Publications by authors named "Martin Sill"

85 Publications

Integrated Molecular-Morphologic Meningioma Classification: A Multicenter Retrospective Analysis, Retrospectively and Prospectively Validated.

J Clin Oncol 2021 Dec 7;39(34):3839-3852. Epub 2021 Oct 7.

Department of Neurosurgery, NYU Langone Hospital, New York, NY.

Purpose: Meningiomas are the most frequent primary intracranial tumors. Patient outcome varies widely from benign to highly aggressive, ultimately fatal courses. Reliable identification of risk of progression for individual patients is of pivotal importance. However, only biomarkers for highly aggressive tumors are established ( and ), whereas no molecularly based stratification exists for the broad spectrum of patients with low- and intermediate-risk meningioma.

Methods: DNA methylation data and copy-number information were generated for 3,031 meningiomas (2,868 patients), and mutation data for 858 samples. DNA methylation subgroups, copy-number variations (CNVs), mutations, and WHO grading were analyzed. Prediction power for outcome was assessed in a retrospective cohort of 514 patients, validated on a retrospective cohort of 184, and on a prospective cohort of 287 multicenter cases.

Results: Both CNV- and methylation family-based subgrouping independently resulted in increased prediction accuracy of risk of recurrence compared with the WHO classification (c-indexes WHO 2016, CNV, and methylation family 0.699, 0.706, and 0.721, respectively). Merging all risk stratification approaches into an integrated molecular-morphologic score resulted in further substantial increase in accuracy (c-index 0.744). This integrated score consistently provided superior accuracy in all three cohorts, significantly outperforming WHO grading (c-index difference = .005). Besides the overall stratification advantage, the integrated score separates more precisely for risk of progression at the diagnostically challenging interface of WHO grade 1 and grade 2 tumors (hazard ratio 4.34 [2.48-7.57] and 3.34 [1.28-8.72] retrospective and prospective validation cohorts, respectively).

Conclusion: Merging these layers of histologic and molecular data into an integrated, three-tiered score significantly improves the precision in meningioma stratification. Implementation into diagnostic routine informs clinical decision making for patients with meningioma on the basis of robust outcome prediction.
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http://dx.doi.org/10.1200/JCO.21.00784DOI Listing
December 2021

Radiation-induced gliomas represent H3-/IDH-wild type pediatric gliomas with recurrent PDGFRA amplification and loss of CDKN2A/B.

Nat Commun 2021 09 20;12(1):5530. Epub 2021 Sep 20.

Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg University Hospital and German Cancer Resarch Center (DKFZ), Heidelberg, Germany.

Long-term complications such as radiation-induced second malignancies occur in a subset of patients following radiation-therapy, particularly relevant in pediatric patients due to the long follow-up period in case of survival. Radiation-induced gliomas (RIGs) have been reported in patients after treatment with cranial irradiation for various primary malignancies such as acute lymphoblastic leukemia (ALL) and medulloblastoma (MB). We perform comprehensive (epi-) genetic and expression profiling of RIGs arising after cranial irradiation for MB (n = 23) and ALL (n = 9). Our study reveals a unifying molecular signature for the majority of RIGs, with recurrent PDGFRA amplification and loss of CDKN2A/B and an absence of somatic hotspot mutations in genes encoding histone 3 variants or IDH1/2, uncovering diagnostic markers and potentially actionable targets.
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http://dx.doi.org/10.1038/s41467-021-25708-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8452680PMC
September 2021

PATZ1 fusions define a novel molecularly distinct neuroepithelial tumor entity with a broad histological spectrum.

Acta Neuropathol 2021 11 21;142(5):841-857. Epub 2021 Aug 21.

Paediatric and Adolescent Medicine, Department of Clinical Medicine, Aarhus University Hospital, Aarhus, Denmark.

Large-scale molecular profiling studies in recent years have shown that central nervous system (CNS) tumors display a much greater heterogeneity in terms of molecularly distinct entities, cellular origins and genetic drivers than anticipated from histological assessment. DNA methylation profiling has emerged as a useful tool for robust tumor classification, providing new insights into these heterogeneous molecular classes. This is particularly true for rare CNS tumors with a broad morphological spectrum, which are not possible to assign as separate entities based on histological similarity alone. Here, we describe a molecularly distinct subset of predominantly pediatric CNS neoplasms (n = 60) that harbor PATZ1 fusions. The original histological diagnoses of these tumors covered a wide spectrum of tumor types and malignancy grades. While the single most common diagnosis was glioblastoma (GBM), clinical data of the PATZ1-fused tumors showed a better prognosis than typical GBM, despite frequent relapses. RNA sequencing revealed recurrent MN1:PATZ1 or EWSR1:PATZ1 fusions related to (often extensive) copy number variations on chromosome 22, where PATZ1 and the two fusion partners are located. These fusions have individually been reported in a number of glial/glioneuronal tumors, as well as extracranial sarcomas. We show here that they are more common than previously acknowledged, and together define a biologically distinct CNS tumor type with high expression of neural development markers such as PAX2, GATA2 and IGF2. Drug screening performed on the MN1:PATZ1 fusion-bearing KS-1 brain tumor cell line revealed preliminary candidates for further study. In summary, PATZ1 fusions define a molecular class of histologically polyphenotypic neuroepithelial tumors, which show an intermediate prognosis under current treatment regimens.
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http://dx.doi.org/10.1007/s00401-021-02354-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8500868PMC
November 2021

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

Acta Neuropathol 2021 11 5;142(5):827-839. Epub 2021 Aug 5.

