Major discoveries in the biology of nervous system tumors have raised the question of how non‐histological data such as molecular information can be incorporated into the next World Health ...Organization (WHO) classification of central nervous system tumors. To address this question, a meeting of neuropathologists with expertise in molecular diagnosis was held in Haarlem, the Netherlands, under the sponsorship of the International Society of Neuropathology (ISN). Prior to the meeting, participants solicited input from clinical colleagues in diverse neuro‐oncological specialties. The present “white paper” catalogs the recommendations of the meeting, at which a consensus was reached that incorporation of molecular information into the next WHO classification should follow a set of provided “ISN‐Haarlem” guidelines. Salient recommendations include that (i) diagnostic entities should be defined as narrowly as possible to optimize interobserver reproducibility, clinicopathological predictions and therapeutic planning; (ii) diagnoses should be “layered” with histologic classification, WHO grade and molecular information listed below an “integrated diagnosis”; (iii) determinations should be made for each tumor entity as to whether molecular information is required, suggested or not needed for its definition; (iv) some pediatric entities should be separated from their adult counterparts; (v) input for guiding decisions regarding tumor classification should be solicited from experts in complementary disciplines of neuro‐oncology; and (iv) entity‐specific molecular testing and reporting formats should be followed in diagnostic reports. It is hoped that these guidelines will facilitate the forthcoming update of the fourth edition of the WHO classification of central nervous system tumors.
In the 2016, WHO classification of tumors of the central nervous system, isocitrate dehydrogenase (IDH) mutation is a main classifier for lower grade astrocytomas and IDH‐mutated astrocytomas is now ...regarded as a single group with longer survival. However, the molecular and clinical heterogeneity among IDH mutant lower grade (WHO Grades II/III) astrocytomas have only rarely been investigated. In this study, we recruited 160 IDH mutant lower grade (WHO Grades II/III) astrocytomas, and examined PDGFRA amplification, CDKN2A deletion and CDK4 amplification by FISH analysis, TERT promoter mutation by Sanger sequencing and ATRX loss and p53 expression by immunohistochemistry. We identified PDGFRA amplification, CDKN2A homozygous deletion and CDK4 amplification in 18.8%, 15.0% and 18.1% of our cohort respectively, and these alterations occurred in a mutually exclusive fashion. PDGFRA amplification was associated with shorter PFS (P = 0.0003) and OS (P < 0.0001). In tumors without PDGFRA amplification, CDKN2A homozygous deletion or CDK4 amplification was associated with a shorter OS (P = 0.035). Tumors were divided into three risk groups based on the presence of molecular alterations: high risk (PDGFRA amplification), intermediate risk (CDKN2A deletion or CDK4 amplification) and low risk (neither CDKN2A deletion and CDK4 amplification nor PDGFRA amplification). These three risk groups were significantly different in overall survival with mean survivals of 40.5, 62.9 and 71.5 months. The high‐risk group also demonstrated a shorter PFS compared to intermediate‐ (P = 0.036) and low‐risk (P < 0.0001) groups. One limitation of this study is the relatively short follow‐up period, a common confounding factor for studies on low‐grade tumors. Our data illustrate that IDH mutant lower grade astrocytomas is not a homogeneous group and should be molecularly stratified for risk.
The failure to develop effective therapies for pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively ...assess the extent to which this was present in these tumors through subclonal genomic analyses and to determine whether distinct tumor subpopulations may interact to promote tumorigenesis by generating subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced tumors revealed multiple tumor subclones, spatially and temporally coexisting in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations that we propose cooperate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in <1% of cells, abrogate DNA repair and confer increased invasion and migration on neighboring cells, in vitro and in vivo, through chemokine signaling and modulation of integrins. These data indicate that even rare tumor subpopulations may exert profound effects on tumorigenesis as a whole and may represent a new avenue for therapeutic development. Unraveling the mechanisms of subclonal diversity and communication in pGBM and DIPG will be an important step toward overcoming barriers to effective treatments.
