Histone serotonylation regulates ependymoma tumorigenesis.

Bidirectional communication between tumours and neurons has emerged as a key facet of the tumour microenvironment that drives malignancy1,2. Another hallmark feature of cancer is epigenomic dysregulation, in which alterations in gene expression influence cell states and interactions with the tumour microenvironment3. Ependymoma (EPN) is a paediatric brain tumour that relies on epigenomic remodelling to engender malignancy4,5; however, how these epigenetic mechanisms intersect with extrinsic neuronal signalling during EPN tumour progression is unknown. Here we show that the activity of serotonergic neurons regulates EPN tumorigenesis, and that serotonin itself also serves as an activating modification on histones. We found that inhibiting histone serotonylation blocks EPN tumorigenesis and regulates the expression of a core set of developmental transcription factors. High-throughput, in vivo screening of these transcription factors revealed that ETV5 promotes EPN tumorigenesis and functions by enhancing repressive chromatin states. Neuropeptide Y (NPY) is one of the genes repressed by ETV5, and its overexpression suppresses EPN tumour progression and tumour-associated network hyperactivity through synaptic remodelling. Collectively, this study identifies histone serotonylation as a key driver of EPN tumorigenesis, and also reveals how neuronal signalling, neuro-epigenomics and developmental programs are intertwined to drive malignancy in brain cancer.

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.

Outcomes for children with recurrent/refractory atypical teratoid rhabdoid tumor: A single-institution study with molecular correlation.

BACKGROUND: Survival data for recurrent pediatric atypical teratoid rhabdoid tumor (ATRT) and its association to molecular groups are extremely limited. METHODS: Single-institution retrospective study of 64 children less than 21 years old with recurrent or treatment-refractory (progressive disease [PD]) ATRT treated at St. Jude Hospital from January 2000 to December 2020. Demographic, clinicopathologic, treatment, molecular grouping (SHH, TYR, and MYC) and germline data were collected. Progression-free survival (PFS2: time from PD to subsequent first progression) and overall survival (OSpostPD: time from PD to death/last follow-up) were estimated by Kaplan-Meier analysis. RESULTS: Median age at and time from initial diagnosis to PD were 2.1 years (range: 0.5-17.9 years) and 5.4 months (range: 0.5-125.6 months), respectively. Only five of 64 children (7.8%) are alive at median follow-up of 10.9 (range: 4.2-18.1) years from PD. The 2/5-year PFS2 and OSpostPD were 3.1% (±1.8%)/1.6% (±1.1%) and 20.3% (±4.8%)/7.3% (±3.5%), respectively. Children with TYR group (n = 10) had a better OSpostPD compared to those with MYC (n = 11) (2-year survival estimates: 60.0% ± 14.3% vs. 18.2% ± 9.5%; p = .019), or those with SHH (n = 21; 4.8% ± 3.3%; p = .014). In univariate analyses, OSpostPD was better with older age at diagnosis (p = .037), female gender (p = .008), and metastatic site of PD compared to local or combined sites of PD (p < .001). Two-year OSpostPD for patients receiving any salvage therapy (n = 39) post PD was 33.3% ± 7.3%. CONCLUSIONS: Children with recurrent/refractory ATRT have dismal outcomes. Older age at diagnosis, female gender, TYR group, and metastatic site of PD were associated with relatively longer survival in our study.

The molecular characteristics of low-grade and high-grade areas in desmoplastic infantile astrocytoma/ganglioglioma.

