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SIOP Free Paper Session (FPS)
Session Type
SIOP Free Paper Session (FPS)
Room
Hall 6
Date
17.10.2020, Saturday
Session Time
08:00 AM - 09:30 AM

RAPID MOLECULAR SUBGROUPING OF MEDULLOBLASTOMA BASED ON DNA METHYLATION BY NANOPORE SEQUENCING

Session Type
SIOP Free Paper Session (FPS)
Date
17.10.2020, Saturday
Session Time
08:00 AM - 09:30 AM
Room
Hall 6
Lecture Time
08:00 AM - 08:10 AM

Abstract

Background and Aims

Medulloblastoma (MB) can be classified into four molecular subgroups (WNT group, SHH group, group 3, and group 4). The gold standard of assignment of molecular subgroup through DNA methylation profiling uses Illumina EPIC array. However, this tool has some limitation in terms of cost and timing, in order to get the results soon enough for clinical use. We present an alternative DNA methylation assay based on nanopore sequencing efficient for rapid, cheaper, and reliable subgrouping of clinical MB samples.

Methods

Low-depth whole genome with long-read single-molecule nanopore sequencing was used to simultaneously assess copy number profile and MB subgrouping based on DNA methylation. The DNA methylation data generated by Nanopore sequencing were compared to a publicly available reference cohort comprising over 2,800 brain tumors including the four subgroups of MB (Capper et al. Nature ; 2018) to generate a score that estimates a confidence with a tumor group assignment.

Results

Among the 24 MB analyzed with nanopore sequencing (six WNT, nine SHH, five group 3, and four group 4), all of them were classified in the appropriate subgroup established by expression-based Nanostring subgrouping. In addition to the subgrouping, we also examine the genomic profile. Furthermore, all previously identified clinically relevant genomic rearrangements (mostly MYC and MYCN amplifications) were also detected with our assay.

Conclusions

Here we are confirming the full reliability of nanopore sequencing as a novel rapid and cheap assay for methylation-based MB subgrouping. We now plan to implement this technology to other embryonal tumors of the central nervous system.

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POOR SURVIVAL IN DNA-REPLICATION REPAIR DEFICIENT (RRD) MEDULLOBLASTOMA AND CNS-EMBRYONAL TUMORS IS RELATED TO HIGH-RISK GENOMIC FEATURES: A REPORT FROM THE INTERNATIONAL RRD CONSORTIUM

Abstract

Background and Aims

Mutations in mismatch repair (MMR) and DNA-polymerase (POL) genes lead to DNA replication repair deficiency (RRD), resulting in a growing group of previously under-recognized childhood brain tumors. Medulloblastoma and embryonal tumors are rarely reported. Their biological and clinical significance is unknown.

Methods

Clinical details for children <18 years with embryonal tumors, registered in the International RRD Consortium, were retrieved from their primary physicians. Cases were centrally reviewed, and high-throughput sequencing performed on the Illumina platform. Medulloblastoma subgrouping was performed using Nano-string and DNA-methylation arrays. Genomic signature profiles were extracted. Clinical variables and genomic data were analysed using the SPSS and R-statistical environments.

Results

Twenty-six tumors were centrally reviewed to confirm medulloblastoma (n=18), embryonal-tumor, NOS (n=5), and three glioblastoma (excluded). Embryonal tumors were observed at median age of 7-years (IQR: 5;11). All but one exhibited clinical cues of germline RRD (café-au-lait macule, or a significant family history), 60% had consanguinity, with synchronous/metachronous tumors reported in 40%.

Medulloblastomas with RRD exhibited anaplastic histology (50%), SHH-subgroup (78%), majority with TP53-mutation (50% among SHH, and 80% overall). Importantly, 70% harboured POLE/POLD1 mutations, resulting in median tumor mutation burden of 164 mut/mb. POL-mutated tumors were significantly ultra-hypermutated (>100 mut/mb) than tumors with MMR-deficiency alone (p=0.015). Despite having localized disease at diagnosis, the median survival for the entire cohort was 17-months (95% CI: 10 to 23). Predicted 3 year overall survival was 37% for medulloblastoma, with no survivors among other embryonal tumors.