Reports on pediatric low‐grade diffuse glioma WHO‐grade II (DG2) suggest an impaired survival rate, but lack conclusive results for genetically defined DG2‐entities. We analyzed the natural history, ...treatment and prognosis of DG2 and investigated which genetically defined sub‐entities proved unfavorable for survival. Within the prospectively registered, population‐based German/Swiss SIOP‐LGG 2004 cohort 100 patients (age 0.8‐17.8 years, 4% neurofibromatosis NF1) were diagnosed with a DG2. Following biopsy (41%) or variable extent of resection (59%), 65 patients received no adjuvant treatment. Radiologic progression or severe neurologic symptoms prompted chemotherapy (n = 18) or radiotherapy (n = 17). Multiple lines of salvage treatment were necessary for 19/35 patients. Five years event‐free survival dropped to 0.44, while 5 years overall survival was 0.90 (median observation time 8.3 years). Extensive genetic profiling of 65/100 DG2 identified Histone3‐K27M‐mutation in 4, IDH1‐mutation in 11, BRAF‐V600‐mutation in 12, KIAA1549‐BRAF‐fusions in 6 patients, while the remaining 32 tumor tissues did not show alterations of these genes. Progression to malignant glioma occurred in 12 cases of all genetically defined subgroups within a range of 0.5 to 10.8 years, except for tumors carrying KIAA1549‐BRAF‐fusions. Histone3‐K27M‐mutant tumors proved uniformly fatal within 0.6 to 2.4 years. The current LGG treatment strategy seems appropriate for all DG2‐entities, with the exemption of Histone3‐K27M‐mutant tumors that require a HGG‐related treatment strategy. Our data confirm the importance to genetically define pediatric low‐grade diffuse gliomas for proper treatment decisions and risk assessment.
What's new?
Pediatric low‐grade diffuse gliomas histologically resemble their adult counterparts, but they differ greatly in terms of genetics, clinical behavior, and prognosis. Here, the authors investigated genetic mutations in 65 pediatric grade 2 diffuse gliomas (DG2), looking for a correlation with long‐term outcome. All 4 tumors carrying the K27M mutation in the Histone3 gene were fatal. Conversely, none of the 6 tumors carrying a particular duplication of the BRAF gene, called the KIAA1549‐BRAF fusion, progressed to malignant glioma. The authors characterized the tumor genetics with respect to prognosis, age at onset, and response to treatment.
Telomerase reverse transcriptase (TERT) promoter (pTERT) mutation has often been described as a late event in gliomagenesis and it has been suggested as a prognostic biomarker in gliomas other than ...1p19q codeleted tumors. However, the characteristics of isocitrate dehydrogenase (IDH) wild type (wt) (IDHwt), pTERTwt glioblastomas are not well known. We recruited 72 adult IDHwt, pTERTwt glioblastomas and performed methylation profiling, targeted sequencing, and fluorescence in situ hybridization (FISH) for TERT structural rearrangement and ALT (alternative lengthening of telomeres). There was no significant difference in overall survival (OS) between our cohort and a the Cancer Genome Atlas (TCGA) cohort of IDHwt, pTERT mutant (mut) glioblastomas, suggesting that pTERT mutation on its own is not a prognostic factor among IDHwt glioblastomas. Epigenetically, the tumors clustered into classic‐like (11%), mesenchymal‐like (32%), and LGm6‐glioblastoma (GBM) (57%), the latter far exceeding the corresponding proportion seen in the TCGA cohort of IDHwt, pTERTmut glioblastomas. LGm6‐GBM‐clustered tumors were enriched for platelet derived growth factor receptor alpha (PDGFRA) amplification or mutation (p = 0.008), and contained far fewer epidermal growth factor receptor (EGFR) amplification (p < 0.01), 10p loss (p = 0.001) and 10q loss (p < 0.001) compared with cases not clustered to this group. LGm6‐GBM cases predominantly showed ALT (p = 0.038). In the whole cohort, only 35% cases showed EGFR amplification and no case showed combined chromosome +7/−10. Since the cases were already pTERTwt, so the three molecular properties of EGFR amplification, +7/−10, and pTERT mutation may not cover all IDHwt glioblastomas. Instead, EGFR and PDGFRA amplifications covered 67% and together with their mutations covered 71% of cases of this cohort. Homozygous deletion of cyclin dependent kinase inhibitor 2A (CDKN2A)/B was associated with a worse OS (p = 0.031) and was an independent prognosticator in multivariate analysis (p = 0.032). In conclusion, adult IDHwt, pTERTwt glioblastomas show epigenetic clustering different from IDHwt, pTERTmut glioblastomas, and IDHwt glioblastomas which are pTERTwt may however not show EGFR amplification or +7/−10 in a significant proportion of cases. CDKN2A/B deletion is a poor prognostic biomarker in this group.
Objective
We aimed to characterise glioblastomas of adolescents and young adults (AYAs) that were isocitrate dehydrogenase (IDH) wild type (wt) and H3wt.
