Genome sequencing of pediatric medulloblastoma links catastrophic DNA rearrangements with TP53 mutations.

Genomic rearrangements are thought to occur progressively during tumor development. Recent findings, however, suggest an alternative mechanism, involving massive chromosome rearrangements in a one-step catastrophic event termed chromothripsis. We report the whole-genome sequencing-based analysis of a Sonic-Hedgehog medulloblastoma (SHH-MB) brain tumor from a patient with a germline TP53 mutation (Li-Fraumeni syndrome), uncovering massive, complex chromosome rearrangements. Integrating TP53 status with microarray and deep sequencing-based DNA rearrangement data in additional patients reveals a striking association between TP53 mutation and chromothripsis in SHH-MBs. Analysis of additional tumor entities substantiates a link between TP53 mutation and chromothripsis, and indicates a context-specific role for p53 in catastrophic DNA rearrangements. Among these, we observed a strong association between somatic TP53 mutations and chromothripsis in acute myeloid leukemia. These findings connect p53 status and chromothripsis in specific tumor types, providing a genetic basis for understanding particularly aggressive subtypes of cancer.

Dabrafenib plus Trametinib in Pediatric Glioma with BRAF V600 Mutations.

BACKGROUND: Detection of the BRAF V600E mutation in pediatric low-grade glioma has been associated with a lower response to standard chemotherapy. In previous trials, dabrafenib (both as monotherapy and in combination with trametinib) has shown efficacy in recurrent pediatric low-grade glioma with BRAF V600 mutations, findings that warrant further evaluation of this combination as first-line therapy. METHODS: In this phase 2 trial, patients with pediatric low-grade glioma with BRAF V600 mutations who were scheduled to receive first-line therapy were randomly assigned in a 2:1 ratio to receive dabrafenib plus trametinib or standard chemotherapy (carboplatin plus vincristine). The primary outcome was the independently assessed overall response (complete or partial response) according to the Response Assessment in Neuro-Oncology criteria. Also assessed were the clinical benefit (complete or partial response or stable disease for ≥24 weeks) and progression-free survival. RESULTS: A total of 110 patients underwent randomization (73 to receive dabrafenib plus trametinib and 37 to receive standard chemotherapy). At a median follow-up of 18.9 months, an overall response occurred in 47% of the patients treated with dabrafenib plus trametinib and in 11% of those treated with chemotherapy (risk ratio, 4.31; 95% confidence interval [CI], 1.7 to 11.2; P<0.001). Clinical benefit was observed in 86% of the patients receiving dabrafenib plus trametinib and in 46% receiving chemotherapy (risk ratio, 1.88; 95% CI, 1.3 to 2.7). The median progression-free survival was significantly longer with dabrafenib plus trametinib than with chemotherapy (20.1 months vs. 7.4 months; hazard ratio, 0.31; 95% CI, 0.17 to 0.55; P<0.001). Grade 3 or higher adverse events occurred in 47% of the patients receiving dabrafenib plus trametinib and in 94% of those receiving chemotherapy. CONCLUSIONS: Among pediatric patients with low-grade glioma with BRAF V600 mutations, dabrafenib plus trametinib resulted in significantly more responses, longer progression-free survival, and a better safety profile than standard chemotherapy as first-line therapy. (Funded by Novartis; ClinicalTrials.gov number, NCT02684058.).

Germline Elongator mutations in Sonic Hedgehog medulloblastoma.