Institute of Neuropathology, University of Giessen, Giessen, Germany.

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.
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http://dx.doi.org/10.1007/s00401-021-02356-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8500895PMC
November 2021

Neoplastic and immune single cell transcriptomics define subgroup-specific intra-tumoral heterogeneity of childhood medulloblastoma.

Neuro Oncol 2021 Jun 2. Epub 2021 Jun 2.

Department of Pediatrics Morgan Adams Foundation Pediatric Brain Tumor Research Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado, 80045, USA.

Background: Medulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. We aimed to determine the influence of neoplastic and immune cell diversity on MB biology in patient samples and animal models.

Methods: To better characterize cellular heterogeneity in MB we used single-cell RNA sequencing, immunohistochemistry and deconvolution of transcriptomic data to profile neoplastic and immune populations in patient samples and animal models across childhood MB subgroups.

Results: Neoplastic cells cluster primarily according to individual sample of origin which is influenced by chromosomal copy number variance. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes, including photoreceptor and glutamatergic neuron-like cells in molecular subgroups GP3 and GP4, and a specific nodule-associated neuronally-differentiated subpopulation in subgroup molecular SHH. We definitively chart the spectrum of MB immune cell infiltrates, which include subpopulations that recapitulate developmentally-related neuron-pruning and antigen presenting myeloid cells. MB cellular diversity matching human samples is mirrored in subgroup-specific mouse models of MB.

Conclusions: These findings provide a clearer understanding of the diverse neoplastic and immune cell subpopulations that constitute the MB microenvironment.
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http://dx.doi.org/10.1093/neuonc/noab135DOI Listing
June 2021

DNA methylation-based profiling of bone and soft tissue tumours: a validation study of the 'DKFZ Sarcoma Classifier'.

J Pathol Clin Res 2021 Jul 5;7(4):350-360. Epub 2021 May 5.

Research Department of Pathology, University College London, UCL Cancer Institute, London, UK.

Diagnosing bone and soft tissue neoplasms remains challenging because of the large number of subtypes, many of which lack diagnostic biomarkers. DNA methylation profiles have proven to be a reliable basis for the classification of brain tumours and, following this success, a DNA methylation-based sarcoma classification tool from the Deutsches Krebsforschungszentrum (DKFZ) in Heidelberg has been developed. In this study, we assessed the performance of their classifier on DNA methylation profiles of an independent data set of 986 bone and soft tissue tumours and controls. We found that the 'DKFZ Sarcoma Classifier' was able to produce a diagnostic prediction for 55% of the 986 samples, with 83% of these predictions concordant with the histological diagnosis. On limiting the validation to the 820 cases with histological diagnoses for which the DKFZ Classifier was trained, 61% of cases received a prediction, and the histological diagnosis was concordant with the predicted methylation class in 88% of these cases, findings comparable to those reported in the DKFZ Classifier paper. The classifier performed best when diagnosing mesenchymal chondrosarcomas (CHSs, 88% sensitivity), chordomas (85% sensitivity), and fibrous dysplasia (83% sensitivity). Amongst the subtypes least often classified correctly were clear cell CHSs (14% sensitivity), malignant peripheral nerve sheath tumours (27% sensitivity), and pleomorphic liposarcomas (29% sensitivity). The classifier predictions resulted in revision of the histological diagnosis in six of our cases. We observed that, although a higher tumour purity resulted in a greater likelihood of a prediction being made, it did not correlate with classifier accuracy. Our results show that the DKFZ Classifier represents a powerful research tool for exploring the pathogenesis of sarcoma; with refinement, it has the potential to be a valuable diagnostic tool.
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http://dx.doi.org/10.1002/cjp2.215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8185366PMC
July 2021

Cross-Species Genomics Reveals Oncogenic Dependencies in ZFTA/C11orf95 Fusion-Positive Supratentorial Ependymomas.

Cancer Discov 2021 Sep 20;11(9):2230-2247. Epub 2021 Apr 20.