AIMS: Desmoplastic infantile astrocytomas and gangliogliomas (DIA/DIGs) are rare brain tumours of infancy. A distinctive feature of their histopathology is a combination of low-grade and high-grade features. Most DIA/DIGs can be surgically resected and have a good prognosis. However, high-grade features often dominate recurrent tumours, some of which have a poor outcome. In this study, we test the hypothesis that low-grade and high-grade areas in DIA/DIGs have distinct molecular characteristics. METHODS: Tissue samples from microdissected low-grade and high-grade areas in 12 DIA/DIGs were analysed by DNA methylation profiling, whole exome sequencing, RNA sequencing and immunohistochemistry to search for potential differences at multiple molecular levels. RESULTS: Copy number variants among tumours and between the two morphologically distinct areas were infrequent. No recurrent genetic alterations were identified across the tumour series, and high-grade areas did not have additional genetic alterations to explain their distinct morphology or biological behaviour. However, high-grade areas showed relative hypomethylation in genes downstream of the transcription factors SOX9 and LEF1 and evidence of a core SOX9 transcription network alongside activation of the BMP, WNT and MAPK signalling pathways. CONCLUSIONS: This study contributes to our knowledge of molecular genetic alterations in DIA/DIGs, uncovers molecular differences between the two distinct cell populations in these tumours and suggests potential therapeutic targets among the more proliferative cell population in DIA/DIGs.

Molecular classification and outcome of children with rare CNS embryonal tumors: results from St. Jude Children's Research Hospital including the multi-center SJYC07 and SJMB03 clinical trials.

Methylation profiling has radically transformed our understanding of tumors previously called central nervous system primitive neuro-ectodermal tumors (CNS-PNET). While this marks a momentous step toward defining key differences, reclassification has thrown treatment into disarray. To shed light on response to therapy and guide clinical decision-making, we report outcomes and molecular features of children with CNS-PNETs from two multi-center risk-adapted studies (SJMB03 for patients ≥ 3 years; SJYC07 for patients < 3 years) complemented by a non-protocol institutional cohort. Seventy patients who had a histological diagnosis of CNS-PNET or CNS embryonal tumor from one of the new categories that has supplanted CNS-PNET were included. This cohort was molecularly characterized by DNA methylation profiling (n = 70), whole-exome sequencing (n = 53), RNA sequencing (n = 20), and germline sequencing (n = 28). Clinical characteristics were detailed, and treatment was divided into craniospinal irradiation (CSI)-containing (SJMB03 and SJMB03-like) and CSI-sparing therapy (SJYC07 and SJYC07-like). When the cohort was analyzed in its entirety, no differences were observed in the 5-year survival rates even when CSI-containing therapy was compared to CSI-sparing therapy. However, when analyzed by DNA methylation molecular grouping, significant survival differences were observed, and treatment particulars provided suggestions of therapeutic response. Patients with CNS neuroblastoma with FOXR2 activation (CNS-NB-FOXR2) had a 5-year event-free survival (EFS)/overall survival (OS) of 66.7% ± 19.2%/83.3% ± 15.2%, and CIC rearranged sarcoma (CNS-SARC-CIC) had a 5-year EFS/OS both of 57.1% ± 18.7% with most receiving regimens that contained radiation (focal or CSI) and multidrug chemotherapy. Patients with high-grade neuroepithelial tumor with BCOR alteration (HGNET-BCOR) had abysmal responses to upfront chemotherapy-only regimens (5-year EFS = 0%), but survival extended with salvage radiation after progression [5-year OS = 53.6% ± 20.1%]. Patients with embryonal tumor with multilayered rosettes (ETMR) or high-grade glioma/glioblastoma multiforme (HGG/GBM) did not respond favorably to any modality (5-year EFS/OS = 10.7 ± 5.8%/17.9 ± 7.2%, and 10% ± 9.0%/10% ± 9.0%, respectively). As an accompaniment, we have assembled this data onto an interactive website to allow users to probe and query the cases. By reporting on a carefully matched clinical and molecular cohort, we provide the needed insight for future clinical management.

Subependymal giant cell astrocytomas are characterized by mTORC1 hyperactivation, a very low somatic mutation rate, and a unique gene expression profile.