Cancer genomics has revealed many genes and core molecular processes that contribute to human malignancies, but the genetic and molecular bases of many rare cancers remains unclear. Genetic predisposition accounts for 5 to 10% of cancer diagnoses in children1,2, and genetic events that cooperate with known somatic driver events are poorly understood. Pathogenic germline variants in established cancer predisposition genes have been recently identified in 5% of patients with the malignant brain tumour medulloblastoma3. Here, by analysing all protein-coding genes, we identify and replicate rare germline loss-of-function variants across ELP1 in 14% of paediatric patients with the medulloblastoma subgroup Sonic Hedgehog (MBSHH). ELP1 was the most common medulloblastoma predisposition gene and increased the prevalence of genetic predisposition to 40% among paediatric patients with MBSHH. Parent-offspring and pedigree analyses identified two families with a history of paediatric medulloblastoma. ELP1-associated medulloblastomas were restricted to the molecular SHHα subtype4 and characterized by universal biallelic inactivation of ELP1 owing to somatic loss of chromosome arm 9q. Most ELP1-associated medulloblastomas also exhibited somatic alterations in PTCH1, which suggests that germline ELP1 loss-of-function variants predispose individuals to tumour development in combination with constitutive activation of SHH signalling. ELP1 is the largest subunit of the evolutionarily conserved Elongator complex, which catalyses translational elongation through tRNA modifications at the wobble (U34) position5,6. Tumours from patients with ELP1-associated MBSHH were characterized by a destabilized Elongator complex, loss of Elongator-dependent tRNA modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response, consistent with loss of protein homeostasis due to Elongator deficiency in model systems7-9. Thus, genetic predisposition to proteome instability may be a determinant in the pathogenesis of paediatric brain cancers. These results support investigation of the role of protein homeostasis in other cancer types and potential for therapeutic interference.

The molecular landscape of ETMR at diagnosis and relapse.

Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis1. Here we collected 193 primary ETMRs and 23 matched relapse samples to investigate the genomic landscape of this distinct tumour type. We found that patients with tumours in which the proposed driver C19MC2-4 was not amplified frequently had germline mutations in DICER1 or other microRNA-related aberrations such as somatic amplification of miR-17-92 (also known as MIR17HG). Whole-genome sequencing revealed that tumours had an overall low recurrence of single-nucleotide variants (SNVs), but showed prevalent genomic instability caused by widespread occurrence of R-loop structures. We show that R-loop-associated chromosomal instability can be induced by the loss of DICER1 function. Comparison of primary tumours and matched relapse samples showed a strong conservation of structural variants, but low conservation of SNVs. Moreover, many newly acquired SNVs are associated with a mutational signature related to cisplatin treatment. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.

Synergy of retinoic acid and BH3 mimetics in MYC(N)-driven embryonal nervous system tumours.

BACKGROUND: Certain paediatric nervous system malignancies have dismal prognoses. Retinoic acid (RA) is used in neuroblastoma treatment, and preclinical data indicate potential benefit in selected paediatric brain tumour entities. However, limited single-agent efficacy necessitates combination treatment approaches. METHODS: We performed drug sensitivity profiling of 76 clinically relevant drugs in combination with RA in 16 models (including patient-derived tumouroids) of the most common paediatric nervous system tumours. Drug responses were assessed by viability assays, high-content imaging, and apoptosis assays and RA relevant pathways by RNAseq from treated models and patient samples obtained through the precision oncology programme INFORM (n = 2288). Immunoprecipitation detected BCL-2 family interactions, and zebrafish embryo xenografts were used for in vivo efficacy testing. RESULTS: Group 3 medulloblastoma (MBG3) and neuroblastoma models were highly sensitive to RA treatment. RA induced differentiation and regulated apoptotic genes. RNAseq analysis revealed high expression of BCL2L1 in MBG3 and BCL2 in neuroblastomas. Co-treatments with RA and BCL-2/XL inhibitor navitoclax synergistically decreased viability at clinically achievable concentrations. The combination of RA with navitoclax disrupted the binding of BIM to BCL-XL in MBG3 and to BCL-2 in neuroblastoma, inducing apoptosis in vitro and in vivo. CONCLUSIONS: RA treatment primes MBG3 and NB cells for apoptosis, triggered by navitoclax cotreatment.

LOGGIC/FIREFLY-2: a phase 3, randomized trial of tovorafenib vs. chemotherapy in pediatric and young adult patients with newly diagnosed low-grade glioma harboring an activating RAF alteration.