Department of Neuropathology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

Molecular groups of supratentorial ependymomas comprise tumors with or -involving fusions and fusion-negative subependymoma. However, occasionally supratentorial ependymomas cannot be readily assigned to any of these groups due to lack of detection of a typical fusion and/or ambiguous DNA methylation-based classification. An unbiased approach with a cohort of unprecedented size revealed distinct methylation clusters composed of tumors with ependymal but also various other histologic features containing alternative translocations that shared as a partner gene. Somatic overexpression of -associated fusion genes in the developing cerebral cortex is capable of inducing tumor formation , and cross-species comparative analyses identified as a key downstream regulator of tumorigenesis in all tumors. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating a potential therapeutic vulnerability in ZFTA fusion-positive tumors. SIGNIFICANCE: fusions are a hallmark feature of supratentorial ependymoma. We find that ZFTA acts as a partner for alternative transcriptional activators in oncogenic fusions of supratentorial tumors with various histologic characteristics. Establishing representative mouse models, we identify potential therapeutic targets shared by fusion-positive tumors, such as GLI2..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0963DOI Listing
September 2021

Glioblastomas with primitive neuronal component harbor a distinct methylation and copy-number profile with inactivation of TP53, PTEN, and RB1.

Acta Neuropathol 2021 07 19;142(1):179-189. Epub 2021 Apr 19.

Division of Neuropathology, Institute of Pathology, Basel University Hospital, Basel, Switzerland.

Glioblastoma IDH-wildtype presents with a wide histological spectrum. Some features are so distinctive that they are considered as separate histological variants or patterns for the purpose of classification. However, these usually lack defined (epi-)genetic alterations or profiles correlating with this histology. Here, we describe a molecular subtype with overlap to the unique histological pattern of glioblastoma with primitive neuronal component. Our cohort consists of 63 IDH-wildtype glioblastomas that harbor a characteristic DNA methylation profile. Median age at diagnosis was 59.5 years. Copy-number variations and genetic sequencing revealed frequent alterations in TP53, RB1 and PTEN, with fewer gains of chromosome 7 and homozygous CDKN2A/B deletions than usually described for IDH-wildtype glioblastoma. Gains of chromosome 1 were detected in more than half of the cases. A poorly differentiated phenotype with frequent absence of GFAP expression, high proliferation index and strong staining for p53 and TTF1 often caused misleading histological classification as carcinoma metastasis or primitive neuroectodermal tumor. Clinically, many patients presented with leptomeningeal dissemination and spinal metastasis. Outcome was poor with a median overall survival of only 12 months. Overall, we describe a new molecular subtype of IDH-wildtype glioblastoma with a distinct histological appearance and genetic signature.
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http://dx.doi.org/10.1007/s00401-021-02302-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217054PMC
July 2021

Atypical Teratoid/Rhabdoid Tumor (AT/RT) With Molecular Features of Pleomorphic Xanthoastrocytoma.

Am J Surg Pathol 2021 09;45(9):1228-1234

Institute of Neuropathology.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant central nervous system tumor predominantly occurring in infants that may also arise in older children and adults. Rare secondary AT/RT developing from other tumors such as pleomorphic xanthoastrocytoma (PXA) are on record, but AT/RT presenting with molecular features of PXA have not been described. Here, we report 3 malignant central nervous system tumors in children (10, 13, and 18 y old). All tumors were located in the temporal lobe. In 2 cases, there was no history of a low-grade precursor lesion; in 1 case anaplastic PXA had been diagnosed 3 months earlier. Histopathologically, all tumors were composed of RT cells and showed frank signs of malignancy as well as loss of nuclear SMARCB1/INI1 protein expression. Two cases displayed homozygous deletions of the SMARCB1 region while the third case showed an exon 7 mutation (c.849_850delGT; p.Met283Ilefs*77). Of note, DNA methylation profiles did not group with AT/RT or other tumor entities using the Heidelberg Brain Tumor Classifier (version v11b4). By unsupervised t-distributed stochastic neighbor embedding analysis and hierarchical clustering analysis, however, all tumors clearly grouped with PXA. Genome-wide copy number analysis revealed homozygous CDNK2A/B deletions and gains of whole chromosome 7. BRAF V600E mutations could be demonstrated in all cases. In conclusion, the possibility of AT/RT with molecular features of PXA needs to be taken into account and warrants molecular characterization of AT/RT especially in older children. Since treatments targeting mutated BRAF are available, identification of such cases may also have therapeutic consequences.
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http://dx.doi.org/10.1097/PAS.0000000000001694DOI Listing
September 2021

The age of adult pilocytic astrocytoma cells.

Oncogene 2021 04 17;40(16):2830-2841. Epub 2021 Mar 17.

Group Genome Instability in Tumors, DKFZ, Heidelberg, Germany.

