Genomic profiles of IDH-mutant gliomas: MYCN-amplified IDH-mutant astrocytoma had the worst prognosis

This study aimed to find any ambiguous genetic outlier for “oligodendroglioma, IDH-mutant and 1p/19q-codeleted (O_IDH_mut)” and “astrocytoma, IDH-mutant (A_IDH_mut)” and to redefine the genetic landscape and prognostic factors of IDH-mutant gliomas. Next-generation sequencing (NGS) using a brain tumor-targeted gene panel, methylation profiles, and clinicopathological features were analyzed for O_IDH_mut (n = 74) in 70 patients and for A_IDH_mut (n = 95) in 90 patients. 97.3% of O_IDH_mut and 98.9% of A_IDH_mut displayed a classic genomic landscape. Combined CIC (75.7%) and/or FUBP1 (45.9%) mutations were detected in 93.2% and MGMTp methylation in 95.9% of O_IDH_mut patients. In A_IDH_mut, TP53 mutations were found in 86.3% and combined ATRX (82.1%) and TERTp (6.3%) mutations in 88.4%. Although there were 3 confusing cases, NOS (not otherwise specified) category, based on genetic profiles, but they were clearly classified by combining histopathology and DKFZ methylation classifier algorithms. The patients with MYCN amplification and/or CDKN2A/2B homozygous deletion in the A_IDH_mut category had a worse prognosis than those without these gene alterations and MYCN-amplified A_IDH_mut showed the worst prognosis. However, there was no prognostic genetic marker in O_IDH_mut. In histopathologically or genetically ambiguous cases, methylation profiles can be used as an objective tool to avoid a diagnosis of NOS or NEC (not elsewhere classified), as well as for tumor classification. The authors have not encountered a case of true mixed oligoastrocytoma using an integrated diagnosis of histopathological, genetic and methylation profiles. MYCN amplification, in addition to CDKN2A/2B homozygous deletion, should be included in the genetic criteria for CNS WHO grade 4 A_IDH_mut.


Materials and methods
A total of 169 cases of O_IDH_mut (n = 74) involving 70 patients and A_IDH_mut (n = 95) involving 90 patients were obtained from the archives of the Seoul National University Hospital (SNUH) from July 2018 to September 2022. Primary IDH-mutant astrocytomas were 18% more common than primary oligodendrogliomas (59% vs. 41%) over the last 3 years at a single institute (SNUH) when recurrent cases were excluded. Next-generation sequencing (NGS) using a customized FiRST ("Friendly integrated Research-based Smart Trustworthy") brain tumor-targeted gene panel (BTP) was performed in all cases. A list of genes included in the FiRST BTP is provided in Supplementary Table 1. Two pathologists (SI Kim and SH Park) reviewed the genetics-integrated diagnoses with NGS results. Genetics-integrated diagnoses were made according to the 5th edition of the WHO classifications of the CNS published in 2021 5 . When the genetic features of A_IDH_mut and O_IDH_mut coexisted, methylation profile-based classification using the Deutsches Krebsforschungszentrum (DKFZ) algorithm was performed using Infinium MethylationEPIC 850K BeadChip array platforms.
Immunohistochemical (IHC) study. Tissue Table 2). IHC staining was performed using a standard avidin-biotin-peroxidase method with a BenchMark ULTRA system (Roche Diagnostics, Indianapolis, US). The positive control was known positive tissue, and entrapped positive cells were used. For the negative control, the primary antibody was omitted.  were as follows: methylated forward, 5′-TTT CGA CGT TCG TAG GTT  TTC GC-3′; methylated reverse, 5′-GCA CTC TTC CGA AAA CGA AAC G-3′; unmethylated forward, 5′-TTT  GTG TTT TGA TGT TTG TAG GTT TTT GT-3′; unmethylated reverse, 5′-AAC TCC ACA CTC TTC CAA AAA CAA AAC A-3′. The annealing temperature was set to 64 °C. The obtained polymerase chain reaction products were electrophoresed on 2% agarose gels and visualized under UV illumination after staining with ethidium bromide.
Methylation array analysis and t-SNE clustering with the Illumina 850 K microarray. DNA methylation array analysis was performed for three ambiguous cases of O_IDH_mut using an Infinium Meth-ylationEpic 850 K BeadChip array platform (Illumina, USA). DNA methylation data analysis was performed using R software (R 4.1.3, https:// www.r-proje ct. org/). Our samples were analyzed using the methylation classifier of DKFZ with reference to a diffuse glioma cohort of 551 samples from 10 subclasses 11 . A preprocessing procedure was conducted for raw methylation intensity signals using the R package "methylationArrayAnalysis" (version 1.14.0) 12 . We utilized the "combinedArray" command to merge the different platforms (450 K, EPIC). After filtering and normalization, 365,201 probes remained for subsequent analysis. The 20,000 most variably methylated probes were selected based on the standard deviation to perform unsupervised nonlinear dimension reduction. The resulting distance matrix was used as the input for t-distributed stochastic neighbor embedding analysis (t-SNE; Rtsne package version 0.15). The nondefault parameters used were distance = TRUE, perplexity = 30, θ = 0, and max_iter = 2000. A t-SNE plot was generated using the "ggplot2" package (version 3.3.3) for effective visualization. IDAT files were uploaded to versions v11b4 and v12.5 of the online CNS tumor methylation classifier (https:// www. molec ularn europ athol ogy. org) for classification.
Survival analysis. The median overall survival (OS) and progression-free survival (PFS) were obtained using the R package "survminer" (version 0.4.9). Survival analysis was performed in 70 patients with O_IDH_ mut and 87 patients with A_IDH_mut. As most patients with O_IDH_mut were alive at the time of analysis during 1 to 244-months (median 34.7 months) follow-up period, overall survival (OS) could not be obtained.