BACKGROUND: Pediatric low-grade glioma (pLGG) is essentially a single pathway disease, with most tumors driven by genomic alterations affecting the mitogen-activated protein kinase/ERK (MAPK) pathway, predominantly KIAA1549::BRAF fusions and BRAF V600E mutations. This makes pLGG an ideal candidate for MAPK pathway-targeted treatments. The type I BRAF inhibitor, dabrafenib, in combination with the MEK inhibitor, trametinib, has been approved by the United States Food and Drug Administration for the systemic treatment of BRAF V600E-mutated pLGG. However, this combination is not approved for the treatment of patients with tumors harboring BRAF fusions as type I RAF inhibitors are ineffective in this setting and may paradoxically enhance tumor growth. The type II RAF inhibitor, tovorafenib (formerly DAY101, TAK-580, MLN2480), has shown promising activity and good tolerability in patients with BRAF-altered pLGG in the phase 2 FIREFLY-1 study, with an objective response rate (ORR) per Response Assessment in Neuro-Oncology high-grade glioma (RANO-HGG) criteria of 67%. Tumor response was independent of histologic subtype, BRAF alteration type (fusion vs. mutation), number of prior lines of therapy, and prior MAPK-pathway inhibitor use. METHODS: LOGGIC/FIREFLY-2 is a two-arm, randomized, open-label, multicenter, global, phase 3 trial to evaluate the efficacy, safety, and tolerability of tovorafenib monotherapy vs. current standard of care (SoC) chemotherapy in patients < 25 years of age with pLGG harboring an activating RAF alteration who require first-line systemic therapy. Patients are randomized 1:1 to either tovorafenib, administered once weekly at 420 mg/m2 (not to exceed 600 mg), or investigator's choice of prespecified SoC chemotherapy regimens. The primary objective is to compare ORR between the two treatment arms, as assessed by independent review per RANO-LGG criteria. Secondary objectives include comparisons of progression-free survival, duration of response, safety, neurologic function, and clinical benefit rate. DISCUSSION: The promising tovorafenib activity data, CNS-penetration properties, strong scientific rationale combined with the manageable tolerability and safety profile seen in patients with pLGG led to the SIOPe-BTG-LGG working group to nominate tovorafenib for comparison with SoC chemotherapy in this first-line phase 3 trial. The efficacy, safety, and functional response data generated from the trial may define a new SoC treatment for newly diagnosed pLGG. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05566795. Registered on October 4, 2022.

Combination drug screen identifies synergistic drug interaction of BCL-XL and class I histone deacetylase inhibitors in MYC-amplified medulloblastoma cells.

PURPOSE: Patients with MYC-amplified Group 3 medulloblastoma (MB) (subtype II) show poor progression-free survival rates. Class I histone deacetylase inhibitors (HDACi) are highly effective for the treatment of MYC-amplified MB in vitro and in vivo. Drug combination regimens including class I HDACi may represent an urgently needed novel treatment approach for this high risk disease. METHODS: A medium-throughput in vitro combination drug screen was performed in three MYC-amplified and one non-MYC-amplified MB cell line testing 75 clinically relevant drugs alone and in combination with entinostat. The drug sensitivity score (DSS) was calculated based on metabolic inhibition quantified by CellTiter-Glo. The six top synergistic combination hits were evaluated in a 5 × 5 combination matrix and a seven-ray design. Synergy was validated and characterized by cell counts, caspase-3-like-activity and poly-(ADP-ribose)-polymerase-(PARP)-cleavage. On-target activity of drugs was validated by immunoprecipitation and western blot. BCL-XL dependency of the observed effect was explored with siRNA mediated knockdown of BCL2L1, and selective inhibition with targeted compounds (A-1331852, A-1155463). RESULTS: 20/75 drugs effectively reduced metabolic activity in combination with entinostat in all three MYC-amplified cell lines (DSS ≥ 10). The combination entinostat and navitoclax showed the strongest synergistic interaction across all MYC-amplified cell lines. siRNA mediated knockdown of BCL2L1, as well as targeted inhibition with selective inhibitors showed BCL-XL dependency of the observed effect. Increased cell death was associated with increased caspase-3-like-activity. CONCLUSION: Our study identifies the combination of class I HDACi and BCL-XL inhibitors as a potential new approach for the treatment of MYC-amplified MB cells.

Rebound growth of BRAF mutant pediatric glioma cells after MAPKi withdrawal is associated with MAPK reactivation and secretion of microglia-recruiting cytokines.