Adult pilocytic astrocytomas (PAs) have been regarded as indistinguishable from pediatric PAs in terms of genome-wide expression and methylation patterns. It has been unclear whether adult PAs arise early in life and remain asymptomatic until adulthood, or whether they develop during adulthood. We sought to determine the age and origin of adult human PAs using two types of "marks" in the genomic DNA. First, we analyzed the DNA methylation patterns of adult and pediatric PAs to distinguish between PAs of different anatomic locations (n = 257 PA and control brain tissues). Second, we measured the concentration of nuclear bomb test-derived C in genomic DNA (n = 14 cases), which indicates the time point of the formation of human cell populations. Our data suggest that adult and pediatric PAs developing in the infratentorial brain are closely related and potentially develop from precursor cells early in life, whereas supratentorial PAs might show age and location-specific differences.
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http://dx.doi.org/10.1038/s41388-021-01738-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062266PMC
April 2021

Sarcoma classification by DNA methylation profiling.

Nat Commun 2021 01 21;12(1):498. Epub 2021 Jan 21.

Department of General Pathology, Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany.

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications.
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http://dx.doi.org/10.1038/s41467-020-20603-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7819999PMC
January 2021

Accurate calling of KIAA1549-BRAF fusions from DNA of human brain tumours using methylation array-based copy number and gene panel sequencing data.

Neuropathol Appl Neurobiol 2021 04 17;47(3):406-414. Epub 2021 Jan 17.

Department of Neuropathology, Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany.

Aims: KIAA1549-BRAF fusions occur in certain brain tumours and provide druggable targets due to a constitutive activation of the MAP-kinase pathway. We introduce workflows for calling the KIAA1549-BRAF fusion from DNA methylation array-derived copy number as well as DNA panel sequencing data.

Methods: Copy number profiles were analysed by automated screening and visual verification of a tandem duplication on chromosome 7q34, indicative of the KIAA1549-BRAF fusion. Pilocytic astrocytomas of the ICGC cohort with known fusion status were used for validation. KIAA1549-BRAF fusions were called from DNA panel sequencing data using the fusion callers Manta, Arriba with modified filtering criteria and deFuse. We screened DNA methylation and panel sequencing data of 7790 specimens from brain tumour and sarcoma entities.

Results: We identified the fusion in 337 brain tumours with both DNA methylation and panel sequencing data. Among these, we detected the fusion from copy number data in 84% and from DNA panel sequencing data in more than 90% using Arriba with modified filters. While in 74% the KIAA1549-BRAF fusion was detected from both methylation array-derived copy number and panel sequencing data, in 9% it was detected from copy number data only and in 16% from panel data only. The fusion was almost exclusively found in pilocytic astrocytomas, diffuse leptomeningeal glioneuronal tumours and high-grade astrocytomas with piloid features.

Conclusions: The KIAA1549-BRAF fusion can be reliably detected from either DNA methylation array or DNA panel data. The use of both methods is recommended for the most sensitive detection of this diagnostically and therapeutically important marker.
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http://dx.doi.org/10.1111/nan.12683DOI Listing
April 2021

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

Acta Neuropathol 2021 02 14;141(2):281-290. Epub 2020 Dec 14.

Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN, USA.

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.
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http://dx.doi.org/10.1007/s00401-020-02247-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847462PMC
February 2021

The genetic landscape of choroid plexus tumors in children and adults.

Neuro Oncol 2021 04;23(4):650-660

Institute of Neuropathology, University Hospital Münster, Münster, Germany.

Background: Choroid plexus tumors (CPTs) are intraventricular brain tumors predominantly arising in children but also affecting adults. In most cases, driver mutations have not been identified, although there are reports of frequent chromosome-wide copy-number alterations and TP53 mutations, especially in choroid plexus carcinomas (CPCs).

Methods: DNA methylation profiling and RNA-sequencing was performed in a series of 47 CPTs. Samples comprised 35 choroid plexus papillomas (CPPs), 6 atypical choroid plexus papillomas (aCPPs) and 6 CPCs plus three recurrences thereof. Targeted TP53 and TERT promotor sequencing was performed in all samples. Whole exome sequencing (WES) and linked-read whole genome sequencing (WGS) was performed in 25 and 4 samples, respectively.

Results: Tumors comprised the molecular subgroups "pediatric A" (N=11), "pediatric B" (N=12) and "adult" (N=27). Copy-number alterations mainly represented whole-chromosomal alterations with subgroup-specific enrichments (gains of Chr1, 2 and 21q in "pediatric B" and gains of Chr5 and 9 and loss of Chr21q in "adult"). RNA sequencing yielded a novel CCDC47-PRKCA fusion transcript in one adult choroid plexus papilloma patient with aggressive clinical course; an underlying Chr17 inversion was demonstrated by linked-read WGS. WES and targeted sequencing showed TP53 mutations in 7/47 CPTs (15%), five of which were children. On the contrary, TERT promoter mutations were encountered in 7/28 adult patients (25%) and associated with shorter progression-free survival (log-rank test, p=0.015).

