Molecular Classification of CNS Tumors

#### **Chapter 6**

## The Distribution and Significance of *IDH* Mutations in Gliomas

*Nu Thien Nhat Tran*

#### **Abstract**

In 2009, the discovery of isocitrate dehydrogenase (IDH) mutations in gliomas is a powerful example of understanding of the relationship between tumor genetics and human diseases. IDHs, catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate with production of NADH/NADPH, is the key enzymes in the Krebs cycle. IDH mutations, which occur early in gliomagenesis, change the function of the enzymes, causing them to produce 2–hydroxyglutarate, and to not create NADPH. Gliomas with mutated IDH have improved prediction of patient outcomes compared to its with wild-type IDH. Thus, the WHO Classification of Tumors of the Central Nervous System was revised in 2016 to incorporate molecular biomarkers (including the IDH mutations) – together with classic histological features – in an integrated diagnosis, in order to define distinct glioma entities as precisely as possible. The aim of this chapter is to review the findings on the epidemiology and significance of IDH mutations in human gliomas, from discovery to the current knowledge about their molecular pathogenesis.

**Keywords:** *IDH* mutation, gliomas, Isocitrate dehydrogenase, significance, therapies

#### **1. Introduction**

Isocitrate dehydrogenase (IDH) is a key enzyme in the Krebs cycle and plays an important role in energy metabolism. This enzyme is involved in a number of cellular processes, such as mitochondrial oxidative phosphorylation, regulation of cellular redox status, glutamine metabolism as well as lipogenesis or glucose sensing.

In 2008, Parsons et al. discovered a link between *IDH* mutations and gliomas. After that, further studies showed that *IDH* mutations are not only common but also closely related to the diagnosis, treatment and prognosis of gliomas. Therefore, the WHO classification of Tumors of the Central Nervous System of 2016, gliomas are subdivided based on combined classical histological with molecular markers (including the *IDH* mutations). This reclassification is expected to guide treatment decisions and improve outcome prediction.

The aim of this chapter is to review the findings on the epidemiology and significance of *IDH* mutations, from current knowledge about molecular pathogenesis to the value these mutations in gliomas.

#### **2. IDH enzymes**

#### **2.1 Normal enzymes**

#### *2.1.1 Genetics and classification enzymes*

IDH is a small molecule protein which is mainly distributed in the liver, heart muscle and skeletal muscle. In humans, there are three isozymes of IDH, which differ in subcellular localization, structural organization, allosteric regulation, catalytic mechanism and cofactor requirement. These are IDH1, IDH2 and IDH3.

These isozymes are encoded by five separate genes. IDH1, encoded by *IDH1* gene on 2q33.3, is configured as a homodimer with two enzymatically active sites and most of its activity is detected in the cytosol and in peroxysomes, Main function of IDH1 is believed to be the synthesis of NADPH, required for reducing reactions and for lipid synthesis [1–3].

IDH2, which is found in mitochondrial, encoded by the *IDH2* gene on 15q26.1, in [4]. Similar to IDH1, this enzyme is structured as a homodimer. Recent findings show that the IDH2 may be the main catalyst for the oxidation of isocitrate (ICT) to α-ketoglutarate (α-KG) in the citric acid cycle (TCA) in [5]. IDH3 is composed of three subunits encoded by *IDH3A* (subunit alpha), on 15q25.1-q25.2, *IDH3B* (subunit beta) on 20p13 and *IDH3G* (subunit gamma), on Xq28 [6–8].

IDH3 is a multi-tetrameric enzyme (2α1β1γ) with α − subunits being catalytic and the β- and γ- subunits being believed to be regulatory [9, 10]. Since *IDH3* mutations do not occur with a significant frequency in glioma [11] this chapter focuses on the roles of IDH1 and IDH2 in glioma biology and uses IDH to refer to both IDH1 and IDH2 but not IDH3 (**Figure 1**).

#### *2.1.2 Mechanism and function of IDH enzymes*

IDH exists in NADP-dependent forms [2]. Both IDH1 and IDH2 exist as homodimers, share considerable sequence similarity (70% identity humans). IDH1 is highly expressed in the mammalian liver (IDH1 provides NADPH for peroxisomal fat and cholesterol synthesis) with only moderate to absent expression in other tissues, whereas IDH2 is highly expressed in heart, muscle, and activated lymphocytes [13].

The main function of IDH is to catalyze the oxidative decarboxylation of ICT to α-KG. This reaction also produces a molecule called NADPH, which is necessary for

#### **Figure 1.**

*IDH1 is localized in the cytoplasm and peroxisomes, whereas IDH2 is founded within the mitochondrial matrix [12].*

multiple cellular processes. The NADPH is involved in the breakdown of lipids for energy, and also protects cells from potentially harmful molecules called reactive oxygen species.

By providing mitochondrial NADPH for NADPH-dependent antioxidant enzymes, IDH maintains a pool of reduced glutathione and peroxiredoxin [14]. These molecules protect mitochondria from ROS-mediated oxidative damage, ensuing lipid peroxidation and DNA damage, and from stress induced by heat shock, cadmium, excess fructose, or tumor necrosis factor-α (TNF-α) [13, 14]. These data suggest that IDH is important for cell stress responses, mitochondrial bioenergetics, and macromolecular synthesis to support cell survival and growth.

#### **2.2 Molecular pathogenesis of IDH mutations**

*IDH1* mutations almost occur at Arginine 132 resulting in amino acid exchange, including R132H (most common, 88%), R132C, R132L, R132S, and R132G. *IDH2* mutations typically occur at R140 or R172. Of *IDH2* mutations, R172K is most common. *IDH1* and *IDH2* mutations are mutually exclusive [15].

#### *2.2.1 Enzymatic properties of mutant IDHs*

Mutations in *IDH* are neomorphic gain-of-function mutations, which affect cofactor binding affinity and conformation of the enzymes' active center. When mutated, the enzymes' binding affinity to ICT decreases, while affinity to NADPH increases. Mutations result in a dominant gain-of-function that catalyzes the NADPH-dependent reduction of α -KG to D-2-hydroxyglutarate (D-2HG or R-2HG) but not further carboxylated [16]. D-2HG is a competitive inhibitor of multiple α -KG-dependent dioxygenases, including histone demethylase. As a result, D-2HG makes histone methylation and blocks cell differentiation. Therefore, D-2HG is called oncometabolite.

All *IDH* mutant enzymes produce D-2HG; their allelic frequency, enzymatic property, and association with overall prognosis, however, are markedly different. For example, while the IDH2Arg140 mutation is exclusively found in myeloid malignancies [17], the IDH1Arg132 and IDH2Arg172 mutations are common in gliomas.

In addition, due to essential roles of IDHs in producing cytoplasmic and mitochondrial NADPH, tumor cell survival may also be dependent on basal IDH activities to maintain cytoplasmic and mitochondrial redox homeostasis.

#### *2.2.2 Mutant IDH enzymes control cellular growth*

A large body of evidence indicates that IDH mutation inhibits cell proliferation [18–20]. Theoretically, D-2HG inhibits ATP synthase, resulting in decreased mTOR (mammalian target of rapamycin) signaling and cell growth. Moreover, by inhibiting the FTO (fat mass and obesity-associated) demethylase activity, D-2HG promotes cell-cycle arrest, thereby increasing N6-methyladenosine modification of MYC/CEBPA (CCAAT/enhancer binding protein alpha) transcripts for destabilization and, thus, decreasing proliferative signaling [20].

There is a study in mice indicating that IDH1R132H homozygous expression in neural progenitor cells (NPCs) results in extensive cerebral hemorrhage and perinatal lethality [21]. On molecular levels, high-level accumulation of D-2HG inhibits prolyl-hydroxylation and subsequent maturation of collagen. Immature collagens accumulate, resulting in an aberrantly formed basement membrane and the initiation of an endoplasmic reticulum (ER) stress response. As a result, mice developed hydrocephalus and grossly dilated lateral ventricles.

Collectively, these studies provide strong evidence that IDH mutation targets various signaling pathways to inhibit glial cell proliferation.

#### **2.3 IDH mutation involvement human cancers**

Mutations in *IDH1* and *IDH2* have recently been discovered in CNS cancers like gliomas, and a number of types of leukemia, including acute myeloid leukemia. This discovery has been extended to prostate cancer, intrahepatic cholangiocarcinoma, colon cancer, and thyroid cancer as well since 2009.

Mutations targeting IDH in different types of tumors share four distinct biochemical features. First of all, *IDH* mutations are almost somatic and rarely germline. In addition, predominantly all reported cases have been frameshifts or deletions, whereas nonsense mutations have not been observed in cancer.