INTRODUCTION: Patients with pediatric low-grade gliomas (pLGGs), the most common primary brain tumors in children, can often benefit from MAPK inhibitor (MAPKi) treatment. However, rapid tumor regrowth, also referred to as rebound growth, may occur once treatment is stopped, constituting a significant clinical challenge. METHODS: Four patient-derived pediatric glioma models were investigated to model rebound growth in vitro based on viable cell counts in response to MAPKi treatment and withdrawal. A multi-omics dataset (RNA sequencing and LC-MS/MS based phospho-/proteomics) was generated to investigate possible rebound-driving mechanisms. Following in vitro validation, putative rebound-driving mechanisms were validated in vivo using the BT-40 orthotopic xenograft model. RESULTS: Of the tested models, only a BRAFV600E-driven model (BT-40, with additional CDKN2A/Bdel) showed rebound growth upon MAPKi withdrawal. Using this model, we identified a rapid reactivation of the MAPK pathway upon MAPKi withdrawal in vitro, also confirmed in vivo. Furthermore, transient overactivation of key MAPK molecules at transcriptional (e.g. FOS) and phosphorylation (e.g. pMEK) levels, was observed in vitro. Additionally, we detected increased expression and secretion of cytokines (CCL2, CX3CL1, CXCL10 and CCL7) upon MAPKi treatment, maintained during early withdrawal. While increased cytokine expression did not have tumor cell intrinsic effects, presence of these cytokines in conditioned media led to increased attraction of microglia cells in vitro. CONCLUSION: Taken together, these data indicate rapid MAPK reactivation upon MAPKi withdrawal as a tumor cell intrinsic rebound-driving mechanism. Furthermore, increased secretion of microglia-recruiting cytokines may play a role in treatment response and rebound growth upon withdrawal, warranting further evaluation.

DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

Erdafitinib in patients with advanced solid tumours with FGFR alterations (RAGNAR): an international, single-arm, phase 2 study.

BACKGROUND: FGFR alterations are reported across various malignancies and might act as oncogenic drivers in multiple histologies. Erdafitinib is an oral, selective pan-FGFR tyrosine kinase inhibitor with activity in FGFR-altered advanced urothelial carcinoma. We aimed to evaluate the safety and activity of erdafitinib in previously treated patients with FGFR-altered advanced solid tumours. METHODS: The single-arm, phase 2 RAGNAR study was conducted at 156 investigative centres (hospitals or oncology practices that are qualified oncology study centres) across 15 countries. The study consisted of four cohorts based on tumour histology and patient age; the results reported in this Article are for the primary cohort of the study, defined as the Broad Panel Cohort, which was histology-agnostic. We recruited patients aged 12 years or older with advanced or metastatic tumours of any histology (except urothelial cancer) with predefined FGFR1-4 alterations (mutations or fusions according to local or central testing). Eligible patients had disease progression on at least one previous line of systemic therapy and no alternative standard therapy available to them, and an Eastern Cooperative Oncology Group performance status of 0-1 (or equivalent for adolescents aged 12-17 years). Patients received once-daily oral erdafitinib (8 mg/day with provision for pharmacodynamically guided up-titration to 9 mg/day) on a continuous 21-day cycle until disease progression or intolerable toxicity. The primary endpoint was objective response rate by independent review committee according to Response Evaluation Criteria In Solid Tumors (RECIST), version 1.1, or Response Assessment In Neuro-Oncology (RANO). The primary analysis was conducted on the treated population of the Broad Panel Cohort. This ongoing study is registered with ClinicalTrials.gov, number NCT04083976. FINDINGS: Patients were recruited between Dec 5, 2019, and Feb 15, 2022. Of 217 patients treated with erdafitinib, 97 (45%) patients were female and 120 (55%) were male. The data cutoff was Aug 15, 2022. At a median follow-up of 17·9 months (IQR 13·6-23·9), an objective response was observed in 64 (30% [95% CI 24-36]) of 217 patients across 16 distinct tumour types. The most common grade 3 or higher treatment-emergent adverse events related to erdafitinib were stomatitis (25 [12%]), palmar-plantar erythrodysaesthesia syndrome (12 [6%]), and hyperphosphataemia (11 [5%]). The most commonly occurring serious treatment-related adverse events (grade 3 or higher) were stomatitis in four (2%) patients and diarrhoea in two (1%). There were no treatment-related deaths. INTERPRETATION: RAGNAR results show clinical benefit for erdafitinib in the tumour-agnostic setting in patients with advanced solid tumours with susceptible FGFR alterations who have exhausted other treatment options. These results support the continued development of FGFR inhibitors in patients with advanced solid tumours. FUNDING: Janssen Research & Development.