Ethics approval and consent to participate. The institutional review board (IRB) of Seoul National
University Hospital (SNUH) approved this study (IRB No: 1906-020-1037) and has therefore been performed under the ethical standards set out in the 1964 Declaration of Helsinki and its subsequent amendments. As this study is a retrospective review of anonymized electronic medical records, pathology, and NGS data utilizing a brain tumor-specific somatic gene panel, IRB of SNUH (IRB No: 1906-020-1037) waived the informed consent for this study under the Korean Bioethics and Safety Act.

Comparison of the molecular profiles of sequentially developed tumors.
When comparing the molecular profiles of the 5 pairs of A_IDH_mut and 4 pairs of O_IDH_mut, no significant difference was noted. However, in one case each of A_IDH_mut and O_IDH_mut, the homozygous deletion of CDKN2A/2B was disappeared in the recurrent tumors that was present in the previous tumors. MMR-deficient primary astrocytomas and one temozolomide (TMZ)-induced hypermutated recurrent astrocytoma (grade 4). The first patient was a 28-year-old male who initially had grade 3 A_IDH_mut. He received local RT (50.4 + 10.8 Gy) after initial resection of the tumor and a second RT (45 Gy/25 Fx) at 3 and a half years later when the tumor relapsed as WHO grade 4 A_IDH-mut. He underwent a total of 3 reoperations. This patient was not treated with TMZ. This patient harbored an MLH1 germline mutation (p.Arg687Trp, c.2059C > T), and the tumor exhibited high microsatellite instability (MSI-H), corresponding to Lynch syndrome-associated, MMRdeficient A_IDH_mut. Nevertheless, the patient developed no tumor other than the brain tumor. The third patient initially had grade 3 A_IDH_mut at the age of 32 years. After receiving concurrent chemoradiotherapy (CCRT) with TMZ for 2 years, the tumor recurred, and he underwent a second CCRT. After six months, the recurrent tumor was removed, and A_IDH_mut, grade 4, was diagnosed. NGS of the recurrent tumor revealed hypermutation. Because the patient did not carry a germline mutation in the MMR gene, the case was judged to be TMZ-induced hypermutation.
Study of histologically and genetically confusing cases with DNA methylation profiling. Although most O_IDH_mut cases were not difficult to diagnose, 2.7% (2/74) of O_IDH_mut and 1.1% (1/95) of A_IDH_mut cases in our study showed confounding with respect to genomic profiles and morphology (Figs. 2, 3, and 4). All three cases had TP53 mutations, along with classic genomic signatures of O_IDH_mut; 1p/19q-codeletion, TERTp and CIC, mutations. The first two cases showed histopathological oligodendroglioma-like morphology with minigemistocytic or gliofibrillary oligodendrocytes. Remaining one patient had histopathologically CNS WHO grade 2 astrocytoma as the initial tumor but progressed to CNS WHO grade 3, with an ambiguous appearance with more rounded nuclei in the recurrent tumor (Fig. 4).
We performed a dimensional reduction procedure to visualize the results into t-SNE plot using a reference cohort of adult-type diffuse gliomas and these three cases whown in Table 5. Since the reference cohort data of the t-SNE plot was version v11b4 of DKFZ, one case of 1p/19q-codeleted A_IDH_mut clustered with O_IDH_mut, as mentioned above ( Supplementary Fig. 1, t-SNE plot by v11b4); unfortunately, the t-SNE plot by v12.5 is not shown because we could not obtain the reference data by v12.5.