Second, the vast majority of *IDH* mutations (Mut) are heterozygous with a wildtype (Wt) allele [22, 23]. The existence of wild type-mutant (Wt-Mut) and mutantmutant (Mut-Mut) dimers in addition to wild type-wild type (Wt-Wt) dimer in a cell heterozygous for *IDH* mutation has been reported [24]. An illustration of the three dimer types allele is provided in **Figure 2**.

From what has been mentioned so far, the most likely model is as follows: substitution of two arginine residues on both monomers inactivates both forward oxidative decarboxylation and reverse reductive carboxylation reactions while the presence of one arginine fully inhibits the forward oxidative decarboxylation reaction but changes the product of the reverse reductive carboxylation reaction to be D-2HG instead of ICT.

It is conceivable that the Mut-Mut dimer is totally, while the Wt-Mut dimer increases the production of D-2HG from 2KG through the reverse reaction and does not interconvert ICT and 2KG. Since D-2HG is thought to inactivate 2KG utilizing enzymes, it is possible that it also inhibits the Wt-Wt dimer form and that might explain the dominant negative effect of heterozygous arginine substitution (**Figure 3**) [24, 25].

Third, nearly all *IDH* mutations cause a single amino acid replacement, Arg132 in *IDH1* (into one of six amino acid residues -His, Cys, Leu, Ile, Ser, Gly

**Figure 2.**

*The three dimer types formed in a cancer cell heterozygous for IDH mutation.*

*The Distribution and Significance of* IDH *Mutations in Gliomas DOI: http://dx.doi.org/10.5772/intechopen.97380*

#### **Figure 3.**

*The model gains of function and dominant negative effect exerted by heterozygous IDH mutation. (D)-2HG (d) 2-hydroxyglutarate, ICT: isocitrate, Mut: mutant, Wt: wildtype.*

and Val), as well as Arg172 in *IDH2* (into one of four other residues -Lys, Met, Gly and Trp), and Arg140 in *IDH2* to either Gln or Trp. Rarely *IDH1* mutations also are reported, including R100A in adult glioma, G97D in colon cancer cells and a pediatric glioblastoma. The synthesis of cancer-associated *IDH* mutations in the functional region of the enzyme suggested that these mutations might give the mutant protein with a new and possibly oncogenic enzymatic activity.

Lastly, the mutual exclusivity seen in mutant *IDH1* and *IDH2* alleles in most cases. Obviously, in a cancerous cell transformed by one of these mutant alleles, the forward oxidative decarboxylation reaction catalyzed by the remaining wild-type isoform would still be important for that cell to be able to produce NADPH. In other words, cancerous cells that have mutant IDH2, would still need the wild type IDH1 isoform to catalyze the forward oxidative decarboxylation reaction to produce NADPH. Only rarely, individual tumors have been found to sustain mutations in both the *IDH1* and *IDH2* genes [26].

*IDH* mutations also exhibit three distinct clinical features. First, they exist in a highly restricted tumor spectrum. For example, they occur frequently in low-grade gliomas and secondary glioblastomas (GBM), but rarely in primary GBM. Likewise, they are often found in genetically normal AML. Second, the *IDH* mutations occur at an early stage in tumor formation, and occur the earliest known mutation in glioma. Finally, in glioma, AML and intrahepatic cholangiocarcinoma, *IDH* mutations alone or in combination with other genetic mutations (in the case of AML) are associated with better prognosis.

#### **2.4** *IDH* **mutations in human gliomas**

Glioma stem cells are small numbers of tumor cells that act as stem cells in glial cells. According to the "seed and soil" theory put forward by Paget, if the tumor microenvironment is soil, then glioma stem cells are seeds.

The IDH mutations enhance function in glial tumor cells, leading to the accumulation and secretion of large amounts of the oncometabolite, D-2HG, which ultimately inhibits the catalytic activity of α-KG-dependent dioxygenase, damaging the key steps in angiogenesis, hypoxic stress, and mature differentiation of cells. These processes are closely related to the occurrence and development of tumors. However, researches showed that D-2HG is a weak competitive inhibitor

#### *Central Nervous System Tumors*

of α-KG. Thus, it can only be observed to inhibit the differentiation of glioma stem cells when the accumulation of D-2HG is high. Therefore, the formation of gliomas requires not only seeds (glioma stem cells) but also soil (tumor microenvironment).

It was found that the *IDH* mutations could promote tumorigenesis microenvironment by increasing the expression of VEGF and making it suitable for glioma stem cell growth.

Interestingly, VEGF is initiated transcription by HIF-1α, and hypoxia can cause an increase in VEGF. IDH mutants can modulate VEGF to promote tumor microchip formation by inhibiting HIF-1α degradation. Moreover, quick growth of tumors will rapidly consume the surrounding energy and nutrients. Thus, HIF-1α is a stably expressed surrounding tumor. *IDH* mutations make tumor microenvironments easier to form.

With the appropriate soil, glioma tumor stem cells grow rapidly and continue to invade the surrounding tissues, ultimately accelerating the growth of gliomas.

*IDH* mutations occur in about 80% of all grade II/III gliomas (low-grade gliomas - LGG) and secondary glioblastomas, which progress from the less malignant grade 2 diffuse astrocytomas or grade 3 anaplastic astrocytomas. In contrast, IDH mutations accounted for less than 5% of primary glioblastomas, which arise de novo [27, 28] and approximately 10% of pediatric glioblastomas [26, 29]. This suggests that LGG and secondary GBM are minimally overlapping disease subtypes.

In contrast to diffuse gliomas, *IDH* mutations are rare in many of WHO grade I gliomas, for example gangliogliomas, subependymal giant cell tumors, pilocytic astrocytomas, ependymomas and pleomorphic xanthoastrocytoma.

Aggregate data from multiple preclinical and clinical studies have shown that *IDH* mutations alone are not enough to turn malignant. *IDH* mutations occur early in gliomas formation and often have secondary genetic abnormalities such as mutations in *TP53*, chromosomal region 1p/19q co-deletion or loss of nuclear ATRX reactivity.

These changes relate to the histological classification of the disease. For example, diffuse astrocytomas, mutant *IDH*, often contain *TP53* mutations and lose ATRX. In contrast, almost histologically confirmed IDH mutant oligodendrogliomas have 1p/19q co-delection. In particular, the majority of glioma patients with IDH mutation and 1p/19q co-deletion also had a mutation in the promoter regions of


#### **Table 1.**

*Frequency of IDH mutations in different types of gliomas.*


*The Distribution and Significance of* IDH *Mutations in Gliomas DOI: http://dx.doi.org/10.5772/intechopen.97380*

#### **Table 2.**

*Frequency of specific IDH mutations in gliomas.*

the telomerase reverse transcriptase (*TERT*). These mutations are thought to be mutually exclusive [30–32], thus aiding in distinguishing diffuse astrocytomas from oligodendrogliomas (**Tables 1** and **2**) [38].

#### **3. Clinical indications involving the discovery of IDH-mutated glioma**

#### **3.1 Diagnosis**

The latest WHO classification of CNS tumors using the integrated phenotypic and molecular parameters (including the *IDH* mutation) have re-established the CNS tumors classification. This classification includes glioblastoma, *IDH*-wildtype, *IDH*-mutant or NOS; diffuse astrocytoma, *IDH-*wildtype, *IDH*-mutant or NOS; anaplastic astrocytoma, *IDH*-wildtype, *IDH*-mutant or NOS; oligodendroglioma, *IDH*-mutant and 1p/19q-codeleted or NOS; oligodendroglioma, *IDH*-mutant and 1p/19q-codeleted or NOS; anaplastic oligodendroglioma, *IDH*-mutant and 1p/19qcodeleted or NOS.

There are wo features make *IDH* mutations easily detectable, reliable as biomarkers. First, nearly all tumors carry *IDH* mutations located at specific residues, such as Arg132 in *IDH1* or Arg140 and Arg172 in *IDH2*, which are located in a single exon 4 and can be simply identified through PCR-based amplification and sequencing. Second, antibodies specifically recognizing mutant IDH1R132H protein have been developed, thus it may be identified through conventional immunohistochemistry (IHC). Based on these hypotheses, to determine *IDH* mutations, currently, different methods are available to diagnose this status. They analyze either the nucleotide sequence of the gene (as the direct method) or the altered structure of the protein (as the indirect method).