Conclusion: Pediatric CPTs lack recurrent driver alterations except for TP53, whereas CPTs in adults show TERT promoter mutations or a novel CCDC47-PRKCA gene fusion, being associated with a more unfavorable clinical course.
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http://dx.doi.org/10.1093/neuonc/noaa267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041331PMC
April 2021

An extracellular vesicle-related gene expression signature identifies high-risk patients in medulloblastoma.

Neuro Oncol 2021 04;23(4):586-598

Department of Pediatric Hematology and Oncology, University Children's Hospital Münster, Münster, Germany.

Background: Medulloblastoma (MB) is a malignant brain tumor in childhood. It comprises 4 subgroups with different clinical behaviors. The aim of this study was to characterize the transcriptomic landscape of MB, both at the level of individual tumors as well as in large patient cohorts.

Methods: We used a combination of single-cell transcriptomics, cell culture models and biophysical methods such as nanoparticle tracking analysis and electron microscopy to investigate intercellular communication in the MB tumor niche.

Results: Tumor cells of the sonic hedgehog (SHH)-MB subgroup show a differentiation blockade. These cells undergo extensive metabolic reprogramming. The gene expression profiles of individual tumor cells show a partial convergence with those of tumor-associated glial and immune cells. One possible cause is the transfer of extracellular vesicles (EVs) between cells in the tumor niche. We were able to detect EVs in co-culture models of MB tumor cells and oligodendrocytes. We also identified a gene expression signature, EVS, which shows overlap with the proteome profile of large oncosomes from prostate cancer cells. This signature is also present in MB patient samples. A high EVS expression is one common characteristic of tumors that occur in high-risk patients from different MB subgroups or subtypes.

Conclusions: With EVS, our study uncovered a novel gene expression signature that has a high prognostic significance across MB subgroups.
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http://dx.doi.org/10.1093/neuonc/noaa254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041350PMC
April 2021

Genetic and epigenetic landscape of IDH-wildtype glioblastomas with FGFR3-TACC3 fusions.

Acta Neuropathol Commun 2020 11 9;8(1):186. Epub 2020 Nov 9.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

A subset of glioblastomas (GBMs) harbors potentially druggable oncogenic FGFR3-TACC3 (F3T3) fusions. However, their associated molecular and clinical features are poorly understood. Here we analyze the frequency of F3T3-fusion positivity, its associated genetic and methylation profiles, and its impact on survival in 906 IDH-wildtype GBM patients. We establish an F3T3 prevalence of 4.1% and delineate its associations with cancer signaling pathway alterations. F3T3-positive GBMs had lower tumor mutational and copy-number alteration burdens than F3T3-wildtype GBMs. Although F3T3 fusions were predominantly mutually exclusive with other oncogenic RTK pathway alterations, they did rarely co-occur with EGFR amplification. They were less likely to harbor TP53 alterations. By methylation profiling, they were more likely to be assigned the mesenchymal or RTK II subclass. Despite being older at diagnosis and having similar frequencies of MGMT promoter hypermethylation, patients with F3T3-positive GBMs lived about 8 months longer than those with F3T3-wildtype tumors. While consistent with IDH-wildtype GBM, F3T3-positive GBMs exhibit distinct biological features, underscoring the importance of pursuing molecular studies prior to clinical trial enrollment and targeted treatment.
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http://dx.doi.org/10.1186/s40478-020-01058-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653727PMC
November 2020

A subset of pediatric-type thalamic gliomas share a distinct DNA methylation profile, H3K27me3 loss and frequent alteration of EGFR.

Neuro Oncol 2021 01;23(1):34-43

Department of Neuropathology, Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.

Background: Malignant astrocytic gliomas in children show a remarkable biological and clinical diversity. Small in-frame insertions or missense mutations in the epidermal growth factor receptor gene (EGFR) have recently been identified in a distinct subset of pediatric-type bithalamic gliomas with a unique DNA methylation pattern.

Methods: Here, we investigated an epigenetically homogeneous cohort of malignant gliomas (n = 58) distinct from other subtypes and enriched for pediatric cases and thalamic location, in comparison with this recently identified subtype of pediatric bithalamic gliomas.

Results: EGFR gene amplification was detected in 16/58 (27%) tumors, and missense mutations or small in-frame insertions in EGFR were found in 20/30 tumors with available sequencing data (67%; 5 of them co-occurring with EGFR amplification). Additionally, 8 of the 30 tumors (27%) harbored an H3.1 or H3.3 K27M mutation (6 of them with a concomitant EGFR alteration). All tumors tested showed loss of H3K27me3 staining, with evidence of overexpression of the EZH inhibitory protein (EZHIP) in the H3 wildtype cases. Although some tumors indeed showed a bithalamic growth pattern, a significant proportion of tumors occurred in the unilateral thalamus or in other (predominantly midline) locations.

