A_IDH_mut and O_IDH_mut are the second and third most common adult-type diffuse gliomas, respectively, after GBM. They 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 clustering.
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 level16. The resulting chromatin compaction promotes the CpG island hypermethylation phenotype (glioma CpG island methylator phenotype, gCIMP), resulting in global silencing of tumor-suppressor genes (TSGs)16,17. Lu et al. reported that impaired histone lysine demethylation by accumulation of 2-HG caused by IDH mutation prevents cellular differentiation18. However, the exact mechanism of action of oncometabolites remains unknown. Unexpectedly, Filipski et al. and Habiba et al. found that IDH1 R132H-mutant O_IDH_mut involves H3K27me3 protein loss in 96% (25/26) and 90% (36/40) of cases, respectively, but that non-R132H IDH-mutant O_IDH_mut does not show H3K27me3 loss19,20. As mentioned above, loss of nuclear H3K27me3 is known to increase histone methylation by impairing demethylation of inhibitory histone markers induced by increased 2-HG levels in IDH mutant gliomas18. We believe that 2-HG may play a pivotal role in histone demethylation. CpG island DNA methylation in the nonpromoter region of IDH mutant cells, that is, DNA methylation of the CCCTC-binding factor (CTCF) binding site, results in loss of the insulated site, intercepts the topographically associated domain (TAD) boundary, and leads to an active enhancer in genes, such as PDGFRA21.
Dysfunction of tumor-suppressor genes, TP53 in astrocytoma, CIC and FUBP1 in oligodendroglioma
IDH mutations are an early oncogenic molecular change in IDH-mutant gliomas. Mutation of IDH can cause oncogene (oncometabolite)-induced cellular senescence22. Constitutive activation of RB1, TP53, and CDKN2A is a well-known growth arrest signaling pathway that produces hypoproliferative senescent cells23. Senescent glioma cells adopt a different path to survive. Tumor cells need to block senescence-inducing TSGs, such as TP53, RB1, and CDKN2A/2B, and 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 oligodendrogliomas24, 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 factors24. 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 growth24.
FUBP1 mutations are reported in approximately 15% of O_IDH_mut cases25, 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 DNA-binding 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 hematopoiesis26,27. FUBP1 is also a long-tail cancer driver that cooperates with other tumor-suppressor genes28. FUBP1 is a posttranscriptional regulator of N6-methyladenosine (m6A) RNA methylation, translation, mRNA stability, and splicing. Its loss leads to global changes in RNA splicing and widespread expression of aberrant driver isoforms28. Therefore, somatic alteration of FUBP1 (FUBP1−/−) contributes to neoplastic transformation via aberrant RNA splicing and m6A methylation28. 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), including 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 gliomas29. 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) complex30. However, TERTp mutation may be a second genetic event following oncogenic activation, such as IDH mutation in diffuse gliomas, BRAF V600E mutation in thyroid cancer, and FGFR3 mutation in urothelial carcinoma29,31,32.
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 expression31. Inhibition or inactivation of CIC by mutation is associated with TERTp mutation and increased TERT mRNA expression in O_IDH_mut33. TERTp and ATRX mutations are mutually exclusive, suggesting that they have equivalent TMMs34; 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 factors35. 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 cancers36. Genome-wide DNA methylation patterns can be used to subclassify brain tumors, which correlate with mutational status, DNA copy-number aberrations, and gene expression signatures37,38.
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 itself39. MGMTp methylation is a better prognostic factor for gliomas40 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 astrocytomas42. 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 tumors43. A summary 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.