Survival analysis. Kaplan-Meier survival analysis of the OS and PFS of patients with A_IDH_mut revealed
significantly worse survival with increasing grade (Fig. 5A, B).
Patients with A_IDH_mut with CDKN2A/2B homozygous deletion or MYCN amplification had significantly worse survival than those with A_IDH_mut without these two gene alterations (Fig. 5C-F). These results correspond to previous studies and cIMPACT-NOW updates 5 and 6 6,13-15 . However, PDGFRA amplification and Table 4. Genetic profile of 11 cases of ATRX-intact astrocytoma, IDH-mutant. VAF variant allele frequency.

Discussion
A_IDH_mut and O_IDH_mut have characteristic histopathological, genetic and epigenetic profiles. Although it is well known that morphologically ambiguous cases exist, they can be diagnosed as A_IDH_mut or O_IDH_ mut through a genetics-integrated diagnosis. This study aimed to determine whether mixed oligoastrocytoma occurs and whether "NOS" cases can be eliminated using genetics-integrated diagnostics and DKFZ methylation classifier.

Function of oncometabolites produced by IDH mutation. The oncometabolite 2-hydroxyglutarate
(2-HG), which is produced by IDH1/2 mutations, is a competitive inhibitor of multiple alpha-ketoglutarate (a-KG)-dependent dioxygenases. 2-HG induces a wide range of histone demethylases at the promoter level and the ten eleven translocation (TET) family of dioxygenases of 5-methylcytosine (5mC) at the gene level 16   can cause oncogene (oncometabolite)-induced cellular senescence 22 . Constitutive activation of RB1, TP53, and CDKN2A is a well-known growth arrest signaling pathway that produces hypoproliferative senescent cells 23 . Senescent glioma cells adopt a different path to survive. Tumor cells need to block senescence-inducing TSGs, such as TP53, RB1, and CDKN2A/2B, so that can bypass senescence-induced tumor cell apoptosis. These molecular mechanisms may induce simultaneous TP53 mutations in A_IDH_mut and CIC and/or FUBP1 mutations in O_IDH_mut to obtain proliferative activity and to avoid cellular senescence. CIC mutations are found in approximately 70% of oligodendrogliomas 24 , and we found them in 75.7% of our O_IDH_mut tumors. CIC is an important tumor suppressor that acts through transcriptional repression of target genes, including the polyoma enhancer activator 3 (PEA3) subfamily of E26 transformation-specific (ETS) transcription factors 24 . CIC is a transcriptional repressor that recruits histone deacetylations. CIC inactivation by mutations or deletion increases the level of histone acetylation, leading to transcription of EGFR/RAS/MAPK pathway components, promoting mitogen-independent tumor growth 24 . www.nature.com/scientificreports/ FUBP1 mutations are reported in approximately 15% of O_IDH_mut cases 25 , but they were found in 45.9% of O_IDH_mut cases in our series, including intronic mutations in 9.5%. Only four novel FUBP1 mutations were identified in the present study (Table 2). FUBP1 was first described in 1994 as a single-stranded DNAbinding protein that binds to a noncoding, single-stranded far upstream element (FUSE) 2.5 kb upstream of the MYC promoter. FUBP1 binds to the transcription factor IIH (TFIIH), activates the MYC oncogene, and directly represses p21 in normal hematopoiesis 26,27 . FUBP1 is also a long-tail cancer driver that cooperates with other tumor-suppressor genes 28 . FUBP1 is a posttranscriptional regulator of N6-methyladenosine (m 6 A) RNA methylation, translation, mRNA stability, and splicing. Its loss leads to global changes in RNA splicing and widespread expression of aberrant driver isoforms 28 . Therefore, somatic alteration of FUBP1 (FUBP1 −/− ) contributes to neoplastic transformation via aberrant RNA splicing and m 6 A methylation 28 . FUBP1 missense, nonsense, silent mutations, whole-gene deletions, frameshift deletions, and insertions have been observed in oligodendrogliomas 7 .
Telomere lengthening by ATRX and TERTp mutations. Cancer involves rapidly proliferating cells that result in telomere shortening until the maximal number of cell divisions is reached ("Hayflick limit") 22 . If replication proceeds, tumor cells gain chromosomal instability and eventually undergo apoptosis. For immortal growth, glioma cells exhibit telomere maintenance mechanisms (TMMs), which are TERTp mutation in O_IDH_mut and IDH-wildtype GBM and ATRX mutation in the majority of astrocytomas and histone-mutant pediatric-type high-grade gliomas 29 . TERT allows stabilization and elongation of telomeres. Altered length of telomeres (ALT) is another TMM that is induced by dysfunction of the ATRX/death-associated protein 6 (DAXX) complex 30 . However, TERTp mutation may be a second genetic event following oncogenic activation,   29,31,32 . In our study, TERTp mutations were found in 100% of O_IDH_mut and 6.3% of A_IDH_mut cases, while ATRX mutations were present in 82.1% of A_IDH_mut cases but not in oligodendrogliomas. TERT has many functions, both canonical and noncanonical. The most significant canonical function of TERT is telomere lengthening, and the most important noncanonical functions of TERT are reduction of apoptosis, regulation of chromatin structure and gene expression 31 . Inhibition or inactivation of CIC by mutation is associated with TERTp mutation and increased TERT mRNA expression in O_IDH_mut 33 . TERTp and ATRX mutations are mutually exclusive, suggesting that they have equivalent TMMs 34 ; however, questions of why O_IDH_mut involves TERTp mutation rather than ATRX mutation and most A_IDH_mut have ATRX mutations, not TERTp mutations, remain. In patients with A-IDH-mut, there was no difference in OS or PFS between the those with ATRX mutation and TERTp mutation (Supplementary Fig. 2A,B). ATRX-or TERTp-wildtype in patients with A_IDH_mut were associated with worse OS (p = 0.046) but did not affect PFS ( Supplementary Fig. 2C-F).
MGMTp methylation. DNA methylation patterns are generally stable and unique in differentiated cells; however, methylation profiles can be altered by extrinsic or intrinsic factors 35 . Both hypermethylation and hypomethylation play essential roles in long-term gene regulation and reactivation of oncogenes, TSG inhibition, deregulation of mRNA expression, mutagenesis, or alteration of functional chromosomal stability in cancers 36 . Genome-wide DNA methylation patterns can be used to subclassify brain tumors, which correlate with mutational status, DNA copy-number aberrations, and gene expression signatures 37,38 .
MGMT is a key DNA repair enzyme that removes mutagenic methyl groups from the P6 position of guanine by transferring it to the cysteine acceptor site of the protein itself 39 . MGMTp methylation is a better prognostic factor for gliomas 40 and was eventually confirmed as a predictive marker for alkylating agents by European Organization for Research and Treatment (EORTC) 41 . MGMTp methylation belongs to the glioma CIMP family and was found in 95.9% of our O_IDH_mut series, and loss of nuclear H3K27me3 can be generated using IDH mutation-induced demethyltransferase blockade. According to Horbinski et al. 's study, the average beta values of O_IDH_mut of all 147 MGMTp CpG sites were significantly higher at 44.9%, and transcriptionally sensitive regions were 69% more methylated in O_IDH_mut. However, none were significantly more methylated in astrocytomas 42 . It is curious why MGMTp methylation appears to be less frequent (73.7%) in A_IDH_mut than in O_IDH_mut.