Practical guidelines are available for detection of *IDH* mutations with molecular genetics techniques. In this regard, crucial aspects are the availability of tumor tissue, the tumor cell content and the quality of the respective genomic DNA (gDNA). Among them, conventional Sanger sequencing is a relatively inexpensive method and therefore is widely used in laboratories. As a consequence, it becomes the "gold standard" for the detection of *IDH* mutations. Beside, alternative methods to assess

the *IDH* mutation status exist. They include derived cleaved amplified polymorphic sequence (dCAPS), PCR-based restriction length polymorphism assays, cold PCR high resolution melting (HRM), post-PCR fluorescence melting curve analysis (FMCA) and SNaPshot assays. These are new methods and unapproved for clinical use in determining IDH status.

As the indirect method to confirm *IDH* status, immunohistochemistry of the *IDH1* mutant proteins is used. IHC using IDH1 R132H mutation-specific antibody detects *IDH1* mutation. However, this method can miss about 10% of gliomas carrying an *IDH1* mutation and all gliomas with an *IDH2* mutation [39]. It is conceivable that, when the IHC is negative for IDH1 R132H, the tumor can carry the IDH1 mutation in another location or the IDH2 mutation. In this case, subsequent genetic analysis is recommended.

All of the above methods have in common the need for tissue samples. Thus, surgery or biopsy of the tumor is necessary. This is a diagnostic difficulty. Therefore, recently, studies on non-invasive methods are being carried out, in which diagnosis by magnetic resonance spectroscopy (MRS) and amide proton transfer-weighted (APTw) have been shown to be promising [40–42]. In IDH mutant gliomas, D-2HG accumulates to sufficient levels as a brain metabolite, which renders its visibility on MRS. Therefore, this may provide crucial longitudinal data for the determination of disease progression and therapy response.

Identification of *IDH* status allows differential diagnosis between gliomas and non-neoplastic CNS lesions (astrocytoma or therapy-induced changes), between gliomas and non-glial CNS tumors, and within glioma subtypes. As discussed above, *IDH* status may be used to differentiate primary from secondary glioblastomas. In addition, IDH status associated with 1p/19q co-deletion became the key in the diagnosis of oligodendroglioma.

#### **3.2 Prognostic**

Generally, *IDH* mutations are associated with a better outcome than other types of mutations [43–45]. In 2008, Parsons et al. reported that mutations in IDH1 occurred in most secondary GBM, and were related with better overall survival (OS) [46]. Similar trends were reported in variety studies using different datasets [29]. For example, in a prospective translational cohort study of the German Glioma Network, patients with anaplastic astrocytoma carry *IDH1* wild-type exhibited a worse overall survival rate than patients with glioblastomas with *IDH1* mutation [47] *IDH*-mutated astrocytomas harboring ATRX mutation also were shown to form a subgroup of astrocytomas with a favorable prognosis [48].

Furthermore, in the SongTao study, *IDH* mutations were associated with prolonged PFS together with MGMT promoter methylation and 1p/19q codeletion and a higher rate of objective response to temozolomide in secondary glioblastomas [43]. Even in primary glioblastomas, *IDH1*/2 mutations define a subgroup of tumors of long-term survival patients [49].

In 2009, using a large clinical dataset, Yan et al. reported that GBM patients with *IDH* mutations tended to prolonged median OS compared with patients carrying *IDH* wild-type GBM. Similar findings were also observed in patients with anaplastic astrocytoma.

The median OS was 65 months for gliomas patients with *IDH* mutant, compared with 20 months for those with IDH wild-type. Furthermore, the progression-free survival (PFS) was also improved among GBM patients with IDH mutations compared with other patients [29].

Extensive meta-analysis (2,190 cases) confirmed *IDH* mutation as a prognostic biomarker of gliomas [44]. Many other studies have shown that *IDH* mutations are an independent prognostic marker for improved PFS and OS in patients with grade III gliomas [47, 50–52].

Several studies have explained that the favorable prognosis of IDH mutant gliomas is due to their increased sensitivity to chemotherapy and radiotherapy [47, 53]. IDH mutant gliomas likely harbour defects in multiple DNA repair pathways, which render them vulnerable to radiotherapy- or chemotherapy-induced DNA damage [54, 55]. These findings indicate that IDH mutation could serve as an important predictive factor for treatment response among glioma patients.

#### *3.2.1 Novel therapies*

Glioma is the most frequent brain tumor and has a notably high mortality and disability rate. For its complex pathogenesis, the surgical and drug-assisted treatments do not seem to be effective. Therefore, it is of great significance to find new targets for diagnosis and treatment. The detection of IDH mutations in gliomas offers bases to research new therapies.

Some studies indicated that IDH-mutated gliomas maintain the IDH-mutated allele even after acquiring oncogenic driver mutations [56, 57]. This may show that IDH-mutated gliomas may remain vulnerable to the targeted therapies developed specifically for IDH mutations even at progression or after malignant transformation to higher grade glioma. The therapeutic effects may be further enhanced by combining different targeted therapies or with traditional chemotherapeutics or radiation.

#### *3.2.2 IDH-mutated inhibitors*

Since the neomorphic activity of IDH mutants is correlated with malignant transformation, direct targeting of the mutant enzymes becomes a heavily pursued strategy.

Over the past decade, several attempts have been made to find and develop small molecular compounds that directly inhibit the IDH-mutated enzymes. Some synthetic inhibitors reported as AGI-5198, ivosidenib (AG-120) and vorasidenib (AG-881), demonstrated effective and safe in treating IDH-mutated myeloid malignancies and solid tumors, including glioma [58–60]. BAY1436032, another IDH-mutant inhibitor, had shown tumor-suppressing effects as experimental therapeutics for the treatment of AML and astrocytoma in animal models [61, 62]. Recently, ivosidenib and vorasidenib have been approved by the Food and Drug Administration as a therapeutic option for IDH-mutated AML.

Despite the promising success of the IDH-mutated inhibitors, a number of studies have indicated the potential limitations of their application. As discussed above, IDH-mutated enzymes enhance sensitivity to chemotherapy and radiotherapy. So that, using these inhibitors reduces D-2HG production and relieves the burden on the multiple DNA repair pathways, resulting in chemoresistance. For example, AGI-5198 might increase their resistance to genotoxic therapies, such as radiation and chemo agents [63, 64].

Overall, targeting IDH-mutated activity is a straightforward strategy and has shown efficacy gliomas in humans. However, whether inhibition of mutant IDH and subsequent reduction in D-2HG production are sufficient to halt tumor growth in gliomas and other solid tumors remains unclear. In addition, whether these drugs will cross the blood brain barrier for admission to IDH mutant glioma cells is a question that requires further studies.

#### *3.2.3 Targeting redox homoeostasis*

Redox homeostasis has been reported to be greatly affected by IDH mutations, notably elevated levels of oxidative stress. Targeting redox homeostasis may be effective in gliomas with IDH mutations. In fact, in IDH-mutated gliomas, the synthesis of NAD is largely compromised. As a result, tumor cells rely on a path of salvation to create NAD. Consequently, the IDH-mutated gliomas cells can be extremely sensitive to the blockade of the salvage pathway.

In addition, one study demonstrated that levels of glutamate, glutamine and glutathione decreased in tumor regions in patients with IDH-mutated glioma, compared with levels in contralateral regions. Furthermore, the glutathione level negatively correlates with the level of D-2HG, suggesting that glutathione is required for IDH-mutated cells to maintain redox homoeostasis [65]. An animal preclinical study has shown that inhibiting glutamine metabolism using the glutaminase inhibitor CB-839 leads to impaired redox homoeostasis and makes IDHmutated glioma sensitivity to radiotherapy [66].

Since the disruption of redox homoeostasis results in potent cytotoxicity accompanied by tumor suppression, current therapeutic compounds are mostly at the preclinical stage and show considerable systemic toxicity. Nevertheless, developing the next generation of therapeutic compounds with both potency and selectivity will be of great help for targeting redox imbalance in IDH-mutated malignancies.

#### *3.2.4 Immunotherapies*

With evidence that IDH mutation is an early event in tumorigenesis and is present homogenously in all glioma tumor cells at specific codons. These mutations are ideal immunotherapy targets.

In fact, there are increasing evidences that the IDH mutation might play critical roles in altering the immunological microenvironment of the tumor, as shown by an inhibition of tumor-infiltrating lymphocytes, cytotoxic T cells and natural killer cells [67, 68]. Additionally, the presence of IDH mutation correlates with a decrease in the expression of PD-L1 (Programmed Death-Ligand 1). Decreased expression of PD-L1 in IDH-mutated gliomas implies a stronger T cell activation, because PD-L1 is a cellular surface protein that modulates the immune system and promotes selftolerance through inhibition T cell activity [69].

The combination with the IDH-mutated inhibitors shows an improvement in the efficacy of PD-1-resistant derived immunotherapy, which induces intracellular CD4 + T-cell proliferation. The result is a reduction in tumor size and a prolonged survival. Further studies are currently under investigation, promising to bring positive results.