Conclusions

This is the largest cohort of replication repair deficient medulloblastoma reported till date. The tumors demonstrate anaplasia, belong to SHH-subgroup, harbour somatic mutations in TP53 and/or POLE/POLD1, are hypermutated, and have very poor survival with current chemo-irradiation approaches. These biologically unique tumors, discovered through a robust international collaboration, expand the spectrum of high-risk TP53-mutant SHH-medulloblastoma and SHH-medulloblastoma with germline predisposition, and need novel strategies for treatment.

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PROGNOSTIC FACTORS OF TREATMENT IN CHILDREN WITH MEDULLOBLASTOMA OF MOLECULAR SUBGROUP 3.

Session Type
SIOP Free Paper Session (FPS)
Date
17.10.2020, Saturday
Session Time
08:00 AM - 09:30 AM
Room
Hall 6
Lecture Time
08:20 AM - 08:30 AM
Authors

Abstract

Background and Aims

Medulloblastoma is a heterogeneous tumor.Molecular subgroup 3 is characterized by a dismal prognosis.Aim:to evaluate the treatment results among the patients of molecular subgroup 3 and to determine the factors affecting the prognosis.

Methods

We analyzed 103patients with medulloblastoma of molecular subgroup 3.In all the patients removal of the tumor was performed.Most of them had subtotal resection.All the patients got chemoradiotherapy according to the HIT protocol.Patients with stage M0 and classic variant got a reduced dose of craniospinal irradiation;those with MYC amplification and anaplastic/large cell medulloblastoma got CSI in standard doses.There were 32girls and 71boys.Most patients were over 3 years old:75compared to 28younger than 3.38 had M0,62-М+ and 3-Mx stage.MYC amplification was found in 19patients.MYCN amplification-in 4 patients.Classic medulloblastoma was in 68patients,anaplastic/large cell-in 35.

Results

50(49%)are alive and 53(51%)died.Three patients(2.9%) had a secondary tumor:glioblastoma,anaplastic astrocytoma,osteosarcoma.The median observation time was 59 months(4 to 276 months).The five-year PFS was 0.52±0.05,the five-year OS was 0.51±0.04,and the 20-year OS was 0.47±0.05.The median survival was 84months, and the median PFS was 43months(4 to 276 months).There were no significant variations of PFS depending on the sex and age.The treatment results depended on the histological subtype:for classic medulloblastoma, the five-year PFS was 0.59;for the anaplastic/large cell subtypes-0.39(р=0.03833).The presence of metastases significantly affected the survival:PFS for stage M0 was 0.76;for stage М+,0.38 (р=0.00124).Patients with MYC amplification had a significantly worse survival compared to MYCN patients and those without MYC amplification:0.1, 0.75,and 0.61(р=0.00001).3patients with MYC amplification are alive:2 are recurrence-free, and 1 had a tumor recurrence after the treatment.Two recurrence-free patients had MGMT methylation detected.Among the patients with MYCN amplification,3 with classic medulloblastoma are alive,and one with anaplastic medulloblastoma died.

Conclusions

The results of treatment depended on the tumor subtype, presence of metastases, MYC amplification and MGMT methylation. In the absence of unfavorable factors, the survival was the same as in molecular subgroup4.

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REPLICATION REPAIR DEFICIENT MOUSE MODELS PROVIDE INSIGHT ON HYPERMUTANT BRAIN TUMOURS, MECHANISMS OF IMMUNE EVASION, AND COMBINATORIAL IMMUNOTHERAPY.

Abstract

Background and Aims

Replication repair deficiency (RRD) is the leading cause of hypermutant brain tumours in children. RRD is caused by defects in one of four mismatch repair (MMR) genes and mutations in POLE or POLD1. Such tumours are resistant to common therapeutic agents and animal models are needed to study RRD in vivo and test novel therapies like immune checkpoint inhibitors (ICIs).