Gene dosage effect in gliomas.
In this study, higher grade A_IDH_mut showed significantly more CNVs than lower grade A_IDH_mut (Supplementary Table 3). A comprehensive genomic landscape revealed the gene dosage effect of gliomas, with more gene mutations resulting in more aggressive tumors 43 . A summary Table 5. The histologically and genetically confusing cases with DNA methylation profiling, which are TP53mutant oligodendrogliomas and 1p/19q-deleted astrocytoma, IDH-mutant. DOD death of disease. www.nature.com/scientificreports/ of the genomic features and possible effect of the molecular changes in O_IDH_mut and A_IDH_mut in our study is shown in Fig. 8.

Conclusion
The combined oncometabolite 2-HG induced by IDH mutations, TSG blockade, and TMM are important drivers and diagnostic hallmark of IDH-mutant gliomas. Despite reliable histological or molecular characteristics of our IDH-mutant gliomas, there were doubts in three cases because of the mixed features of O_IDH_mut and A_IDH_mut. The methylation classifier of DKFZ (v.12.5) matched perfectly with either O_IDH_mut or A_IDH_ mut, and no genetically or epigenetically ambiguous IDH-mutant adult-type diffuse gliomas were observed. These valuable features should be recognized in differential diagnosis and genotype-specific therapeutic strategy. Finally, patients with MYCN-amplified A_IDH_mut had the worst prognosis, strongly suggesting that MYCN amplification, in addition to CDKN2A/2B homozygous deletion, should be included in the genetic criteria for grade 4 A_IDH_mut.  The hazard plot of various grade and genetic parameters in A_IDH_mut and O_IDH_mut. In A_IDH_mut, grade and CDKN2A/2B were statistically significant and MYCN-amplified tumor also were statistical significance, however, PTEN loss and PDGFRA amplification did not affect on the biological behavior of A_IDH_mut. In O_IDH_mut, the higher grade the higher HR, but it does not have statistical significance in this study (OS overall survival, N number of cases, HR hazard ratio, MVP microvascular proliferation).

Data availability
The datasets generated and/or analysed during the current study are available in the Gene Expression Omnibus (GEO) repository under accession number GSE222423 (https:// www. ncbi. nlm. nih. gov/ geo/ query/ acc. cgi? acc= GSE22 2423).