Subependymal giant-cell astrocytomas (SEGAs) are slow-growing brain tumors that are a hallmark feature seen in 5-10% of patients with Tuberous Sclerosis Complex (TSC). Though histologically benign, they can cause serious neurologic symptoms, leading to death if untreated. SEGAs consistently show biallelic loss of TSC1 or TSC2. Herein, we aimed to define other somatic events beyond TSC1/TSC2 loss and identify potential transcriptional drivers that contribute to SEGA formation. Paired tumor-normal whole-exome sequencing was performed on 21 resected SEGAs from 20 TSC patients. Pathogenic variants in TSC1/TSC2 were identified in 19/21 (90%) SEGAs. Copy neutral loss of heterozygosity (size range: 2.2-46 Mb) was seen in 76% (16/21) of SEGAs (44% chr9q and 56% chr16p). An average of 1.4 other somatic variants (range 0-7) per tumor were identified, unlikely of pathogenic significance. Whole transcriptome RNA-sequencing analyses revealed 190 common differentially expressed genes in SEGA (n = 16, 13 from a prior study) in pairwise comparison to each of: low grade diffuse gliomas (n = 530) and glioblastoma (n = 171) from The Cancer Genome Atlas (TCGA) consortium, ganglioglioma (n = 10), TSC cortical tubers (n = 15), and multiple normal tissues. Among these, homeobox transcription factors (TFs) HMX3, HMX2, VAX1, SIX3; and TFs IRF6 and EOMES were all expressed >12-fold higher in SEGAs (FDR/q-value < 0.05). Immunohistochemistry supported the specificity of IRF6, VAX1, SIX3 for SEGAs in comparison to other tumor entities and normal brain. We conclude that SEGAs have an extremely low somatic mutation rate, suggesting that TSC1/TSC2 loss is sufficient to drive tumor growth. The unique and highly expressed SEGA-specific TFs likely reflect the neuroepithelial cell of origin, and may also contribute to the transcriptional and epigenetic state that enables SEGA growth following two-hit loss of TSC1 or TSC2 and mTORC1 activation.

Clear cell meningiomas are defined by a highly distinct DNA methylation profile and mutations in SMARCE1.

Clear cell meningioma represents an uncommon variant of meningioma that typically affects children and young adults. Although an enrichment of loss-of-function mutations in the SMARCE1 gene has been reported for this subtype, comprehensive molecular investigations are lacking. Here we describe a molecularly distinct subset of tumors (n = 31), initially identified through genome-wide DNA methylation screening among a cohort of 3093 meningiomas, of which most were diagnosed histologically as clear cell meningioma. This cohort was further supplemented by an additional 11 histologically diagnosed clear cell meningiomas for analysis (n = 42). Targeted DNA sequencing revealed SMARCE1 mutations in 33/34 analyzed samples, accompanied by a nuclear loss of expression determined via immunohistochemistry and a decreased SMARCE1 transcript expression in the tumor cells. Analysis of time to progression or recurrence of patients within the clear cell meningioma group (n = 14) in comparison to those with meningioma WHO grade 2 (n = 220) revealed a similar outcome and support the assignment of WHO grade 2 to these tumors. Our findings indicate the existence of a highly distinct epigenetic signature of clear cell meningiomas, separate from all other variants of meningiomas, with recurrent mutations in the SMARCE1 gene. This suggests that these tumors may arise from a different precursor cell population than the broad spectrum of the other meningioma subtypes.

Recurrent fusions in PLAGL1 define a distinct subset of pediatric-type supratentorial neuroepithelial tumors.

Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur along the entire neuroaxis. In recent years, extensive (epi-)genomic profiling efforts have identified several molecular groups of ependymoma that are characterized by distinct molecular alterations and/or patterns. Based on unsupervised visualization of a large cohort of genome-wide DNA methylation data, we identified a highly distinct group of pediatric-type tumors (n = 40) forming a cluster separate from all established CNS tumor types, of which a high proportion were histopathologically diagnosed as ependymoma. RNA sequencing revealed recurrent fusions involving the pleomorphic adenoma gene-like 1 (PLAGL1) gene in 19 of 20 of the samples analyzed, with the most common fusion being EWSR1:PLAGL1 (n = 13). Five tumors showed a PLAGL1:FOXO1 fusion and one a PLAGL1:EP300 fusion. High transcript levels of PLAGL1 were noted in these tumors, with concurrent overexpression of the imprinted genes H19 and IGF2, which are regulated by PLAGL1. Histopathological review of cases with sufficient material (n = 16) demonstrated a broad morphological spectrum of tumors with predominant ependymoma-like features. Immunohistochemically, tumors were GFAP positive and OLIG2- and SOX10 negative. In 3/16 of the cases, a dot-like positivity for EMA was detected. All tumors in our series were located in the supratentorial compartment. Median age of the patients at the time of diagnosis was 6.2 years. Median progression-free survival was 35 months (for 11 patients with data available). In summary, our findings suggest the existence of a novel group of supratentorial neuroepithelial tumors that are characterized by recurrent PLAGL1 fusions and enriched for pediatric patients.

Patient-derived orthotopic xenografts of pediatric brain tumors: a St. Jude resource.

Pediatric brain tumors are the leading cause of cancer-related death in children. Patient-derived orthotopic xenografts (PDOX) of childhood brain tumors have recently emerged as a biologically faithful vehicle for testing novel and more effective therapies. Herein, we provide the histopathological and molecular analysis of 37 novel PDOX models generated from pediatric brain tumor patients treated at St. Jude Children's Research Hospital. Using a combination of histopathology, whole-genome and whole-exome sequencing, RNA-sequencing, and DNA methylation arrays, we demonstrate the overall fidelity and inter-tumoral molecular heterogeneity of pediatric brain tumor PDOX models. These models represent frequent as well as rare childhood brain tumor entities, including medulloblastoma, ependymoma, atypical teratoid rhabdoid tumor, and embryonal tumor with multi-layer rosettes. PDOX models will be valuable platforms for evaluating novel therapies and conducting pre-clinical trials to accelerate progress in the treatment of brain tumors in children. All described PDOX models and associated datasets can be explored using an interactive web-based portal and will be made freely available to the research community upon request.

Risk-adapted therapy and biological heterogeneity in pineoblastoma: integrated clinico-pathological analysis from the prospective, multi-center SJMB03 and SJYC07 trials.

Pineoblastoma is a rare embryonal tumor of childhood that is conventionally treated with high-dose craniospinal irradiation (CSI). Multi-dimensional molecular evaluation of pineoblastoma and associated intertumoral heterogeneity is lacking. Herein, we report outcomes and molecular features of children with pineoblastoma from two multi-center, risk-adapted trials (SJMB03 for patients ≥ 3 years; SJYC07 for patients < 3 years) complemented by a non-protocol institutional cohort. The clinical cohort consisted of 58 patients with histologically diagnosed pineoblastoma (SJMB03 = 30, SJYC07 = 12, non-protocol = 16, including 12 managed with SJMB03-like therapy). The SJMB03 protocol comprised risk-adapted CSI (average-risk = 23.4 Gy, high-risk = 36 Gy) with radiation boost to the primary site and adjuvant chemotherapy. The SJYC07 protocol consisted of induction chemotherapy, consolidation with focal radiation (intermediate-risk) or chemotherapy (high-risk), and metronomic maintenance therapy. The molecular cohort comprised 43 pineal parenchymal tumors profiled by DNA methylation array (n = 43), whole-exome sequencing (n = 26), and RNA-sequencing (n = 16). Respective 5-year progression-free survival rates for patients with average-risk or high-risk disease on SJMB03 or SJMB03-like therapy were 100% and 56.5 ± 10.3% (P = 0.007); respective 2-year progression-free survival rates for those with intermediate-risk or high-risk disease on SJYC07 were 14.3 ± 13.2% and 0% (P = 0.375). Of patients with average-risk disease treated with SJMB03/SJMB03-like therapy, 17/18 survived without progression. DNA-methylation analysis revealed four clinically relevant pineoblastoma subgroups: PB-A, PB-B, PB-B-like, and PB-FOXR2. Pineoblastoma subgroups differed in age at diagnosis, propensity for metastasis, cytogenetics, and clinical outcomes. Alterations in the miRNA-processing pathway genes DICER1, DROSHA, and DGCR8 were recurrent and mutually exclusive in PB-B and PB-B-like subgroups; PB-FOXR2 samples universally overexpressed the FOXR2 proto-oncogene. Our findings suggest superior outcome amongst older children with average-risk pineoblastoma treated with reduced-dose CSI. The identification of biologically and clinically distinct pineoblastoma subgroups warrants consideration of future molecularly-driven treatment protocols for this rare pediatric brain tumor entity.