Materials and Methods
Fifty such patients ...(aged 16–32) were studied by methylation profiling, targeted sequencing and targeted RNA‐seq.
Results
Tumours predominantly clustered into three methylation classes according to the terminology of Capper et al. (2018): (anaplastic) pleomorphic xanthoastrocytoma (PXA) (21 cases), GBM_midline (15 cases) and glioblastoma RTK/mesenchymal (seven cases). Two cases clustered with ANA_PA, four cases with LGG classes and one with GBM_MYCN. Only fifteen cases reached a calibrated score >0.84 when the cases were uploaded to DKFZ Classifier. GBM_midline‐clustered tumours had a poorer overall survival (OS) compared with the PXA‐clustered tumours (p = 0.030). LGG‐clustered cases had a significantly better survival than GBM_midline‐clustered tumours and glioblastoma RTK/mesenchymal‐clustered tumours. Only 13/21 (62%) of PXA‐clustered cases were BRAF V600E mutated. Most GBM_midline‐clustered cases were not located in the midline. GBM_midline‐clustered cases were characterised by PDGFRA amplification/mutation (73.3%), mutations of mismatch repair genes (40.0%), and all showed H3K27me3 and EZH1P loss, and an unmethylated MGMT promoter. Across the whole cohort, MGMT promoter methylation and wt TERT promoter were favourable prognosticators. Mismatch repair gene mutations were poor prognosticators and together with methylation class and MGMT methylation, maintained their significance in multivariate analyses. BRAF mutation was a good prognosticator in the PXA‐clustered tumours.
Conclusion
Methylation profiling is a useful tool in the diagnosis and prognostication of AYA glioblastomas, and the methylation classes have distinct molecular characteristics. The usual molecular diagnostic criteria for adult IDHwt glioblastoma should be applied with caution within the AYA age group.
We studied a cohort of IDH‐wildtype, H3‐wildtype glioblastomas of adolescents and young adults (AYA) with genome‐wide DNA methylation profiling, targeted sequencing and TERT promoter sequencing. We found that AYA glioblastomas can be separated into clinically significant groups by methylation profiling and these groups have distinct molecular features. TERT promoter mutation, EGFR amplification and combined whole chromosome +7/−10 are not common in this cohort. And, MGMT methylation, TERTp mutation and MMR mutation are prognostic significance.
Giant cell glioblastoma (gcGBM) is a rare histological variant of GBM, accounting for about 1% of all GBM. The prognosis is poor generally though gcGBM does slightly better than the other ...IDH‐wild‐type GBM. Because of the rarity of the cases, there has been no comprehensive molecular analysis of gcGBM. Previously, single‐gene study identified genetic changes in TP53, PTEN and TERT promoter mutation in gcGBM. In this report, we performed whole‐exome sequencing (WES) to identify somatically acquired mutations and copy number variations (CNVs) in 10 gcGBM genomes. We also examined TERT promoter mutation and MGMT methylation in our cohort. On top of the reported mutations, WES revealed ATRX, PIK3R1, RB1 and SETD2 as the recurrent mutations in gcGBM. Notably, one tumor harbored a mutation in MutS homolog 6 (MSH6) that is a key mismatch repair (MMR) gene. This tumor demonstrated hypermutation phenotype and showed an increased number of somatic mutations. TERT promoter mutation and MGMT methylation were observed in 20% and 40% of our samples, respectively. In conclusion, we described relevant mutation profiling for developing future targeted therapies in gcGBM.
Accumulating evidence suggests that microRNAs (miRNAs) are over‐ or under‐expressed in tumors, and abnormalities in miRNA expression may contribute to carcinogenesis. MiR‐383 was previously ...identified as one of the under‐expresssed miRNAs in medulloblastoma (MB) by miRNA expression profiling. Quantitative reverse transcription polymerase chain reaction (RT‐PCR)‐based miRNA assays showed an enrichment of miR‐383 in normal brain. Based on these data, we speculated that miR‐383 is important in MB pathogenesis. In this study, we demonstrated significant downregulation of miR‐383 in 23/29 (79%) MB samples and 7/7 (100%) MB cells lines. Ectopic expression of miR‐383 in MB cells led to suppression of cell growth, cell accumulation at sub‐G1 phase and alteration of apoptosis‐related proteins. By transcriptomic analysis and computational algorithms, we identified peroxiredoxin 3 (PRDX3) as a target gene of miR‐383. Luciferase reporter assay confirmed that miR‐383 negatively regulated PRDX3 by interaction between miR‐383 and complementary sequences in the 3′ UTR of PRDX3. MiR‐383 repressed PRDX3 at transcriptional and translational levels as revealed by quantitative RT‐PCR and Western blot analysis. Furthermore, depletion of PRDX3 by siRNAs resulted in similar effects as observed in miR‐383‐transfected cells. In conclusion, miR‐383 acts as a regulator controlling cell growth of MB, at least in part, through targeting PRDX3.
