Molecular landscape of pediatric type IDH wildtype, H3 wildtype hemispheric glioblastomas.

The WHO (2021) Classification classified a group of pediatric-type high-grade gliomas as IDH wildtype, H3 wildtype but as of currently, they are characterized only by negative molecular features of IDH and H3. We recruited 35 cases of pediatric IDH wildtype and H3 wildtype hemispheric glioblastomas. We evaluated them with genome-wide methylation profiling, targeted sequencing, RNAseq, TERT promoter sequencing, and FISH. The median survival of the cohort was 27.6 months. With Capper et al.'s36 methylation groups as a map, the cases were found to be epigenetically heterogeneous and were clustered in proximity or overlay of methylation groups PXA-like (n = 8), LGG-like (n = 10), GBM_MYCN (n = 9), GBM_midline (n = 5), and GBM_RTKIII (n = 3). Histology of the tumors in these groups was not different from regular glioblastomas. Methylation groups were not associated with OS. We were unable to identify groups specifically characterized by EGFR or PDGFRA amplification as proposed by other authors. EGFR, PDGFRA, and MYCN amplifications were not correlated with OS. 4/9 cases of the GBM_MYCN cluster did not show MYCN amplification; the group was also enriched for EGFR amplification (4/9 cases) and the two biomarkers overlapped in two cases. Overall, PDGFRA amplification was found in only four cases and they were not restricted to any groups. Cases in proximity to GBM_midline were all hemispheric and showed loss of H3K27me3 staining. Fusion genes ALK/NTRK/ROS1/MET characteristic of infantile glioblastomas were not identified in 17 cases successfully sequenced. BRAF V600E was only found in the PXA group but CDKN2A deletion could be found in other methylation groups. PXA-like cases did not show PXA histological features similar to findings by other authors. No case showed TERT promoter mutation. Mutations of mismatch repair (MMR) genes were poor prognosticators in single (p ≤ 0.001) but not in multivariate analyses (p = 0.229). MGMT had no survival significance in this cohort. Of the other common biomarkers, only TP53 and ATRX mutations were significant poor prognosticators and only TP53 mutation was significant after multivariate analyses (p = 0.024). We conclude that IDH wildtype, H3 wildtype pediatric hemispheric glioblastomas are molecularly heterogeneous and in routine practice, TP53, ATRX, and MMR status could profitably be screened for risk stratification in laboratories without ready access to methylation profiling.

Mismatch repair proteins PMS2 and MLH1 can further refine molecular stratification of IDH-mutant lower grade astrocytomas.

The diagnostic role of Isocitrate Dehydrogenase (IDH) mutation status in adult lower grade astrocytomas was first formally presented within the WHO Classification of Tumours of the Central Nervous System (2016). IDH-mutant astrocytomas are not as common as IDH-wildtype astrocytomas but are of better prognosis. Our previous study provided an evident that IDH-mutant lower grade astrocytomas is not a homogeneous group and could be further stratified by PDGFRA amplification, CDK4 amplification and CDKN2A deletion. In this study, we detected the expressions of DNA mismatch repair (MMR) proteins (PMS2, MLH1, MSH2, MSH6) and PD-L1 by immunohistochemistry in 147 IDH-mutant lower grade astrocytomas and explored their clinical relevance. The loss of was identified in 28.6%, 1.4%, 8.8% and 13.6%, respectively. PD-L1 expression was detected in 1.4% of this cohort. Survival analysis revealed that loss of PMS2 was correlated with shorter OS (p < 0.001) and PFS (p = 0.005). Loss of PMS2 or MLH1 was associated with shorter OS (p < 0.001) and PFS (p = 0.008). In IDH-mutant lower grade astrocytomas without CDKN2A deletion, loss of PMS2 was associated with poorer OS (p < 0.001) and PFS (p = 0.001). Furthermore, among IDH-mutant lower grade astrocytomas lacking the three biomarkers (PDGFRA, CDK4 and CDKN2A), loss of PMS2 was also associated with a poorer OS (p < 0.001) and PFS (p = 0.003). Our data illustrated the potential application of MMR genes in stratification of IDH-mutant lower grade astrocytomas without PDGFRA, CDK4 and CDKN2A copy number alterations.

IDH mutant lower grade (WHO Grades II/III) astrocytomas can be stratified for risk by CDKN2A, CDK4 and PDGFRA copy number alterations.