#### *3.2.5 Vaccines*

Vaccination is the most effective measure of disease prevention and control. In many low-grade glioma patients, the spontaneous immune response to IDH1 mutation has been found [70]. The use of the self-immune response to tumor treatment has also been a heavily researched subject in recent years and provides evidences that is worth the wait. For example, in animal experiments, it was found that the vaccine not only was able to prevent from IDH1 mutant cells growing in the brain, but also did not destroy the normal physiological function of the IDH1 enzyme [70].

Specifically, a phase 1 clinical trial is ongoing to confirm the safety and therapeutic efficacy of the IDH1 R132H mutant peptide vaccine (NOA-16)

#### *The Distribution and Significance of* IDH *Mutations in Gliomas DOI: http://dx.doi.org/10.5772/intechopen.97380*

in newly diagnosed grade III and IV gliomas with IDH1 mutation. The first reported results demonstrated the safety and immunogenicity of NOA-16, with 80% of patients having mutation-specific T cell immune responses, and 87% of the patients displaying humoral immune responses; no deaths have been reported [71].

It is difficult to completely remove gliomas by surgery and drugs, so they often recur. Moreover, the recurrent gliomas after clearance generally tend to be more resistant and invasive. Vaccines can play a maintenance role in these cases. So finding a suitable vaccine will greatly benefit patients and help them escape the magic spell of glioma recurrence.

#### *3.2.6 Other therapies*

In addition to the treatments outlined above, there are other methods base on vulnerability of IDH-mutant cells to NAD+ depletion, hypoxia-inducible factor-1? (HIF-1?) pathway of IDH mutation or mammalian target of rapamycin (mTOR) signaling pathway. These are all new methods, are preclinical models and promise to bring about a change in treatment for gliomas with IDH mutations.

It is generally known that trials of IDH mutant inhibitors, vaccines, immunotherapies and so on in IDH mutant gliomas and recurrent gliomas have been conducted. Meanwhile, old drugs for other tumors have also been developed to treat gliomas with IDH mutations, such as azacitidine, nivolumab, and temozolomide.

In summary, targeting the distinctive vulnerabilities of IDH-mutated glioma has been shown to be successful, as cancer cells are less likely to compensate for the loss of essential biological pathways. However, development of further studies is needed for more convincing evidence to apply these novel therapies to treatment.

#### **4. Conclusion**

The discovery of the IDH mutation not only adds to the landscape of glioma genetics but also supports diagnosis and prognosis. For IDH-mutated gliomas, numerous attempts have been made to define selective and effective therapeutics that target the biological signatures, with the aim of improving standard treatments.

From the above mentioned biological bases, IDH mutation is an important target for the prevention and treatment of gliomas. However, due to the short and uncertain clinical trial duration, most clinical trials of vaccines, IDH inhibitors or other methods are still underway. Much research still needs to be completed. However, we believe that the great potential of these new treatments offers hope in patients with gliomas.

Finally, a major obstacle in IDH-mutated glioma is that the critical oncogenic drivers of this disease remain controversial. One of the main questions remains the molecular pathogenesis of WHO grade II and III gliomas without IDH mutations, which often do not show changes in genes typically associated with gliomas. In-depth investigation of critical molecular pathways will be of great importance to develop highly potent and selectivity treatment.

*Central Nervous System Tumors*

#### **Author details**

Nu Thien Nhat Tran Thu Duc City Hospital, Ho Chi Minh City, Viet Nam

\*Address all correspondence to: nhat24.10@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Distribution and Significance of* IDH *Mutations in Gliomas DOI: http://dx.doi.org/10.5772/intechopen.97380*

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## **Chapter 7** CNS High Grade Glioma

*Liam Chen*

### **Abstract**

Since the publication of the 2016 edition of the WHO Classification of CNS Tumors, advances in neuropathology have enhanced our understanding of the molecular underpinnings of CNS tumors, providing new elements to refine their classification and improve pathological diagnosis of these neoplasms. This chapter will review the highlights of the updated recommendations which provide guidance for how even in the absence of histopathological characteristics of the highest malignancy grade, molecular markers can be used to reach a diagnosis of glioblastoma, IDH–wild-type or astrocytoma, IDH-mutant, grade IV. These changes have important implications for the management of patients with CNS tumors in current neuro-oncology practice.

**Keywords:** astrocytoma, oligodendroglioma, glioblastoma, IDH-mutant glioma, molecular pathology

#### **1. Introduction**

The 2016 WHO classification divided the glial tumors into these categories: diffuse astrocytic and oligodendroglial tumors, other astrocytic tumors, ependymal tumors, and other gliomas [1]. It for the first time broke a nearly century-old tradition of classifying CNS tumors based merely on concepts of histogenesis and histological features by incorporating well-established molecular parameters into the classification of different gliomas. Further refinements of the classification were subsequently proposed by the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy — Not Officially WHO (cIMPACT- NOW) [2–8]. The chapter will review the highlights of the published cIMPACT-NOW updates and discusses their implications for the management of patients with CNS tumors in current neuro-oncology practice. We will focus on the adult high grade CNS glioma. Infant and pediatric high grade gliomas are beyond the scope of our discussion, but viewers can refer to some of a few excellent reviews [9, 10] published recently.

#### **2. Grading of CNS tumors**

The taxonomy and grading of neoplasms have evolved over time as clinical studies have become more sophisticated. Moreover, as the field of bioinformatics has exploded, and statistical analysis has become more powerful, pathologist, clinicians and scientists have employed these tools to better stratify nervous system tumors. In most cases, tumor classifications and grading schemes are based on


#### **Table 1.** *WHO grading system.*

retrospective studies of patients with a given tumor. Data from different pathologic features such as number of mitotic figures per 10 high powered fields (HPF) (400x magnification), nuclear anaplasia, presence or absence of necrosis, growth pattern, and specific histology are analyzed using complex multivariate analysis models to determine which factors represent statistically significant independent risk factors for aggressive behavior. In many cases, two or three of the most important features are used to determine different grades of a given tumor. For example, one criterion of the grading scheme of anaplastic oligodendroglioma, IDH-mutant and 1p/19qcodeleted, requires a mitotic count. Based on a series of studies, neuropathologists and physician collaborators determined that tumors with greater than 6 mitoses per 10 HPF were shown to have a worse prognosis, and were thereby designated "anaplastic" oligodendroglioma, WHO Grade 3. The current WHO grading scheme of CNS neoplasms is summarized in **Table 1**. Obviously, this is a gross oversimplification, especially considering the prognosis of tumors, given the advances in chemotherapy, radiotherapy, and diagnosis.

#### **3. Astrocytoma, IDH-mutant**

IDH-mutant astrocytoma is a diffusely infiltrating astrocytoma with a mutation in either *IDH1* or *IDH2* gene. This tumor most commonly affects young adults and occurs throughout the CNS, but is preferentially located in the frontal lobes. This is similar to the preferential localization of IDH-mutant and 1p/19q-codeleted oligodendroglioma and supports the hypothesis that these gliomas develop from a distinct population of precursor cells [11]. Seizures are common presenting symptom. MRI studies usually show T1-hypodensityh and T2-hyperintesnsity, with enlargement of the areas involved early in the evolution of the tumor. Gadolinium enhancement is not common in low-grade diffuse astrocytoma, but tends to appear during tumor progression as diffuse astrocytomas have an intrinsic capacity for malignant progression to IDH-mutant anaplastic astrocytoma and eventually to IDH-mutant glioblastoma.

#### *CNS High Grade Glioma DOI: http://dx.doi.org/10.5772/intechopen.99984*

Fibrillary astrocytoma is the classic type of diffuse astrocytoma. Another variant is the gemistocytic astrocytoma that is characterized by the presence of a conspicuous proportion of gemistocytic neoplastic astrocytes. The gemistocytes have plump, glassy, eosinophilic cell bodies and eccentric nuclei. Nevertheless, both types carry mutations in *IDH* genes. Glioma-associated IDH mutations impart a gain-of-function phenotype to the respective metabolic enzymes IDH1 and IDH2, which overproduce the oncometabolite 2-hydorxylutarate [12]. The physiological consequences of 2-hydroxyglutarate overproduction are widespread, including profound effects on cellular epigenomic states and gene regulation. Specifically, IDH mutations induce G-CIMP, by which widespread hypermethylation in gene promoter regions silences the expression of several important cellular differentiation factors. In this way, IDH mutation and C-CIMP are thought to maintain glioma cells of origin in stem cell-like physiological states inherently more prone to self-renewal and tumorigenesis. The vast majority of IDH-mutant diffuse astrocytomas also harbor loss-of-function mutations in *TP53* and *ATRX*. *ATRX* encodes an essential chromatin-binding protein, and its deficiency has been associated with epigenomic dysregulation and telomere dysfunction. In particular, *ATRX* mutations seem to induce an abnormal telomere maintenance mechanism known as alternative lengthening of telomeres. *ATRX* mutations and alternative lengthening of telomeres are mutually exclusive with activating mutations in the *TRET* gene, which encodes the catalytic component of telomerase. Interestingly, TERT mutations are found in the vast majority of oligodendrogliomas and IDH-wildtype glioblastomas [13]. ATRX deficiency has also been associated with generalized genomic instability, which can induce p53-dependent cell death. Therefore, *TP53* mutations in diffuse astrocytoma may enable tumor cell survival in the setting of ATRX loss.