Conclusions: Our findings present a distinct molecular class of pediatric-type malignant gliomas largely overlapping with the recently reported bithalamic gliomas characterized by EGFR alteration, but additionally showing a broader spectrum of EGFR alterations and tumor localization. Global H3K27me3 loss in this group appears to be mediated by either H3 K27 mutation or EZHIP overexpression. EGFR inhibition may represent a potential therapeutic strategy in these highly aggressive gliomas.
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http://dx.doi.org/10.1093/neuonc/noaa251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7850075PMC
January 2021

Drivers underpinning the malignant transformation of giant cell tumour of bone.

J Pathol 2020 12 6;252(4):433-440. Epub 2020 Oct 6.

Department of Pathology (research), University College London Cancer Institute, London, UK.

The rare benign giant cell tumour of bone (GCTB) is defined by an almost unique mutation in the H3.3 family of histone genes H3-3A or H3-3B; however, the same mutation is occasionally found in primary malignant bone tumours which share many features with the benign variant. Moreover, lung metastases can occur despite the absence of malignant histological features in either the primary or metastatic lesions. Herein we investigated the genetic events of 17 GCTBs including benign and malignant variants and the methylation profiles of 122 bone tumour samples including GCTBs. Benign GCTBs possessed few somatic alterations and no other known drivers besides the H3.3 mutation, whereas all malignant tumours harboured at least one additional driver mutation and exhibited genomic features resembling osteosarcomas, including high mutational burden, additional driver event(s), and a high degree of aneuploidy. The H3.3 mutation was found to predate the development of aneuploidy. In contrast to osteosarcomas, malignant H3.3-mutated tumours were enriched for a variety of alterations involving TERT, other than amplification, suggesting telomere dysfunction in the transformation of benign to malignant GCTB. DNA sequencing of the benign metastasising GCTB revealed no additional driver alterations; polyclonal seeding in the lung was identified, implying that the metastatic lesions represent an embolic event. Unsupervised clustering of DNA methylation profiles revealed that malignant H3.3-mutated tumours are distinct from their benign counterpart, and other bone tumours. Differential methylation analysis identified CCND1, encoding cyclin D1, as a plausible cancer driver gene in these tumours because hypermethylation of the CCND1 promoter was specific for GCTBs. We report here the genomic and methylation patterns underlying the rare clinical phenomena of benign metastasising and malignant transformation of GCTB and show how the combination of genomic and epigenomic findings could potentially distinguish benign from malignant GCTBs, thereby predicting aggressive behaviour in challenging diagnostic cases. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432151PMC
December 2020

Molecular tumor classification using DNA methylome analysis.

Hum Mol Genet 2020 10;29(R2):R205-R213

Division of Translational Medical Oncology, Section Translational Cancer Epigenomics, German Cancer Research Center (DKFZ), National Center for Tumor Diseases (NCT) Heidelberg, 69120 Heidelberg, Germany.

Tumor classifiers based on molecular patterns promise to define and reliably classify tumor entities. The high tissue- and cell type-specificity of DNA methylation, as well as its high stability, makes DNA methylation an ideal choice for the development of tumor classifiers. Herein, we review existing tumor classifiers using DNA methylome analysis and will provide an overview on their emerging impact on cancer classification, the detection of novel cancer subentities and patient stratification with a focus on brain tumors, sarcomas and hematopoietic malignancies. Furthermore, we provide an outlook on the enormous potential of DNA methylome analysis to complement classical histopathological and genetic diagnostics, including the emerging field of epigenomic analysis in liquid biopsies.
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http://dx.doi.org/10.1093/hmg/ddaa147DOI Listing
October 2020

Nonmetastatic Medulloblastoma of Early Childhood: Results From the Prospective Clinical Trial HIT-2000 and An Extended Validation Cohort.

J Clin Oncol 2020 06 24;38(18):2028-2040. Epub 2020 Apr 24.

Department of Neuropathology, Institute for Pathology, Hannover Medical School, Hannover, Germany.

Purpose: The HIT-2000-BIS4 trial aimed to avoid highly detrimental craniospinal irradiation (CSI) in children < 4 years of age with nonmetastatic medulloblastoma by systemic chemotherapy, intraventricular methotrexate, and risk-adapted local radiotherapy.

Patients And Methods: From 2001-2011, 87 patients received systemic chemotherapy and intraventricular methotrexate. Until 2006, CSI was reserved for nonresponse or progression. After 2006, local radiotherapy was introduced for nonresponders or patients with classic medulloblastoma (CMB) or large-cell/anaplastic medulloblastoma (LCA). DNA methylation profiles of infantile sonic hedgehog-activated medulloblastoma (SHH-INF) were subdivided into iSHH-I and iSHH-II subtypes in the HIT-2000-BIS4 cohort and a validation cohort (n = 71) from the HIT group and Russia.