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.

Identification of subsets of IDH-mutant glioblastomas with distinct epigenetic and copy number alterations and stratified clinical risks.

BACKGROUND: IDH-mutant glioblastoma is classified by the 2016 CNS WHO as a group with good prognosis. However, the actual number of cases examined in the literature is relatively small. We hypothesize that IDH-mutant glioblastoma is not a uniform group and should be further stratified. METHODS: We conducted methylation profiles and estimated copy number variations of 57 IDH-mutant glioblastomas. RESULTS: Our results showed that 59.6% and 40.4% of tumors belonged to glioma-CpG island methylator phenotype (G-CIMP)-high and G-CIMP-low methylation subgroups, respectively. G-CIMP-low subgroup was associated with significantly worse overall survival (OS) as compared to G-CIMP-high (P = .005). CDKN2A deletion (42.1%) was the most common gene copy number variation, and was significantly associated with G-CIMP-low subgroup (P = .004). Other frequent copy number changes included mesenchymal-epithelial transition (MET) (5.3%), CCND2 (19.3%), PDGFRA (14.0%), CDK4 (12.3%), and EGFR (12.3%) amplification. Both CDKN2A deletion (P = .036) and MET amplification (P < .001) were associated with poor OS in IDH-mutant glioblastomas. Combined epigenetic signature and gene copy number variations separated IDH-mutant glioblastomas into Group 1 (G-CIMP-high), Group 2 (G-CIMP-low without CDKN2A nor MET alteration), and Group 3 (G-CIMP-low with CDKN2A and/or MET alteration). Survival analysis revealed Groups 1 and 2 exhibited a favorable OS (median survival: 619 d [20.6 mo] and 655 d [21.8 mo], respectively). Group 3 exhibited a significant shorter OS (median survival: 252 d [8.4 mo]). Multivariable analysis confirmed the independent prognostic significance of our Groups. CONCLUSIONS: IDH-mutant glioblastomas should be stratified for risk with combined epigenetic signature and CDKN2A/MET status and some cases have poor outcome.

Whole-exome sequencing revealed mutational profiles of giant cell glioblastomas.

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.

Pediatric low-grade gliomas can be molecularly stratified for risk.

Pediatric low-grade gliomas (PLGGs) consist of a number of entities with overlapping histological features. PLGGs have much better prognosis than the adult counterparts, but a significant proportion of PLGGs suffers from tumor progression and recurrence. It has been shown that pediatric and adult low-grade gliomas are molecularly distinct. Yet the clinical significance of some of newer biomarkers discovered by genomic studies has not been fully investigated. In this study, we evaluated in a large cohort of 289 PLGGs a list of biomarkers and examined their clinical relevance. TERT promoter (TERTp), H3F3A and BRAF V600E mutations were detected by direct sequencing. ATRX nuclear loss was examined by immunohistochemistry. CDKN2A deletion, KIAA1549-BRAF fusion, and MYB amplification were determined by fluorescence in situ hybridization (FISH). TERTp, H3F3A, and BRAF V600E mutations were identified in 2.5, 6.4, and 7.4% of PLGGs, respectively. ATRX loss was found in 4.9% of PLGGs. CDKN2A deletion, KIAA1549-BRAF fusion and MYB amplification were detected in 8.8, 32.0 and 10.6% of PLGGs, respectively. Survival analysis revealed that TERTp mutation, H3F3A mutation, and ATRX loss were significantly associated with poor PFS (p < 0.0001, p < 0.0001, and p = 0.0002) and OS (p < 0.0001, p < 0.0001, and p < 0.0001). BRAF V600E was associated with shorter PFS (p = 0.011) and OS (p = 0.032) in a subset of PLGGs. KIAA1549-BRAF fusion was a good prognostic marker for longer PFS (p = 0.0017) and OS (p = 0.0029). MYB amplification was also a favorable marker for a longer PFS (p = 0.040). Importantly, we showed that these molecular biomarkers can be used to stratify PLGGs into low- (KIAA1549-BRAF fusion or MYB amplification), intermediate-I (BRAF V600E and/or CDKN2A deletion), intermediate-II (no biomarker), and high-risk (TERTp or H3F3A mutation or ATRX loss) groups with distinct PFS (p < 0.0001) and OS (p < 0.0001). This scheme should aid in clinical decision-making.