Multiple studies have identified homozygous deletion of *CDKN2A/B* as a marker of poor prognosis in patients with IDH-mutant diffuse astrocytic gliomas [6]. Thus,

#### **Figure 1.**

*Neuropathological features of high grade gliomas. Hypercellularity, nuclear pleomorphism, significant proliferative activity (A) and a mutation in IDH1 R132H as detected by immunohistochemistry (B) are essential features of astrocytoma, IDH-mutant, grade 3. A hypercellular band of cells tracing the border of necrotic zones in what is known as pseudopalisading necrosis (C) and/or microvascular proliferation (D) are required histological characteristics for diagnosis of glioblastoma, grade 4.*

IDH-mutant astrocytomas that lack significant mitotic activity, histologic anaplasia, microvascular proliferation, necrosis and *CDKN2A/B* homozygous deletion are referred to as Astrocytoma, IDH-mutant, grade 2. Patients with these tumors have a median overall survival greater than 10 years. An IDH-mutant astrocytoma that contains elevated mitotic activity and histologic anaplasia, yet lacks microvascular proliferation, necrosis and *CDKN2A/B* homozygous deletion, currently fits into the designation of Astrocytoma, IDH-mutant, grade 3 (**Figure 1A**-**B**). Recognizing that no validated published criteria exist for mitotic count cut-off values for grading IDH-mutant astrocytomas, "significant" mitotic from grade 2 tumors. Most neuropathologists use a threshold of ≥2 mitoses within the entire specimen, or one mitosis in very small biopsies, while large specimens may require more. Lastly, IDHmutant astrocytomas with microvascular proliferation or necrosis or CDKN2A/B homozygous deletion, or any combination of these features, correspond to WHO grade 4.

#### **4. Glioblastoma, IDH-wildtype**

By far, the most frequent malignant brain tumor in adults is glioblastoma, accounting for approximately 15% of all intracranial neoplasms and approximately 45-50% of all primary malignant brain tumors. The annual incidence of glioblastoma in the USA, adjusted to the United States Standard Population, is 3.19 cases per 100, 000 population. It preferentially affects older adults, with peak incidence occurring in patients aged 55-85 years. A series of environmental and genetic factors have been studies as potential causes of glioblastoma. To date, the only validated risk factor associations are an increased risk after ionizing radiation to the head and neck and a decreased risk among individuals with a history of allergies and atopic disease [14]. Glioblastoma is most often centered in the subcortical white matter of the cerebral hemispheres. Glioblastoma is particularly notorious for its rapid invasion of neighboring brain structures. Infiltration occurs most readily along white matter tracts, but can also involve cortical and deep gray structures. When infiltration extends through the corpus callosum, with subsequent growth in the contralateral hemisphere, the result can be a bilateral, symmetrical lesion (so called butterfly glioma). The symptoms depend largely on the tumor location, primarily manifesting as focal neurological deficits and edema with increase in intracranial pressure. As many as half of all patients are diagnosed after an inaugural seizure. On MRI, glioblastomas are irregularly shaped and have a ring-shaped zone of contrast enhancement around a dark, central area of necrosis.

Glioblastoma is typically a highly cellular glioma, usually composed of poorly differentiated, sometimes pleomorphic tumor cells with nuclear atypia and brisk mitotic activity. Tumor necrosis is a fundamental feature of glioblastoma. Palisading form, which consists of multiple, small, irregularly shaped band-like or serpiginous foci surrounded by radially oriented, densely packed glioma cells, is a histological hallmark of glioblastoma (**Figure 1C**). The other histological hallmark is microvascular proliferation (**Figure 1D**). Glioblastomas are among the most vascularized of all human tumors. Hypoxia is a major driving force of glioblastoma angiogenesis and leads to intracellular stabilization of the master regulator HIF1A [15]. HIF1A accumulation leads to transcriptional activation of over a hundred of hypoxia-regulated genes encoding proteins that control angiogenesis. Among them, VEGFA seems to be the most important mediator of glioma-associated vascular functions; it is primarily produced by perinecrotic palisading cells as a consequence of cellular stress such as hypoxia and hypoglycaemia. Therapeutic blocking of VEGFA by monoclonal antibodies is effective to target small, immature vessels and

#### *CNS High Grade Glioma DOI: http://dx.doi.org/10.5772/intechopen.99984*

lead to vascular normalization accompanied by improved perfusion and oxygenation [16]. On light microscopy, microvascular proliferation typically presents as socalled glomeruloid tufts of multilayered mitotically active endothelial cells together with smooth muscle cells/pericytes. Another less common form is hypertrophic proliferating endothelial cells within medium-sized vessels.

Multiple studies have concluded that a substantial subset of IDH-wildtype diffuse or anaplastic astrocytomas in adults has an aggressive clinical course, with overall patient survival time almost equal to the patients with IDH-mutant glioblastoma, WHO grade 4 [17, 18]. Thus, cIMPACT has reached a consensus that despite the WHO grade 2 or 3 histology, IDH-wildtype diffuse astrocytic tumors would follow an aggressive clinical course and considered as an entity equivalent to glioblastoma if they have the genotype of epidermal growth factor receptor (EGFR) amplification and/or combined whole chromosome seven gain and whole chromosome ten loss (+7/−10) and/or TERT promoter mutation. Although these tumors possess so-called "GBM genotypes", there has been a reluctance to designate the tumor as a glioblastoma in the absence of histological features including palisading necrosis and microvascular proliferation. cIMPACT has thus reached consensus on the designation of diffuse astrocytic glioma, IDH-wild type, with molecular features of glioblastoma, WHO grade 4 as the most appropriate terminology at this time, since this conveys the histologic, molecular and clinical features of glioblastoma that does not alter the long-standing histologic definition [4]. Glioblastoma is highly resistant to therapy, with only modest survival increases achieved in a minority of patients, even after aggressive surgical resection, external beam radiation therapy, and maximum tolerated doses of chemotherapy. MGMT promoter methylation is the only predictive biomarker for the efficacy of and response to alkylating and methylating chemotherapy agents in glioblastoma [19].

### **5. Oligodendroglioma**

While astrocytoma represents roughly 80-90% of all gliomas, oligodendroglioma, the second most common primary CNS tumor, represents only about 5-6% of all gliomas, with peak incidence in patients aged 35-44 years [20]. Approximately two-thirds of patients present with seizures. The frontal lobe is the most common location. They are characterized by cortex and white-matter based proliferations of neoplastic cells morphologically resembling oligodendrocytes which have a characteristic "fried-egg" appearance (a delayed fixation tissue artifact seen on H&E permanent sections, and rarely observed on frozen section), delicate "chicken wire" vasculature, mucoid/cystic degeneration and microcalcifications. The current WHO classification of oligodendroglioma requires demonstration of *IDH1* or *IDH2* mutation, typically by immunohistochemistry using the mutation-specific antibody against R132H-mutant IDH1 (followed by DNA genotyping when R132H-mutant IDH1 immunostaining is negative), as well as demonstration of 1p/19q codeletion by FISH or molecular genetic testing. Mutations in the *CIC* gene on 19q13.2 and *FUBP1* gene on 1p31.1, among other genes on 1p and 19q, may contribute to the distinctive biology of 1p/19q codeletion [21]. Unlike IDH-mutant diffuse astrocytomas, oligodendroglioma usually lacks wide-spread nuclear p53 staining, a finding consistent with the mutual exclusivity of *TP53* mutation and 1p/19q deletion [22]. In addition, oligodendrogliomas lack *ATRX* mutation but virtually always carry activating mutations in the *TERT* promoter region, leading to increased expression of TERT [23]. In this manner, 1p/19q testing can be skipped if an IDH-mutant tumor appears clearly astrocytic and the ATRX/p53 immunohistochemistry results are consistent with an astrocytic genotype (*ATRX* and/or *TP53* mutations).