Neurosurgical morbidity in pediatric supratentorial midline low-grade glioma: Results from the German LGG studies.

Surgical resection is a mainstay of treatment for pediatric low-grade glioma (LGG) within all current therapy algorithms, yet associated morbidity is scarcely reported. As supratentorial midline (SML) interventions are particularly challenging, we investigated the frequency of neurosurgical complications/new impairments aiming to identify their risk factors. Records were retrospectively analyzed from 318 patients with SML-LGG from successive German multicenter LGG studies, undergoing surgery between May 1998 and June 2020. Exactly 537 operations (230 resections, 167 biopsies, 140 nontumor procedures) were performed in 318 patients (54% male, median age: 7.6 years at diagnosis, 9.5 years at operation, 11% NF1, 42.5% optic pathway glioma). Surgical mortality rate was 0.93%. Applying the Drake classification, postoperative surgical morbidity was observed following 254/537 (47.3%) and medical morbidity following 97/537 (18.1%) patients with a 40.1% 30-day persistence rate for newly developed neurological deficits (65/162). Neuroendocrine impairment affected 53/318 patients (16.7%), visual deterioration 34/318 (10.7%). Postsurgical morbidity was associated with patient age <3 years at operation, tumor volume ≥80 cm3 , presence of hydrocephalus, complete resection, surgery in centers with less than median reported tumor-related procedures and during the earlier study period between 1998 and 2006, while the neurosurgical approach, tumor location, NF1 status or previous nonsurgical treatment were not. Neurosurgery-associated morbidity was frequent in pediatric patients with SML-LGG undergoing surgery in the German LGG-studies. We identified patient- and institution-associated factors that may increase the risk for complications. We advocate that local multidisciplinary teams consider the planned extent of resection and surgical skills.

Identification of low and very high-risk patients with non-WNT/non-SHH medulloblastoma by improved clinico-molecular stratification of the HIT2000 and I-HIT-MED cohorts.

Molecular groups of medulloblastoma (MB) are well established. Novel risk stratification parameters include Group 3/4 (non-WNT/non-SHH) methylation subgroups I-VIII or whole-chromosomal aberration (WCA) phenotypes. This study investigates the integration of clinical and molecular parameters to improve risk stratification of non-WNT/non-SHH MB. Non-WNT/non-SHH MB from the HIT2000 study and the HIT-MED registries were selected based on availability of DNA-methylation profiling data. MYC or MYCN amplification and WCA of chromosomes 7, 8, and 11 were inferred from methylation array-based copy number profiles. In total, 403 non-WNT/non-SHH MB were identified, 346/403 (86%) had a methylation class family Group 3/4 methylation score (classifier v11b6) ≥ 0.9, and 294/346 (73%) were included in the risk stratification modeling based on Group 3 or 4 score (v11b6) ≥ 0.8 and subgroup I-VIII score (mb_g34) ≥ 0.8. Group 3 MB (5y-PFS, survival estimation ± standard deviation: 41.4 ± 4.6%; 5y-OS: 48.8 ± 5.0%) showed poorer survival compared to Group 4 (5y-PFS: 68.2 ± 3.7%; 5y-OS: 84.8 ± 2.8%). Subgroups II (5y-PFS: 27.6 ± 8.2%) and III (5y-PFS: 37.5 ± 7.9%) showed the poorest and subgroup VI (5y-PFS: 76.6 ± 7.9%), VII (5y-PFS: 75.9 ± 7.2%), and VIII (5y-PFS: 66.6 ± 5.8%) the best survival. Multivariate analysis revealed subgroup in combination with WCA phenotype to best predict risk of progression and death. The integration of clinical (age, M and R status) and molecular (MYC/N, subgroup, WCA phenotype) variables identified a low-risk stratum with a 5y-PFS of 94 ± 5.7 and a very high-risk stratum with a 5y-PFS of 29 ± 6.1%. Validation in an international MB cohort confirmed the combined stratification scheme with 82.1 ± 6.0% 5y-PFS in the low and 47.5 ± 4.1% in very high-risk groups, and outperformed the clinical model. These newly identified clinico-molecular low-risk and very high-risk strata, accounting for 6%, and 21% of non-WNT/non-SHH MB patients, respectively, may improve future treatment stratification.

Glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA): a molecularly distinct brain tumor type with recurrent NTRK gene fusions.

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors.

Class I HDAC inhibition reduces DNA damage repair capacity of MYC-amplified medulloblastoma cells.

PURPOSE: MYC-driven Group 3 medulloblastoma (MB) (subtype II) is a highly aggressive childhood brain tumor. Sensitivity of MYC-driven MB to class I histone deacetylase inhibitors (HDACi) has been previously demonstrated in vitro and in vivo. In this study we characterize the transcriptional effects of class I HDACi in MYC-driven MB and explore beneficial drug combinations. METHODS: MYC-amplified Group 3 MB cells (HD-MB03) were treated with class I HDACi entinostat. Changes in the gene expression profile were quantified on a microarray. Bioinformatic assessment led to the identification of pathways affected by entinostat treatment. Five drugs interfering with these pathways (olaparib, idasanutlin, ribociclib, selinexor, vinblastine) were tested for synergy with entinostat in WST-8 metabolic activity assays in a 5 × 5 combination matrix design. Synergy was validated in cell count and flow cytometry experiments. The effect of entinostat and olaparib on DNA damage was evaluated by γH2A.X quantification in immunoblotting, fluorescence microscopy and flow cytometry. RESULTS: Entinostat treatment changed the expression of genes involved in 22 pathways, including downregulation of DNA damage response. The PARP1 inhibitors olaparib and pamiparib showed synergy with entinostat selectively in MYC-amplified MB cells, leading to increased cell death, decreased viability and increased formation of double strand breaks, as well as increased sensitivity to additional induction of DNA damage by doxorubicin. Non-MYC-amplified MB cells and normal human fibroblasts were not susceptible to this triple treatment. CONCLUSION: Our study identifies the combination of entinostat with olaparib as a new potential therapeutic approach for MYC-driven Group 3 MB.

Generation of patient-derived pediatric pilocytic astrocytoma in-vitro models using SV40 large T: evaluation of a modeling workflow.

PURPOSE: Although pediatric low-grade gliomas (pLGG) are the most common pediatric brain tumors, patient-derived cell lines reflecting pLGG biology in culture are scarce. This also applies to the most common pLGG subtype pilocytic astrocytoma (PA). Conventional cell culture approaches adapted from higher-grade tumors fail in PA due to oncogene-induced senescence (OIS) driving tumor cells into arrest. Here, we describe a PA modeling workflow using the Simian Virus large T antigen (SV40-TAg) to circumvent OIS. METHODS: 18 pLGG tissue samples (17 (94%) histological and/or molecular diagnosis PA) were mechanically dissociated. Tumor cell positive-selection using A2B5 was perfomed in 8/18 (44%) cases. All primary cell suspensions were seeded in Neural Stem Cell Medium (NSM) and Astrocyte Basal Medium (ABM). Resulting short-term cultures were infected with SV40-TAg lentivirus. Detection of tumor specific alterations (BRAF-duplication and BRAF V600E-mutation) by digital droplet PCR (ddPCR) at defined time points allowed for determination of tumor cell fraction (TCF) and evaluation of the workflow. DNA-methylation profiling and gene-panel sequencing were used for molecular profiling of primary samples. RESULTS: Primary cell suspensions had a mean TCF of 55% (+/- 23% (SD)). No sample in NSM (0/18) and ten samples in ABM (10/18) were successfully transduced. Three of these ten (30%) converted into long-term pLGG cell lines (TCF 100%), while TCF declined to 0% (outgrowth of microenvironmental cells) in 7/10 (70%) cultures. Young patient age was associated with successful model establishment. CONCLUSION: A subset of primary PA cultures can be converted into long-term cell lines using SV40-TAg depending on sample intrinsic (patient age) and extrinsic workflow-related (e.g. type of medium, successful transduction) parameters. Careful monitoring of sample-intrinsic and extrinsic factors optimizes the process.