Methods

To model RRD brain tumours specifically, we engineered a Pole mutant mouse model harbouring the S459F mutation (PoleS459F). We combined PoleS459F mice with conditional Msh2 knockout (Msh2LoxP) and Nestin-cre mice. Resulting tumors were characterized and subsequently used to test therapies including immune checkpoint inhibitors.

Results

All Nestin-cre+Msh2LoxP/LoxPPoleS459F/+ mice rapidly succumbed to posterior fossa brain tumours between 8.6 and 12.4 weeks. Importantly, tumours exhibited hallmark “ultrahypermutation” (~350 mutations/Mb) and the corresponding signatures characteristic of human combined MMR and POLE-proofreading glioblastoma. Interestingly, Nestin-cre+Msh2LoxP/LoxPPoleS459F/S459F mice failed to establish normal cerebella, suggesting such mutational loads may not support normal brain development. Furthermore, OLIG2-cre+Msh2LoxP/LoxPPoleS459F/+ mice failed to develop tumors.

Tumors transplanted into syngeneic vs immunocompromised animals egrafted well orthotopically in the mouse hindbrain but significantly less efficiently when engrafted subcutaneously. Furthermore, immunocompromised and subcutaneous tumors revealed striking differences in mutational burden and clonal architecture, suggestive of nonautonomous immunoediting. Finally, anti-PD1 was sufficient to treat subcutaneously engrafted tumors in immunocompetent animals.

Conclusions

This first mouse model of immunocompetent, hypermutant brain tumors can be used to uncover unique characteristics of RRD tumour evolution and allow for immune based therapeutic preclinical testing. Experiments to assess combinational ICIs and other therapeutic interventions in orthotopically transplanted tumors will also be presented.

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HIF-2ALPHA: A NEW THERAPEUTIC TARGET IN PEDIATRIC HIGH-GRADE GLIOMA.

Session Type
SIOP Free Paper Session (FPS)
Date
17.10.2020, Saturday
Session Time
08:00 AM - 09:30 AM
Room
Hall 6
Lecture Time
08:40 AM - 08:50 AM

Abstract

Background and Aims

Pediatric high-grade gliomas (pHGGs) are aggressive and incurable tumors, reasons why there is a pressing need for novel therapeutic approaches to treat them. Proactive translational studies wish to go further discovering new targetable proteins in these tumors, especially those participating to the modulation of microenvironmental extrinsic features. To do so, we were focusing on specific actors involved in hypoxic tumor environment. We looked, first, to Hypoxia-Inducible factors (HIFs) expression in a pHGG cohort to see their potential prognostic and outcome impacts. In a second step, we tested the anti-HIFs strategies.

Methods

We designed this work to determine in a collection of 45 diagnostic FFPE pHGGs (17 DIPGs, 4 gliomatosis cerebri and 24 thalamic/hemispheric tumors) and 7 relapsing samples the expression of HIF-1alpha and HIF-2alpha by immunohistochemistry and its correlation with cell metabolism (HRMAS technique). We also checked HIFs’ expression in 6 PDCLs (H3.3 mutated or wild-type). We focused on HIF-2alpha to study its role, its induction in normoxic and hypoxic microenvironments and its transcriptional targets using RNAseq, metabolomics and ChipSeq analyses. Complementary functional analyses were performed using inhibition strategies with commercialized and non-commercialized antagonists to evaluate antiproliferative and pro-apoptotic effects.

Results

We confirmed hyperexpression of HIF-1alpha in 61% of diagnostic samples and HIF-2alpha in 40%. All relapses were acquiring a HIF-2alpha hyperexpression associated to metabolomic resistant features. Only HIF-2alpha was linked to a worst outcome, reason why we focused on it. HIF-2alpha is involved in pHGG stemness features in culture and has a direct link with cell metabolism adaptation and specific interactions with histones. The use of inhibitors in a single and combinatorial strategy targeting HIF-2alpha plus another hypoxic biomarkers was highly effective for cell arrest and apoptosis and stopped cell migration.

Conclusions

Our results confirmed the central role of HIF-2alpha, which seems to be a major biomarker to be targeted in pHGGs.

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