There is no WHO grade 1 variant of oligodendroglioma. WHO grade 2 oligodendroglioma is defined by a diffusely infiltrating, slow-growing glioma without evidence of increased mitotic activity (a few mitoses are permitted), endothelial proliferation, or necrosis. In contrast, WHO grade 3 anaplastic oligodendroglioma is defined by histological features of anaplasia. In particular, microvascular proliferation and/or increased mitotic activity (there is debate among experts, although classic studies have indicated a cutoff of ≥6 mitoses/10 HPF) have been suggested to be of important indicators of anaplasia in oligodendroglioma. Interestingly, contrast enhancement has been detected in <20% of WHO grade 2, but in >70% of grade 3 anaplastic oligodendrogliomas [24]. Thus lack of contrast enhancement does not exclude anaplastic oligodendroglioma. 1p/19q codeletion has been found to be associated with better therapeutic response and longer survival in patients treated with adjuvant radiotherapy and chemotherapy [25]. Not surprisingly, long-term follow-up data indicate higher median overall survival times (>10 years) for patients with anaplastic oligodendrogliomas who were treated with combined radiotherapy and chemotherapy.

Finally, to make matters even slight more complicated, there is a class of tumors that combines the histologic features of both astrocytomas and oligodendrogliomas. The existence of these entities is hotly contested; however, as of the 2016 iteration of the WHO classification, there is a grade II oligoastrocytoma, NOS and a grade III anaplastic oligoastrocytoma, NOS. They usually manifest in adult patients, with preferential localization in the cerebral hemispheres. Again, NOS indicates that molecular testing could not be completed or is inconclusive.

#### **6. Diffuse midline glioma, H3 K27M-mutant**

By definition, this entity is an infiltrative midline glioma with predominantly astrocytic differentiation and a K27M mutation in either *H3F3A* or *HIST1H3B/C* [3]. K27M mutations affecting H3.3 (encoded by *H3F3A*) are about three times as prevalent as the same mutation in histone variant H3.1 (occurring in *HIST1H3B* or *HIST1H3C).* Notably, these mutations are not exclusive to diffuse midline gliomas. Over the past few years, a number of tumors that are not diffuse midline gliomas have been reported with the same H3 K27M mutation, including ependymomas, pilocytic astrocytomas, pediatric diffuse astrocytomas, and gangliogliomas. Therefore these mutations cannot be used in and of themselves to define a diffuse midline glioma, H3 K27M-mutant.

It predominates in children but can also be seen in adults, with the most common locations being brain stem, thalamus, and spinal cord. Classic clinical symptoms include the triad of multiple cranial neuropathies, long tract signs, and ataxia, typically developing over a short period of time (1-2 months). The prognosis is poor, despite current therapies, with a 2-year survival rate of <10%. Correspondingly, H3 K27M-mutant diffuse midline glioma is WHO grade 4. The mere grading criterion of an entity based on a specific mutation means the histological features do not predict the outcome. Indeed, about 10% pontine examples lack mitotic figures, microvascular proliferation, and necrosis, and thus are histologically consistent with WHO grade 2. The remaining cases are histologically high grade, with 25% containing mitotic figures and the remainder containing mitotic figures as well as foci of necrosis and microvascular proliferation. The use of H3 K27M-mutant specific immunohistochemistry is useful to identify the mutation and specifically for diagnosis of diffuse midline glioma, H3 K27M-mutant. The K27M substitution results in a decrease in H3K27me3 (trimethylated), thought to be due to inhibition of PRC2 activity [26]. Antibody that has also been used to guide diagnosis of these

tumors is against H3 K27me3. H3 K27me3 immunohistochemistry, however, should only be used in conjunction with H3K27M immunohistochemistry, since loss of H3 K27me3 expression is by itself not specific.

#### **7. Other astrocytic tumors**

Pleomorphic xanthoastrocytoma is rare (constituting <1% of all astrocytic neoplasms) and most commonly affects children and young adults, with a median patient age at diagnosis of 22 years [27]. A superficial location, involving the leptomeninges and cerebrum is characteristic of this neoplasm. Approximately 98% of cases occur supratentorially, most commonly in the temporal lobe. Due to the superficial cerebral location of the lesion, many patients present with a fairly long history of seizures. On MRI, the solid portion of the tumor is hyperintense on T2 FLAIR images. Postcontrast enhancement is moderate or strong.

The adjective "pleomorphic" refers to the variable histological appearance of the tumor, in which spindled cells are intermingled with mononucleated or multinucleated giant astrocytes. The term "xanthoastrocytoma" refers to the presence of large, often multinucleated xanthomatous cells that have intracellular accumulation of lipids. Granular bodies are a nearly invariable finding. Focal collections of small lymphocytes are also frequent. The third histological hallmark of pleomorphic xanthoastrocytoma is the presence of reticulin fibers. Despite its alarming histological appearance, pleomorphic xanthoastrocytoma has relatively favorable prognosis compared with diffusely infiltrative astrocytoma, with 70.9% recurrence-free and 90.4% overall survival rates at 5 years, corresponding to WHO grade 2. Patients with anaplastic pleomorphic xanthoastrocytoma have significantly worse survival than those whose tumors show <5 mitoses per 10 HPFs. Necrosis may be present. *BRAF V600E* mutations occurs in approximately 50-78% of cases [28]. The frequency of *BRAF V600E* mutation is lower among anaplastic pleomorphic xanthoastrocytoma than among WHO grade 2 pleomorphic xanthoastrocytoma, but the prognostic significance of the mutation is unknown.

#### **8. Ependymal tumors**

Ependymomas are tumors that can arise anywhere along the ependymal-lined ventricular spaces of the neuraxis, including the brain and spinal cord. Like the other gliomas, this is a heterogeneous class of tumors that ranges from benign (subependymoma and myxopapillary ependymoma) to malignant (anaplastic ependymoma). Due to their location, even biologically benign examples can cause malignant clinical sequelae, including recurrent obstructive hydrocephalus and even death. The age of the patient seems to affect prognosis, with adults doing better than children, probably due in part to a predominant spinal cord involvement in adults. Histologically, these tumors are characterized by solid or pseudo-papillary proliferations of small to medium sized, hyperchromatic, oval to spindled cells with conspicuous pseudo-rosettes (cells palisading/lining up like a picket fence around a central capillary) and/or, less commonly, true ependymal rosettes (cells palisading around a hollow canal in an attempt to recapitulate the embryonic central canal). There are three histologic variants of WHO grade II ependymoma: papillary, clear cell (which tends to be biologically more aggressive), and tanycytic. However, the criteria for defining anaplastic ependymoma are not as well developed as those of astrocytomas as no association between grade and biological behavior or survival has been definitively established.

Advances in the understanding of the biological basis and molecular characteristics of ependymal tumors have prompted the cIMPACT-NOW group to recommend a new classification. Separation of ependymal tumors by anatomic site is an important principle of the new classification and was prompted by methylome profiling data to indicate that molecular groups of ependymal tumors in the posterior fossa and supratentorial and spinal compartments are distinct [8]. A supratentorial ependymoma characterized by a *C11orf95-RELA* fusion gene accounts for approximately 70% of all childhood supratentorial tumors and a lower proportion of such ependymomas in adult patients [29]. It forms in the context of chromothripsis, a shattering and reassembly of the genome that rearranges genes and produces oncogenic gene products. Rarely, *C11orf95* or *RELA* can be fused with other genes as a result of chromothripsis. *RELA* fusion-positive ependymomas show constitutive activation of the NF-kappaB pathway. Immunohistochemistry to assess the expression of L1CAM correlates well with the presence of a *RELA* fusion in these tumors. Importantly, *RELA* fusion-positive ependymomas have been reported to have the worst outcome among the supratentorial ependymomas [30].

#### **9. Conclusions**

Since 2016, ongoing discoveries in molecular pathology have advanced our understanding of many of the entities organized under the WHO 2016 classification. Most of these changes carry important implications for clinical practice and for the design and interpretation of clinical trials. It is almost certain that our understanding of the biology of CNS tumors will continue to expand at a rapid pace. Thus continuation of the efforts of optimal (evidence-based, balanced, rapid) and timely translation of novel insights into clinical diagnostics, is the ultimate goal to provide the best possible care to CNS tumor patients.

#### **Conflict of interest**

The author declares no conflict of interest.