Recurrence is a major complication of some meningiomas. Although there were many studies on biomarkers associated with higher grades or increased aggressiveness, few studies specifically examined ...longitudinal samples of primary meningiomas and recurrences from the same patients for molecular life history. We studied 99 primary and recurrent meningiomas from 42 patients by FISH for 22q, 1q, 1p, 3p, 5q, 6q, 10p, 10q, 14q, 18q, CDKN2A/B homozygous deletion, ALT (Alternative Lengthening of Telomere), TERT re‐arrangement, targeted sequencing and TERTp sequencing. Although NF2 mutation and 22q were well known to be aetiological events in meningiomas, we found that in these paired meningiomas, combining the two events resulted in an NF2/22q group (57 tumors from 25 patients) which were almost mutually exclusive with those cases without these two changes (42 tumors from 17 patients) for NF2/22q. No other molecular changes were totally unique to NF2/22q or non‐NF2/22q tumors. For molecular evolution, NF2/22q meningiomas had higher cytogenetic abnormalities than non‐NF2/22q meningiomas (p = 0.003). Most of the cytogenetic changes in NF2/22q meningiomas were present from the outset whereas for non‐NF2/22q meningiomas, cytogenetic events were uncommon in the primary tumors and most were acquired in recurrences. For non‐NF2/22q tumors, CDKN2A/B homozygous deletion, 1q gain, 18p loss, 3p loss, and ALT were preferentially found in recurrences. Mutations were largely conserved between primary and recurrent tumors. Phylogenetic trees showed 11/11 patients with multiple recurrent tumors had a conserved evolutionary pattern. We conclude that for molecular life history, NF2 and 22q should be regarded as a group. NF2/22q recurring meningiomas showed more cytogenetic abnormalities in the primary tumors, whereas non‐NF2/22q meningiomas showed CDKN2A/B deletion and other cytogenetic abnormalities and ALT at recurrences. Although chromosome 1p loss is a known poor prognostic marker in meningiomas, it was also associated with a shorter TBR (time between resection) in this cohort (p = 0.002).
Recent genomic studies have shed light on the biology and inter-tumoral heterogeneity underlying pineal parenchymal tumors, in particular pineoblastomas (PBs) and pineal parenchymal tumors of ...intermediate differentiation (PPTIDs). Previous reports, however, had modest sample sizes and lacked the power to integrate molecular and clinical findings. The different proposed molecular group structures also highlighted a need to reach consensus on a robust and relevant classification system. We performed a meta-analysis on 221 patients with molecularly characterized PBs and PPTIDs. DNA methylation profiles were analyzed through complementary bioinformatic approaches and molecular subgrouping was harmonized. Demographic, clinical, and genomic features of patients and samples from these pineal tumor groups were annotated. Four clinically and biologically relevant consensus PB groups were defined: PB-miRNA1 (
n
= 96), PB-miRNA2 (
n
= 23), PB-MYC/FOXR2 (
n
= 34), and PB-RB1 (
n
= 25). A final molecularly distinct group, designated PPTID (
n
= 43), comprised histological PPTID and PBs. Genomic and transcriptomic profiling allowed the characterization of oncogenic drivers for individual tumor groups, specifically, alterations in the microRNA processing pathway in PB-miRNA1/2,
MYC
amplification and
FOXR2
overexpression in PB-MYC/FOXR2,
RB1
alteration in PB-RB1, and
KBTBD4
insertion in PPTID. Age at diagnosis, sex predilection, and metastatic status varied significantly among tumor groups. While patients with PB-miRNA2 and PPTID had superior outcome, survival was intermediate for patients with PB-miRNA1, and dismal for those with PB-MYC/FOXR2 or PB-RB1. Reduced-dose CSI was adequate for patients with average-risk, PB-miRNA1/2 disease. We systematically interrogated the clinical and molecular heterogeneity within pineal parenchymal tumors and proposed a consensus nomenclature for disease groups, laying the groundwork for future studies as well as routine use in tumor diagnostic classification and clinical trial stratification.
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