Results: Five years after diagnosis, patients with desmoplastic medulloblastoma (DMB) or medulloblastoma with extensive nodularity (MBEN; n = 42) had 93% progression-free survival (5y-PFS), 100% overall survival (5y-OS), and 93% CSI-free (5y-CSI-free) survival. Patients with CMB/LCA (n = 45) had 37% 5y-PFS, 62% 5y-OS, and 39% 5y-CSI-free survival. Local radiotherapy did not improve survival in patients with CMB/LCA. All DMB/MBEN assessed by DNA methylation profiling belonged to the SHH-INF subgroup. Group 3 patients (5y-PFS, 36%; n = 14) relapsed more frequently than the SHH-INF group (5y-PFS, 93%; n = 28) or group 4 patients (5y-PFS, 83%; n = 6; < .001). SHH-INF split into iSHH-I and iSHH-II subtypes in HIT-2000-BIS4 and the validation cohort, without prognostic impact (5y-PFS: iSHH-I, 73%, iSHH-II, 83%; = .25; n = 99). Intelligence quotient (IQ) was significantly lower in patients after CSI (mean IQ, 90 [no radiotherapy], 74 [CSI]; = .012).

Conclusion: Systemic chemotherapy and intraventricular methotrexate led to favorable survival in both iSHH subtypes of SHH-activated DMB/MBEN with acceptable neurotoxicity. Survival in patients with non-wingless (WNT)/non-SHH disease with CMB/LCA was not improved by local radiotherapy. Patients with group 4 disease had more favorable survival rates than those with group 3 medulloblastoma.
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http://dx.doi.org/10.1200/JCO.19.03057DOI Listing
June 2020

Infant High-Grade Gliomas Comprise Multiple Subgroups Characterized by Novel Targetable Gene Fusions and Favorable Outcomes.

Cancer Discov 2020 07 1;10(7):942-963. Epub 2020 Apr 1.

Department of Neuropathology, University Hospital Hamburg-Eppendorf, and Research Institute Children's Cancer Center, Hamburg, Germany.

Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an "intrinsic" spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ( = 31), ( = 21), ( = 9), and ( = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management. SIGNIFICANCE: Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of , or gene fusions. Kinase fusion-positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype...
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http://dx.doi.org/10.1158/2159-8290.CD-19-1030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8313225PMC
July 2020

Machine learning workflows to estimate class probabilities for precision cancer diagnostics on DNA methylation microarray data.

Nat Protoc 2020 02 13;15(2):479-512. Epub 2020 Jan 13.

Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.

DNA methylation data-based precision cancer diagnostics is emerging as the state of the art for molecular tumor classification. Standards for choosing statistical methods with regard to well-calibrated probability estimates for these typically highly multiclass classification tasks are still lacking. To support this choice, we evaluated well-established machine learning (ML) classifiers including random forests (RFs), elastic net (ELNET), support vector machines (SVMs) and boosted trees in combination with post-processing algorithms and developed ML workflows that allow for unbiased class probability (CP) estimation. Calibrators included ridge-penalized multinomial logistic regression (MR) and Platt scaling by fitting logistic regression (LR) and Firth's penalized LR. We compared these workflows on a recently published brain tumor 450k DNA methylation cohort of 2,801 samples with 91 diagnostic categories using a 5 × 5-fold nested cross-validation scheme and demonstrated their generalizability on external data from The Cancer Genome Atlas. ELNET was the top stand-alone classifier with the best calibration profiles. The best overall two-stage workflow was MR-calibrated SVM with linear kernels closely followed by ridge-calibrated tuned RF. For calibration, MR was the most effective regardless of the primary classifier. The protocols developed as a result of these comparisons provide valuable guidance on choosing ML workflows and their tuning to generate well-calibrated CP estimates for precision diagnostics using DNA methylation data. Computation times vary depending on the ML algorithm from <15 min to 5 d using multi-core desktop PCs. Detailed scripts in the open-source R language are freely available on GitHub, targeting users with intermediate experience in bioinformatics and statistics and using R with Bioconductor extensions.
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http://dx.doi.org/10.1038/s41596-019-0251-6DOI Listing
February 2020

The molecular landscape of ETMR at diagnosis and relapse.

Nature 2019 12 4;576(7786):274-280. Epub 2019 Dec 4.

Department of Pathology, St Jude Children's Research Hospital, Memphis, TN, USA.

Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis. 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 C19MC 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.
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http://dx.doi.org/10.1038/s41586-019-1815-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908757PMC
December 2019

DNA methylation-based profiling for paediatric CNS tumour diagnosis and treatment: a population-based study.

Lancet Child Adolesc Health 2020 02 27;4(2):121-130. Epub 2019 Nov 27.

Department of Neuropathology, The Walton Centre NHS Foundation Trust, Liverpool, UK.

Background: Marked variation exists in the use of genomic data in tumour diagnosis, and optimal integration with conventional diagnostic technology remains uncertain despite several studies reporting improved diagnostic accuracy, selection for targeted treatments, and stratification for trials. Our aim was to assess the added value of molecular profiling in routine clinical practice and the impact on conventional and experimental treatments.