#### **Author details**

Liam Chen University of Minnesota Medical School, Minneapolis, USA

\*Address all correspondence to: llchen@umn.edu

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*CNS High Grade Glioma DOI: http://dx.doi.org/10.5772/intechopen.99984*

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#### **Chapter 8**

## Molecular Classification of Diffuse Gliomas

*Kanwalpreet Kaur*

#### **Abstract**

In 2016 WHO classification of CNS tumors genotypic and phenotypic parameters were integrated to define a new nomenclature of diffuse gliomas on the basis of presence or absence of isocitrate dehydrogenase mutations. This resulted in more homogenous and narrowly defined categories with better accuracy of prognostic information, thus, playing a crucial role in patient management. Broadly, astrocytomas are now histologically and genetically distinct with IDH-mutant, ATRX-mutant, 1p/19q-intact and oligodendroglial tumors has IDH-mutant, ATRXwildtype and 1p/19q-codeleted profile. Glioblastoma are now classified into primary and secondary on the basis of IDH mutations independent of clinical history.

**Keywords:** Diffuse glioma, astrocytoma, oligodendroglioma, IDH, 1p/19q codeletion, ATRX, TERT, EGFR, PTEN, CDKN2A, MGMT, H3F3K27M

#### **1. Introduction**

In 2016 World Health Organization (WHO) classification of tumors of central nervous system, there was a paradigm deviation from earlier morphology based classification of gliomas to a new classification and nomenclature by integrating the molecular and histomorphological parameters. This approach provided finely defined diagnostic categories resulting in better correlation with prognostic and treatment parameters. Now diffuse gliomas whether astrocytoma or oligodendroglioma are grouped together on the basis of their shared *IDH1* or *IDH2* mutation status. Oligodendrogliomas also show 1p/19q codeletion. So, in 2016 diffuse glioma category comprise of WHO grade II and III astrocyctic tumors, grade II and III oligodendroglioma and grade IV glioblastoma, IDH mutant and wildtype. Continuing evolving knowledge on pathology of glioma led to a pediatric midline glioma with mutations in histone H3 genes to be also included with these adult diffuse gliomas. This excludes astrocytoma with circumscribed growth pattern and lacking IDH mutations i.e. pilocycytic astrocytoma, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma. So, diffuse astrocytoma and oligodendrogliomas are now nosologically more similar than are diffuse astrocytoma and pilocytic astrocytoma and family tree of tumors is being redrawn [1].

This new classification was testament to rapid advancement in the field of molecular biology and reducing cost, easy availability for masses in the present times. Now there is possibility of detecting some of these mutations on immunohistochemistry. This journey from discovery of isocitrate dehydrogenase mutations peculiar to gliomas leading to their radical reclassification and new taxonomy based on the presence or absence of mutations happened over a period of just 8 years [2].

#### **2. Principle mutations**

#### **2.1 Isocitrate dehydrogenase mutations**

Metabolism in cancer cells is rewired compared to normal cells since challenge is production more building blocks for proteins, nucleic acids rather than production of more ATP molecules as cell fuel. But very few tumors show mutations in genes directly involved in metabolic pathways. Isocitrate dehydrogenase (IDH) is an enzyme in the tricarboxylic acid (TCA) cycle of aerobic respiration and catalyzes the oxidative carboxylation converting isocitrate to α –ketoglutarate (α –KT). It exists in 3 isoforms i.e. IDH1, IDH2 and IDH3; isoenzymes are multiple forms of an enzyme catalyzing the same reaction but differ in amino acid sequence and kinetic properties (**Table 1**).

Only IDH 3 is a part of TCA cycle and dependent on nicotinamide adenine dinucleotide (NAD) as co factor [3–5].

Isocitrate + NAD<sup>+</sup> **→** α-ketoglutarate + CO2 + NADH+ H+ .

IDH 1 is found in cytosol and peroxisomes while IDH 2 in mitochondria,both using nicotinamide adenine dinucleotide phosphate (NADP) as cofactor. They both catalyze reversible reaction and prevent oxidative damage by generating NADPH [2].

Isocitrate + NADP+ ⇄ α -ketoglutarate + CO2 + NADPH+ H+ .

In diffuse gliomas, heterozygous mutations seen in cytosolic IDH1 or mitochondrial IDH2 are considered as driver mutations [1–6]. Active site of both enzymes is formed by many arginine residues which is a polar amino acid. It forms hydrophilic bonds with isocitrate which is negatively charged. Most common mutation in diffuse gliomas is heterozygous point mutation at nucleotide position 395 of the *IDH1* replaces guanine by adenine G395A resulting in replacement of arginine by histidine (less polar amino acid) at amino acid residue 132 of the protein (R132H). However, new enzyme IDH1-R132H homodimer is not completely inactive despite losing a critical substrate-binding amino acid residue. It gets a neomorphic activity resulting in reducing α-KG to D-2-hydroxyglutarate (D-2-HG) and oxidizing NADPH to NADP+ [3–5].

This mutation was first discovered in 2008 when next generation sequencing was used to study 22,661 protein coding genes in 22 glioblastomas and 5 of them


#### **Table 1.**

*Properties of three isoforms of IDH.*

#### *Molecular Classification of Diffuse Gliomas DOI: http://dx.doi.org/10.5772/intechopen.98296*

showed expressed IDH mutations all at this same codon [2]. Subsequently, Yan and colleagues (26) analyzed IDH1 and IDH2 loci of nearly 1,000 central nervous system (CNS) tumors and found mutually exclusive mutations of IDH1 or IDH2 in more than 70% of WHO grade II and III astrocytomas and oligodendrogliomas and in secondary GBMs that developed from these lower-grade lesions [6]. Since then, numerous studies throughout the world substantiated the similar findings with heterozygous mutations in IDH 1 or less commonly in IDH2 been identified in nearly 74% diffuse astrocytoma WHO grade II, 59% anaplastic astrocytoma WHO grade III, all secondary glioblastoma, 76% oligodendroglioma WHO grade II and 67% anaplastic oligodendroglioma WHO grade III. Most common hot spot mutations identified are R132C, R132S, R132G, R132L in IDH1 and R172K, R172M, R172W and R172G in IDH2 [1, 5].

Mechanism of oncogenesis of IDH is still under research. These IDH mutations result in metabolic dysregulation in tumor glial cells affecting glucose sensing, glutamine metabolism, lipogenesis, and regulation of cellular redox status. In normal cells, IDH 1and 2 wild type (wt) are an important source of NADPH. They are responsible for nearly 65% of total NADPH production in the cytoplasm of IDH wildtype (IDHwt) glioblastoma [3–5]. Glioma cells overexpressing IDH1-R132H or other mutations in IDH 1 or 2 had decreased NADPH levels so consequently, levels of reactive oxygen species (ROS) increased and GSH decreased. Apart from role of ROS, increased production of D-2-HG also has its implications on the tumor cell. D-2-HG and α-KG are structurally similar; only differ for the C2-linked oxygen atom in α-KG, which is replaced by a hydroxyl group in D-2-HG. So D-2-HG causes competitive inhibition of α-KG–dependent dioxygenases thus exerting its direct oncogenic effects. Approximately 60 dioxygenases regulate diverse and important cellular processes by hydroxylating target acceptor proteins by using α-KG as the donor substrate. Important among these are prolyl hydroxylases that regulate hypoxia-inducible factor (HIF) 1α, chromatin-modifying enzymes like histone N-methyl-lysine demethylases and ten-eleven translocations (TET) 5-methylcytosine hydroxylases. The global histone demethylation by D-2-HG causes hypermethylation at a number of gene loci forming glioma-CpG island methylation phenotype and affect chromatin modification. D-2-HG also inhibits TET-mediated 5-methylcytosine hydroxylases levels which affect the expression of many regulatory proteins and possibly tumor suppressors that also contribute to tumorigenesis [5].

IDH mt gliomas can occur anywhere in the central nervous system but are preferentially located supratentorially in the frontal lobes [1, 7, 8]. Hence it is hypothesized IDH mt gliomas arise from a neural precursor population that is spatially and temporally restricted in the brain.