Correction to: Risk-adapted therapy and biological heterogeneity in pineoblastoma: integrated clinico-pathological analysis from the prospective, multi-center SJMB03 and SJYC07 trials.

The original version of this article unfortunately contained a typesetting error in Fig 3c. The corrected Fig. 3 is given in the following page.

Molecular subgrouping of primary pineal parenchymal tumors reveals distinct subtypes correlated with clinical parameters and genetic alterations.

Tumors of the pineal region comprise several different entities with distinct clinical and histopathological features. Whereas some entities predominantly affect adults, pineoblastoma (PB) constitutes a highly aggressive malignancy of childhood with a poor outcome. PBs mainly arise sporadically, but may also occur in the context of cancer predisposition syndromes including DICER1 and RB1 germline mutation. With this study, we investigate clinico-pathological subgroups of pineal tumors and further characterize their biological features. We performed genome-wide DNA methylation analysis in 195 tumors of the pineal region and 20 normal pineal gland controls. Copy-number profiles were obtained from DNA methylation data; gene panel sequencing was added for 93 tumors and analysis was further complemented by miRNA sequencing for 22 tumor samples. Unsupervised clustering based on DNA methylation profiling separated known subgroups, like pineocytoma, pineal parenchymal tumor of intermediate differentiation, papillary tumor of the pineal region and PB, and further distinct subtypes within these groups, including three subtypes within the core PB subgroup. The novel molecular subgroup Pin-RB includes cases of trilateral retinoblastoma as well as sporadic pineal tumors with RB1 alterations, and displays similarities with retinoblastoma. Distinct clinical associations discriminate the second novel molecular subgroup PB-MYC from other PB cases. Alterations within the miRNA processing pathway (affecting DROSHA, DGCR8 or DICER1) are found in about two thirds of cases in the three core PB subtypes. Methylation profiling revealed biologically distinct groups of pineal tumors with specific clinical and molecular features. Our findings provide a foundation for further clinical as well as molecular and functional characterization of PB and other pineal tumors, including the role of miRNA processing defects in oncogenesis.

Isomorphic diffuse glioma is a morphologically and molecularly distinct tumour entity with recurrent gene fusions of MYBL1 or MYB and a benign disease course.