Class I HDAC inhibitor entinostat synergizes with PLK1 inhibitors in MYC-amplified medulloblastoma cells.

PURPOSE: We and others have demonstrated that MYC-amplified medulloblastoma (MB) cells are susceptible to class I histone deacetylase inhibitor (HDACi) treatment. However, single drug treatment with HDACi has shown limited clinical efficacy. We hypothesized that addition of a second compound acting synergistically with HDACi may enhance efficacy. METHODS: We used a gene expression dataset to identify PLK1 as a second target in MB cells and validated the relevance of PLK1 in MB. We measured cell metabolic activity, viability, and cycle progression in MB cells after treatment with PLK1-specific inhibitors (PLK1i). Chou-Talalay synergy calculations were used to determine the nature of class I HDACi entinostat and PLK1i interaction which was validated. Finally, the clinical potential of the combination was assessed in the in vivo experiment. RESULTS: MYC-amplified tumor cells are highly sensitive towards treatment with ATP-competitive PLK1i as a monotherapy. Entinostat and PLK1i in combination act synergistically in MYC-driven MB cells, exerting cytotoxic effects at clinically relevant concentrations. The downstream effect is exerted via MYC-related pathways, pointing out the potential of MYC amplification as a clinically feasible predictive biomarker for patient selection. While entinostat significantly extended survival of mice implanted with orthotopic MYC-amplified MB PDX, there was no evidence of the improvement of survival when treating the animals with the combination. CONCLUSION: The combination of entinostat and PLK1i showed synergistic interaction in vitro, but not in vivo. Therefore, further screening of blood-brain barrier penetrating PLK1i is warranted to determine the true potential of the combination as no on-target activity was observed after PLK1i volasertib treatment in vivo.

Beyond germline genetic testing - heterozygous pathogenic variants in PMS2 in two children with Osteosarcoma and Ependymoma.

BACKGROUND: Lynch syndrome (LS) is not considered part of childhood cancer predisposition syndromes. CASE PRESENTATION: Analysis of a pediatric osteosarcoma (OS) displayed hypermutation (16.8), alternative lengthening of telomeres (ALT), loss of PMS2 expression in tumor tissue (retained in non-neoplastic cells), PMS2 loss of heterozygosity (LOH), and high-degree of microsatellite instability (MSI) tested by PCR. A heterozygous duplication c.1076dup p.(Leu359Phefs*6) in exon 10 of NM_000535.6:PMS2 was detected by SNV analysis in peripheral blood, confirming diagnosis of LS in the patient. The tumor molecular features suggest LS-associated development of OS. In a second case, whole-genome sequencing identified a heterozygous SNV c.1 A > T p.? in exon 1 of PMS2 in tumor and germline material of a girl with ependymoma. Tumor analysis displayed evidence for ALT and low mutational burden (0.6), PMS2 expression was retained, MSI was low. Multiplex ligation-dependent probe amplification identified no additional PMS2 variant and germline MSI testing did not reveal increased gMSI ratios in the patient´s lymphocytes. Thus, CMMRD was most closely excluded and our data do not suggest that ependymoma was related to LS in the child. CONCLUSIONS: Our data suggest that the LS cancer spectrum may include childhood cancer. The importance of LS in pediatric cancers necessitates prospective data collection. Comprehensive molecular workup of tumor samples is necessary to explore the causal role of germline genetic variants.

iTReX: Interactive exploration of mono- and combination therapy dose response profiling data.