Methods: This population-based study assessed the diagnostic and clinical use of DNA methylation-based profiling in childhood CNS tumours using two large national cohorts in the UK. In the diagnostic cohort-which included routinely diagnosed CNS tumours between Sept 1, 2016, and Sept 1, 2018-we assessed how the methylation profile altered or refined diagnosis in routine clinical practice and estimated how this would affect standard patient management. For the archival cohort of diagnostically difficult cases, we established how many cases could be solved using modern standard pathology, how many could only be solved using the methylation profile, and how many remained unsolvable.

Findings: Of 484 patients younger than 20 years with CNS tumours, 306 had DNA methylation arrays requested by the neuropathologist and were included in the diagnostic cohort. Molecular profiling added a unique contribution to clinical diagnosis in 107 (35%; 95% CI 30-40) of 306 cases in routine diagnostic practice-providing additional molecular subtyping data in 99 cases, amended the final diagnosis in five cases, and making potentially significant predictions in three cases. We estimated that it could change conventional management in 11 (4%; 95% CI 2-6) of 306 patients. Among 195 historically difficult-to-diagnose tumours in the archival cohort, 99 (51%) could be diagnosed using standard methods, with the addition of methylation profiling solving a further 34 (17%) cases. The remaining 62 (32%) cases were unresolved despite specialist pathology and methylation profiling.

Interpretation: Together, these data provide estimates of the impact that could be expected from routine implementation of genomic profiling into clinical practice, and indicate limitations where additional techniques will be required. We conclude that DNA methylation arrays are a useful diagnostic adjunct for childhood CNS tumours.

Funding: The Brain Tumour Charity, Children with Cancer UK, Great Ormond Street Hospital Children's Charity, Olivia Hodson Cancer Fund, Cancer Research UK, and the National Institute of Health Research.
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http://dx.doi.org/10.1016/S2352-4642(19)30342-6DOI Listing
February 2020

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

Acta Neuropathol 2020 02 25;139(2):243-257. Epub 2019 Nov 25.

Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.

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.
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http://dx.doi.org/10.1007/s00401-019-02101-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275775PMC
February 2020

Desmoplastic myxoid tumor, SMARCB1-mutant: clinical, histopathological and molecular characterization of a pineal region tumor encountered in adolescents and adults.

Acta Neuropathol 2020 02 16;139(2):277-286. Epub 2019 Nov 16.

Institute of Neuropathology, University Hospital Münster, Pottkamp 2, Münster, Germany.

Atypical teratoid/rhabdoid tumor (ATRT) is a highly malignant brain tumor predominantly occurring in infants. Mutations of the SMARCB1 gene are the characteristic genetic lesion. SMARCB1-mutant tumors in adolescents and adults are rare and may show uncommon histopathological and clinical features. Here we report seven SMARCB1-deficient intracranial tumors sharing distinct clinical, histopathological and molecular features. Median age of the four females and three males was 40 years (range 15-61 years). All tumors were located in the pineal region. Histopathologically, these tumors displayed spindled and epithelioid cells embedded in a desmoplastic stroma alternating with a variable extent of a loose myxoid matrix. All cases showed loss of nuclear SMARCB1/INI1 protein expression, expression of EMA and CD34 was frequent and the Ki67/MIB1 proliferation index was low in the majority of cases (median 3%). Three cases displayed heterozygous SMARCB1 deletions and two cases a homozygous SMARCB1 deletion. On sequencing, one tumor showed a 2 bp deletion in exon 4 (c.369_370del) and one a short duplication in exon 3 (c.237_276dup) both resulting in frameshift mutations. Most DNA methylation profiles were not classifiable using the Heidelberg Brain Tumor Classifier (version v11b4). By unsupervised t-SNE analysis and hierarchical clustering analysis, however, all tumors grouped closely together and showed similarities with ATRT-MYC. After a median observation period of 48 months, three patients were alive with stable disease, whereas one patient experienced tumor progression and three patients had succumbed to disease. In conclusion, our series represents an entity with distinct clinical, histopathological and molecular features showing epigenetic similarities with ATRT-MYC. We propose the designation desmoplastic myxoid tumor (DMT), SMARCB1-mutant, for these tumors.
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http://dx.doi.org/10.1007/s00401-019-02094-wDOI Listing
February 2020

Correction to: DNA methylation signature is prognostic of choroid plexus tumor aggressiveness.

Clin Epigenetics 2019 Oct 21;11(1):144. Epub 2019 Oct 21.

Genetics and Genome Biology Program, Hospital for Sick Children, PGCRL, 686 Bay Street, Toronto, Ontario, M5G 0A4, Canada.

After publication of the original article [1], authors have requested to add a 'J' as middle name for Richard Gilbertson. Hence, full name should be Richard J Gilbertson.
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http://dx.doi.org/10.1186/s13148-019-0737-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802296PMC
October 2019
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