The "isomorphic subtype of diffuse astrocytoma" was identified histologically in 2004 as a supratentorial, highly differentiated glioma with low cellularity, low proliferation and focal diffuse brain infiltration. Patients typically had seizures since childhood and all were operated on as adults. To define the position of these lesions among brain tumours, we histologically, molecularly and clinically analysed 26 histologically prototypical isomorphic diffuse gliomas. Immunohistochemically, they were GFAP-positive, MAP2-, OLIG2- and CD34-negative, nuclear ATRX-expression was retained and proliferation was low. All 24 cases sequenced were IDH-wildtype. In cluster analyses of DNA methylation data, isomorphic diffuse gliomas formed a group clearly distinct from other glial/glio-neuronal brain tumours and normal hemispheric tissue, most closely related to paediatric MYB/MYBL1-altered diffuse astrocytomas and angiocentric gliomas. Half of the isomorphic diffuse gliomas had copy number alterations of MYBL1 or MYB (13/25, 52%). Gene fusions of MYBL1 or MYB with various gene partners were identified in 11/22 (50%) and were associated with an increased RNA-expression of the respective MYB-family gene. Integrating copy number alterations and available RNA sequencing data, 20/26 (77%) of isomorphic diffuse gliomas demonstrated MYBL1 (54%) or MYB (23%) alterations. Clinically, 89% of patients were seizure-free after surgery and all had a good outcome. In summary, we here define a distinct benign tumour class belonging to the family of MYB/MYBL1-altered gliomas. Isomorphic diffuse glioma occurs both in children and adults, has a concise morphology, frequent MYBL1 and MYB alterations and a specific DNA methylation profile. As an exclusively histological diagnosis may be very challenging and as paediatric MYB/MYBL1-altered diffuse astrocytomas may have the same gene fusions, we consider DNA methylation profiling very helpful for their identification.

C11orf95-RELA fusions drive oncogenic NF-κB signalling in ependymoma.

Members of the nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signalling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells--the cell of origin of ependymoma--to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.

Diffuse Midline Glioma With Osseous Metastases at Diagnosis: A Case Report.

Extraneural metastasis is extremely rare in pediatric patients with high-grade glioma and carries a grim prognosis. Detection of metastases at initial presentation is even rarer. A 15-year-old adolescent girl presented with paraplegia, urinary retention, and a constellation of systemic symptoms. Imaging showed a fourth ventricular lesion, innumerable intradural lesions, leptomeningeal seeding throughout the neuraxis, and numerous osteoblastic lesions involving the spine, ribs, sternum, pelvis, humerus, and femurs. Pathology confirmed metastatic diffuse midline glioma, H3K27M-mutant. Our patient died 2 weeks after initial presentation. Further work is needed to develop effective treatment strategies for these high-risk patients.

Structure and evolution of double minutes in diagnosis and relapse brain tumors.

Double minute chromosomes are extrachromosomal circular DNA fragments frequently found in brain tumors. To understand their evolution, we characterized the double minutes in paired diagnosis and relapse tumors from a pediatric high-grade glioma and four adult glioblastoma patients. We determined the full structures of the major double minutes using a novel approach combining multiple types of supporting genomic evidence. Among the double minutes identified in the pediatric patient, only one carrying EGFR was maintained at high abundance in both samples, whereas two others were present in only trace amounts at diagnosis but abundant at relapse, and the rest were found either in the relapse sample only or in the diagnosis sample only. For the EGFR-carrying double minutes, we found a secondary somatic deletion in all copies at relapse, after erlotinib treatment. However, the somatic mutation was present at very low frequency at diagnosis, suggesting potential resistance to the EGFR inhibitor. This mutation caused an in-frame RNA transcript to skip exon 16, a novel transcript isoform absent in EST database, as well as about 700 RNA-seq of normal brains that we reviewed. We observed similar patterns involving longitudinal copy number shift of double minutes in another four pairs (diagnosis/relapse) of adult glioblastoma. Overall, in three of five paired tumor samples, we found that although the same oncogenes were amplified at diagnosis and relapse, they were amplified on different double minutes. Our results suggest that double minutes readily evolve, increasing tumor heterogeneity rapidly. Understanding patterns of double minute evolution can shed light on future therapeutic solutions to brain tumors carrying such variants.

H3.3 K27M depletion increases differentiation and extends latency of diffuse intrinsic pontine glioma growth in vivo.

