tolerated dose (MTD) as 10 mg/m2 administered 3x/week, 3 weeks on/1 week off. Herein, we report results of stratum 2, involving children with non-progressive DIPG/DMG using an alternative schedule. Primary objectives were to describe the toxicity profile and define the MTD; secondary objectives were to describe progression-free survival (PFS) and overall survival (OS). PATIENTS AND METHODS: Patients with non-progressive DIPG or H3K27M-mutated thalamic DMG were eligible >14 days following standard radiation therapy only. Panobinostat was given every other day, 3x/week, on alternate weeks. Patients who received at least one dose of panobinostat were evaluable for toxicity. Four dose levels (DL) were evaluated: DL1 (16mg/m2/dose), DL2 (22 mg/m2/dose), DL3 (28 mg/m2/dose) and DL4 (36 mg/m2/dose). Dose escalation was determined by a continuous reassessment method. Correlative studies included pharmacokinetics obtained on course 1, day 1, and day 3 prior to subsequent dosing. RESULTS: Thirty-four eligible patients (median age, 7.6 [3-16] years) were enrolled with 29 evaluable for dose finding; DL1, n=3; DL2, n=10; DL3, n=11; DL4, n=5. The primary toxicities were myelosuppression and gastrointestinal. Eight DLTs occurred: DL2, Grade 3 thrombocytopenia (n=1); DL3, Grade 4 neutropenia (n=3), Grade 4 neutropenia and Grade 4 thrombocytopenia, (n=1); DL4, Grade 2 nausea (n=1), Grade 3 increased ALT (n=1), Grade 4 thrombocytopenia (n=1). Median PFS from drug initiation was 4.4 (1-11.2) months; median OS from diagnosis was 11.7 (4.5-25) months. These did not significantly differ from the PBTC historical cohort (PFS, p-value 0.4967; OS, p-value 0.6457). CONCLUSION: The MTD of panobinostat administered on this schedule to children with non-progressive DIPG/DMG is 22 mg/m2/dose. The primary DLT was myelosuppression. There was no significant improvement in PFS or OS in this cohort.
DIPG-11. DIPGs show robust MHC class I expression but low lymphoid and high myeloid infiltration partly reversible by DNA methyltransferase inhibition
Authors: Mishra Deepak , Wang Jie , Chen Chun-Yu , Zhu Xiaoting , Kumar Shiva Senthil , McHugh Todd , Cripe Timothy , Fouladi Maryam , Drissi Rachid
Published: June 2022

Diffuse intrinsic pontine gliomas (DIPGs) are the most aggressive tumors of the central nervous system in children. Median survival of patients is less than one year post-diagnosis. Radiotherapy remains the only standard treatment but is rarely curative. Immunotherapy is an emerging and promising treatment strategy for children with DIPG. However, general immunotherapy has not lived up to its promise to treat many cancers, including DIPGs, in part due to incomplete understanding of the barriers posed by the tumor microenvironment. We therefore evaluated the immune cell infiltration in a syngeneic mouse model of DIPG and in DIPG tumors collected from patients. We evaluated the expression profiles of T lymphocytes and myeloid cell markers in a cohort of 28 DIPG tumors compared to matched normal tissue specimens. Our data indicate that the expression of MHC I components in DIPG tumors is similar to that of matched normal tissue. Moreover, the well-known immune checkpoint, PD-L1, was not overexpressed in DIPG tumors. Using immunohistochemistry, we demonstrated only rare infiltration of lymphocytes but high enrichment of myeloid cells in tumor tissue. We found similar results in a syngeneic mouse model of DIPG. Collectively, our results indicate that DIPG tumors harbor rare lymphocytes and are enriched in myeloid cells. Recent studies have demonstrated that DNA methyltransferase inhibitors prime some tumors for more effective checkpoint blockade by activating expression of human endogenous retroviruses and sparking a T-cell mediated immune response through a process called “viral mimicry.” We tested the effect of decitabine in a syngeneic mouse model of DIPG. Decitabine reduced the tumor growth kinetics compared to vehicle but was unable to induce the recruitment of lymphoid cells in DIPG tumors. Importantly, we observed a noticeable reduction in the myeloid component of the DIPG microenvironment suggesting a possible role of myeloid cells in tumor growth and progression.
DIPG-14. Neogenin Knockout Stops the Invasion and Dissemination of Diffuse Intrinsic Pontine Gliomasin vivo.
Authors: Sesen Julie , Ghalali Aram , Martinez Tyra , Driscoll Jessica , Fehnel Katie Pricola , Smith Edward
Published: June 2022

INTRODUCTION: Diffuse Intrinsic Pontine Glioma (DIPG) is a devastating high-grade glioma localized in the brainstem that occurs almost exclusively in children. The significant mortality rates of DIPG stem largely from its capacity to invade adjacent normal brain. We have previously published studies identifying the axon guidance factor, neogenin as a key driver regulating brain tumor cell invasion in medulloblastoma, glioblastoma and DIPG in vitro. Here we expand this work with an in vivo model to validate neogenin as promising inducer of DIPG invasion. METHODS: CRISPR-Cas9 system was used to knock-out the neogenin gene in DIPG cells. After validation of the knockout (KO) in vitro by RTqPCR, Western blot, and immunofluroresence, the cells were orthotopically implanted in the pons of nude mice (n=8/group) with a stereotactic frame. Tumor growth and dissemination were monitored using the In Vivo Imaging System (IVIS). Then, brains and spinal cords were collected, and H&E staining was performed. RESULTS:In vitro studies confirmed the KO of neogenin in DIPG cells. In vivo studies revealed that the neogenin total KO group had no progression of the tumor without any dissemination after 4-6 weeks, in marked contrast to the WT group, which exhibited steady growth and widespread dissemination into the spine. Immunohistochemistry confirmed the IVIS results. CONCLUSION: Expanding on previous work, this data suggests that neogenin is a major contributor of DIPG cell invasion and dissemination. The blockade of neogenin in DIPG cells demonstrates the potential utility of neogenin as potential therapeutic target, capable of reducing tumor growth and stopping tumor cell dissemination.