High throughput screening methods, measuring the sensitivity and resistance of tumor cells to drug treatments have been rapidly evolving. Not only do these screens allow correlating response profiles to tumor genomic features for developing novel predictors of treatment response, but they can also add evidence for therapy decision making in precision oncology. Recent analysis methods developed for either assessing single agents or combination drug efficacies enable quantification of dose-response curves with restricted symmetric fit settings. Here, we introduce iTReX, a user-friendly and interactive Shiny/R application, for both the analysis of mono- and combination therapy responses. The application features an extended version of the drug sensitivity score (DSS) based on the integral of an advanced five-parameter dose-response curve model and a differential DSS for combination therapy profiling. Additionally, iTReX includes modules that visualize drug target interaction networks and support the detection of matches between top therapy hits and the sample omics features to enable the identification of druggable targets and biomarkers. iTReX enables the analysis of various quantitative drug or therapy response readouts (e.g. luminescence, fluorescence microscopy) and multiple treatment strategies (drug treatments, radiation). Using iTReX we validate a cost-effective drug combination screening approach and reveal the application's ability to identify potential sample-specific biomarkers based on drug target interaction networks. The iTReX web application is accessible at https://itrex.kitz-heidelberg.de.

DNA methylation-based classifier and gene expression signatures detect BRCAness in osteosarcoma.

Although osteosarcoma (OS) is a rare cancer, it is the most common primary malignant bone tumor in children and adolescents. BRCAness is a phenotypical trait in tumors with a defect in homologous recombination repair, resembling tumors with inactivation of BRCA1/2, rendering these tumors sensitive to poly (ADP)-ribose polymerase inhibitors (PARPi). Recently, OS was shown to exhibit molecular features of BRCAness. Our goal was to develop a method complementing existing genomic methods to aid clinical decision making on administering PARPi in OS patients. OS samples with DNA-methylation data were divided to BRCAness-positive and negative groups based on the degree of their genomic instability (n = 41). Methylation probes were ranked according to decreasing variance difference between two groups. The top 2000 probes were selected for training and cross-validation of the random forest algorithm. Two-thirds of available OS RNA-Seq samples (n = 17) from the top and bottom of the sample list ranked according to genome instability score were subjected to differential expression and, subsequently, to gene set enrichment analysis (GSEA). The combined accuracy of trained random forest was 85% and the average area under the ROC curve (AUC) was 0.95. There were 449 upregulated and 1,079 downregulated genes in the BRCAness-positive group (fdr < 0.05). GSEA of upregulated genes detected enrichment of DNA replication and mismatch repair and homologous recombination signatures (FWER < 0.05). Validation of the BRCAness classifier with an independent OS set (n = 20) collected later in the course of study showed AUC of 0.87 with an accuracy of 90%. GSEA signatures computed for this test set were matching the ones observed in the training set enrichment analysis. In conclusion, we developed a new classifier based on DNA-methylation patterns that detects BRCAness in OS samples with high accuracy. GSEA identified genome instability signatures. Machine-learning and gene expression approaches add new epigenomic and transcriptomic aspects to already established genomic methods for evaluation of BRCAness in osteosarcoma and can be extended to cancers characterized by genome instability.

The early evolutionary landscape of osteosarcoma provides clues for targeted treatment strategies.

Osteosarcomas are aggressive primary tumors of bone that are typically detected in locally advanced stages; however, which genetic mutations drive the cancer before its clinical detection remain unknown. To identify these events, we performed longitudinal genome-sequencing analysis of 12 patients with metastatic or refractory osteosarcoma. Phylogenetic and molecular clock analyses were carried out next to identify actionable mutations, and these were validated by integrating data from additional 153 osteosarcomas and pre-existing functional evidence from mouse PDX models. We found that the earliest and thus clinically most promising mutations affect the cell cycle G1 transition, which is guarded by cyclins D3, E1, and cyclin-dependent kinases 2, 4, and 6. Cell cycle G1 alterations originate no more than a year before the primary tumor is clinically detected and occur in >90% and 50% of patients of the discovery and validation cohorts, respectively. In comparison, other cancer driver mutations could be acquired at any evolutionary stage and often do not become pervasive. Consequently, our data support that the repertoire of actionable mutations present in every osteosarcoma cell is largely limited to cell cycle G1 mutations. Since they occur in mutually exclusive combinations favoring either CDK2 or CDK4/6 pathway activation, we propose a new genomically-based algorithm to direct patients to correct clinical trial options. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.