Histone H3 K27M mutation is the defining molecular feature of the devastating pediatric brain tumor, diffuse intrinsic pontine glioma (DIPG). The prevalence of histone H3 K27M mutations indicates a critical role in DIPGs, but the contribution of the mutation to disease pathogenesis remains unclear. We show that knockdown of this mutation in DIPG xenografts restores K27M-dependent loss of H3K27me3 and delays tumor growth. Comparisons of matched DIPG xenografts with and without K27M knockdown allowed identification of mutation-specific effects on the transcriptome and epigenome. The resulting transcriptional changes recapitulate expression signatures from K27M primary DIPG tumors and are strongly enriched for genes associated with nervous system development. Integrated analysis of ChIP-seq and expression data showed that genes upregulated by the mutation are overrepresented in apparently bivalent promoters. Many of these targets are associated with more immature differentiation states. Expression profiles indicate K27M knockdown decreases proliferation and increases differentiation within lineages represented in DIPG. These data suggest that K27M-mediated loss of H3K27me3 directly regulates a subset of genes by releasing poised promoters, and contributes to tumor phenotype and growth by limiting differentiation. The delayed tumor growth associated with knockdown of H3 K27M provides evidence that this highly recurrent mutation is a relevant therapeutic target.

Correction to: H3.3 K27M depletion increases differentiation and extends latency of diffuse intrinsic pontine glioma growth in vivo.

The original article can be found online.

Dedifferentiation in SDH-Deficient Gastrointestinal Stromal Tumor: A Report With Histologic, Immunophenotypic, and Molecular Characterization.

One-third of gastrointestinal stromal tumors (GISTs) that lack KIT or PDGFRA mutations show succinate dehydrogenase (SDH) mutations or promoter hypermethylation. Most SDH-deficient GISTs occur in the pediatric, adolescent, or young adult setting and have unique features including predilection for the stomach, multinodular plexiform architecture, epithelioid cytology, prominence of lymphovascular invasion, and predilection for nodal metastasis. Dedifferentiation in GIST is a rare histologic change which may occur de novo or secondary to imatinib therapy and is characterized by abrupt transition of well-differentiated (WD) GIST to a subclonal anaplastic process that shows loss of immunohistochemical marks (CD117, DOG1). We describe the case of a previously healthy 18-year-old man who presented with a large gastric wall mass that contained 2 distinct morphologic populations. The first was WD and characterized by sweeping fascicles of bland spindled cells. This population abruptly transitioned to dedifferentiated (DD) foci composed of large sheets of discohesive cells that displayed a spectrum of rhabdoid and epithelioid morphologies with marked pleomorphism and mitotic activity. Immunohistochemically, the tumor showed variable staining in the 2 components with diffuse DOG-1 and CD117 positivity in the WD component and complete absence in the DD foci. SDH-B staining was lost in both components. Whole exome and transcriptome analysis was performed on tissue from both components and both showed an SDHB mutation (c.286G>A) as well as unique mutational burden and copy number profiles. Herein, we describe the first case of a DD SDH-deficient GIST with morphologic, immunophenotypic, and molecular characterization.

Risk-adapted therapy for young children with medulloblastoma (SJYC07): therapeutic and molecular outcomes from a multicentre, phase 2 trial.

BACKGROUND: Young children with medulloblastoma have a poor overall survival compared with older children, due to use of radiation-sparing therapy in young children. Radiotherapy is omitted or reduced in these young patients to spare them from debilitating long-term side-effects. We aimed to estimate event-free survival and define the molecular characteristics associated with progression-free survival in young patients with medulloblastoma using a risk-stratified treatment strategy designed to defer, reduce, or delay radiation exposure. METHODS: In this multicentre, phase 2 trial, we enrolled children younger than 3 years with newly diagnosed medulloblastoma at six centres in the USA and Australia. Children aged 3-5 years with newly diagnosed, non-metastatic medulloblastoma without any high-risk features were also eligible. Eligible patients were required to start therapy within 31 days from definitive surgery, had a Lansky performance score of at least 30, and did not receive previous radiotherapy or chemotherapy. Patients were stratified postoperatively by clinical and histological criteria into low-risk, intermediate-risk, and high-risk treatment groups. All patients received identical induction chemotherapy (methotrexate, vincristine, cisplatin, and cyclophosphamide), with high-risk patients also receiving an additional five doses of vinblastine. Induction was followed by risk-adapted consolidation therapy: low-risk patients received cyclophosphamide (1500 mg/m2 on day 1), etoposide (100 mg/m2 on days 1 and 2), and carboplatin (area under the curve 5 mg/mL per min on day 2) for two 4-week cycles; intermediate-risk patients received focal radiation therapy (54 Gy with a clinical target volume of 5 mm over 6 weeks) to the tumour bed; and high-risk patients received chemotherapy with targeted intravenous topotecan (area under the curve 120-160 ng-h/mL intravenously on days 1-5) and cyclophosphamide (600 mg/m2 intravenously on days 1-5). After consolidation, all patients received maintenance chemotherapy with cyclophosphamide, topotecan, and erlotinib. The coprimary endpoints were event-free survival and patterns of methylation profiling associated with progression-free survival. Outcome and safety analyses were per protocol (all patients who received at least one dose of induction chemotherapy); biological analyses included all patients with tissue available for methylation profiling. This trial is registered with ClinicalTrials.gov, number NCT00602667, and was closed to accrual on April 19, 2017. FINDINGS: Between Nov 27, 2007, and April 19, 2017, we enrolled 81 patients with histologically confirmed medulloblastoma. Accrual to the low-risk group was suspended after an interim analysis on Dec 2, 2015, when the 1-year event-free survival was estimated to be below the stopping rule boundary. After a median follow-up of 5·5 years (IQR 2·7-7·3), 5-year event-free survival was 31·3% (95% CI 19·3-43·3) for the whole cohort, 55·3% (95% CI 33·3-77·3) in the low-risk cohort (n=23) versus 24·6% (3·6-45·6) in the intermediate-risk cohort (n=32; hazard ratio 2·50, 95% CI 1·19-5·27; p=0·016) and 16·7% (3·4-30·0) in the high-risk cohort (n=26; 3·55, 1·66-7·59; p=0·0011; overall p=0·0021). 5-year progression-free survival by methylation subgroup was 51·1% (95% CI 34·6-67·6) in the sonic hedgehog (SHH) subgroup (n=42), 8·3% (95% CI 0·0-24·0%) in the group 3 subgroup (n=24), and 13·3% (95% CI 0·0-37·6%) in the group 4 subgroup (n=10). Within the SHH subgroup, two distinct methylation subtypes were identified and named iSHH-I and iSHH-II. 5-year progression-free survival was 27·8% (95% CI 9·0-46·6; n=21) for iSHH-I and 75·4% (55·0-95·8; n=21) for iSHH-II. The most common adverse events were grade 3-4 febrile neutropenia (48 patients [59%]), neutropenia (21 [26%]), infection with neutropenia (20 [25%]), leucopenia (15 [19%]), vomiting (15 [19%]), and anorexia (13 [16%]). No treatment-related deaths occurred. INTERPRETATION: The risk-adapted approach did not improve event-free survival in young children with medulloblastoma. However, the methylation subgroup analyses showed that the SHH subgroup had improved progression-free survival compared with the group 3 subgroup. Moreover, within the SHH subgroup, the iSHH-II subtype had improved progression-free survival in the absence of radiation, intraventricular chemotherapy, or high-dose chemotherapy compared with the iSHH-I subtype. These findings support the development of a molecularly driven, risk-adapted, treatment approach in future trials in young children with medulloblastoma. FUNDING: American Lebanese Syrian Associated Charities, St Jude Children's Research Hospital, NCI Cancer Center, Alexander and Margaret Stewart Trust, Sontag Foundation, and American Association for Cancer Research.