Since IDH mutations play a key role in tumourogenesis. Their diagnostic and prognostic role is well incorporated into routine neuropathology. IDH 1/2 mutations can be detected by Direct Sanger sequencing, pyrosequencing, allele-specific hybridization polymerase chain reaction (PCR), Real-time PCR, digital droplet PCR and high-throughput next-generation sequencing. IDH1 R132H accounts for nearly 90% of all IDH associated mutations in gliomas, so a monoclonal antibody has been developed against the mutant protein, allowing its use in paraffin-embedded specimens [1]. IHC IDH R132H mutation (clone H09) has shown sensitivity of 94% and a specificity of 100%. Positive staining is strong cytoplasmic and weak nuclear in tumor cells. Endothelial cells, perivascular lymphocytes, residual glial cells should be negative. Weak background staining and staining of macrophages is negative. Normal brain does not show staining for mIDH1 R132H IHC but granular staining in the neurons can be seen due to non specific binding to lipofuscin. One caveat to kept in mind while interpreting IHC is that macrophages can show strong cytoplasmic

granular staining even in IDH wt tumors. IHC is very useful for small samples where quantity of DNA extracted is too low for definite results by sequencing since it can highlight single infiltrating tumor cells [9]. IDH helps to separate gliosis (IDH negative) from low grade astrocytoma (IDH mt), if there is doubt in grade I and grade II glioma, IDH presence indicates we are dealing with grade II astrocytoma and also primary glioblastoma (IDH wt) from secondary glioblastoma (IDH mt). If IHC is negative, DNA sequencing is a must before calling a glioma IDH wt.

In all studies IDH mt gliomas have shown better improved progression free survival, longer time for treatment failure and extended overall survival in each of three treatment arms: radiotherapy, radiotherapy with PCV (procarbazine, lomustine and vincristine) or radiotherapy plus temozolomide [5]. So in addition to traditional good prognostic factors i.e. age < 40 years, lower tumor grade, tumor not crossing midline, absence of neurologic deficit before resection and tumor <6 cm, IDH mutation status has emerged as most important favorable prognostic factor in current times. Some studies have reported median survival of 10.9 years in IDH mt diffuse astrocytomas [1, 2, 6, 10].

Due to role of key role IDH mutations in glioma tumorogenesis, many isocitrate dehydrogenase inhibitors like hydroxypyridin-2-one, bis-imidazole phenol, tetrahydropyrazolopyridine are some of the drug under trails [5].

#### **2.2 1p/19q codeletion**

Nearly 60–80% of oligodendroglial neoplasms show co-deletion of 1p/19q: unbalanced translocation t(1;19)(q10;p10) after which only one copy of the short arm of chromosome 1 and one copy of the long arm of chromosome 19 remain and der (1;19) (q10;p10) is produced. It is hypothesized that translocation creates two derivative chromosomes, der(1;19)(p10;q10) and der(1;19)(q10;p10), and is followed by loss of the derivative chromosome containing 1p and 19q [11–16]. IDH wild type gliomas do not have 1p/19q codeletion.

Polysomy of 1p, 19q or both is also noted in a subset of oligodendrogliomas and has been associated with a poor prognosis, independent of deletion status [12, 13]. Oligodendrogliomas of grades II and III that have 1p/19q co-deletion also have a high frequency of TERT promoter mutations, CIC mutations on the remaining chromosome 1p allele and FUBP1 mutation on the remaining 19q allele (**Figure 1**) [14].

1p/19q codeletion are an essential part of molecular diagnostics of oligodendroglioma. Fluorescence in situ hybridisation (FISH), Cytogenomic microarray (CMA), Loss of heterozygosity and next generation sequencing are used to detect 1p/19q co-deletion. FISH is a reliable and validated most commonly used laboratory technique among these. Normal cells show a 2O2G signal (two test and two control probes, test: control ratio = 1.0). Loss of a test signal yields a 1O2G signal pattern (ratio = 0.5) and represents absolute deletion of a chromosome. Presence of aneuploidy, polyploidy and polysomy affects the interpretation since it become unclear what percentage of nuclei are displaying genuine co-deleted signals, so a ratio is calculated dividing total number of test signals by total number of control signals. Atleast 60 non-overlapping nuclei are counted and ratio < 0.8 should be both chromosomes for 1p/19co-deletion. Ratio of 0.75–0.90 is considered borderline [11].

PCR-LOH analysis can be used for borderline cases, it has better specificity than FISH because it tests for multiple loci in a single assay. But PCR is more labour intensive, requires more tissue, a higher proportion of neoplastic cells (at least about 70%) [11].

#### **Figure 1.**

*Unbalanced whole-arm translocation between chromosomes 1 and 19 [t(1,19)(q10;p10)] resulting in 1p/19q codeletion in glioma(11,15,16).*

The 1p/19q codeletion confers a favorable prognosis and is predictive of responses to alkylating chemotherapy and combination of radiotherapy and chemotherapy [1, 11–16].

#### **2.3 Alpha Thalassemia/Mental Retardation Syndrome X-linked (ATRX)**

Another critical marker that defined molecular classification of gliomas is ATRX gene present on Xq21.1. It is named so since it was first discovered through a study of assessing patients with the X-linked mental retardation syndrome presenting with α-thalassemia, severe psychomotor impairments, urogenital abnormalities, and patterns of characteristic facial dysmorphism. This gene encodes a protein which belongs to a chromatin-remodeling pathway (ATRX-DAXX) and is required for genomic stability by the incorporation of H3.3 at telomeres [17]. ATRX inactivation within gliomas can be due to mutations, deletions, gene fusions, or any combination of these. These mutations induce abnormal telomeres that are characteristic of a telomerase-independent telomere maintenance mechanism termed ALT (alternative lengthening of telomeres) [18].

Numerous studies have shown that ATRX mutations have a strong association with IDH mutations but never with 1p/19q codeletion. This property is exploited as diagnostic marker since ATRX inactivation indicates astrocytic lineage and rules out oligodendroglioma. ATRX mutations can be detected by direct Sanger sequencing, pyrosequencing, allele-specific hybridization polymerase chain reaction (PCR), Real-time PCR and high-throughput next-generation sequencing. ATRX IHC: clone CL0537 when show loss of nuclear expression >90% tumor nuclei is indicative of mutated ATRX. Nuclei of non-neoplastic cells such as endothelia, microglia, lymphocytes and reactive astrocytes are strongly positive and serve as positive internal control. When tumor cells show retained nuclear expression of ATRX IHC it indicates wild type ATRX [1, 17–20].

Low-grade glioma patients with ATRX retention and IDH mutations have lower progression-free survival and overall survival (OS) than tumors with 1p/19q codeletion and IDH mutations and longer time to treatment failure than those patients with IDH mutation and wild-type ATRX (55.6 vs. 31.8 months, respectively). Thus ATRX mutation infer a favorable prognosis to tumor [1, 19, 20].

#### **2.4 TP53**

Mutations of *TP53* are found in over 60–80% of infiltrative astrocytomas, anaplastic astrocytomas and secondary GBMs, yet are rare in oligodendrogliomas. There is a strong association between IDH1 mutation and *TP53* mutation in diffuse astrocytomas, and this combination of mutations is helpful in distinguishing astrocytomas from oligodendroglimas [1].

*TP53* mutations can be analyzed by direct Sanger sequencing, pyrosequencing, PCR, allele-specific hybridization, real-time PCR and high-throughput next-generation sequencing. P53 IHC is also easily available and widely used. Immunostain reacts with both the normal and mutant forms of p53. Wild type P53 is rapidly degraded and has short half-life, hence is not detected by p53 IHC. Mutant p53 degrade more slowly, accumulate within nucleus of tumor cells creating a stable target for IHC. IHC detection of overexpressed protein is thus used as a surrogate method for mutation analysis, But it is not sensitive or specific. Over the last 25 years, studies have shown concordance rates between p53 IHC and TP53 mutation status ranging from 55 to 89% in grade I–IV gliomas [1, 21, 22].

TP53 alterations are usually missense producing stable full-length protein. Nonsense, frameshift, or deletion mutations results in incomplete translation of p53 gene producing a truncated protein product or loss of protein expression. This anomalous p53 structure may not be recognized during p53 IHC analysis resulting in false negativity. Some studies considered >10% tumor nuclei staining as positive for TP53 mutation status while others consider >50% as positive [21, 22].

#### **2.5** *TERT* **promotor mutations**

One of the hallmarks of cancer is its ability to proliferate indefinitely. In normal somatic cells, the number of cell division is limited by the telomere length of chromosomes as it decreases with each replicative cycle. Cancer cells often overcome this limit by activating their telomerase**.** Telomerase consists of an RNA subunit and a reverse transcriptase catalytic subunit (TERT), which adds telomeric repeats to chromosome ends, therefore, maintaining telomere length**.** *TERT* gene on 5p15.33 encodes catalytic active site of telomerase and one of the mechanisms of telomerase activation in gliomas is somatic mutations in the promoter region of *TERT*. Most common mutations are C228T and C250T. The frequency of mutation was nearly 72% of IDH wt glioblastomas and in 95% of IDH mt oligodendrogiomas while relatively low in diffuse astrocytomas and anaplastic astrocytomas (19 and 25%, respectively) [23]. ATRX mutations are mutually exclusive of TERT gene mutations [1].

TERT mutations are detected by sequencing. IDH mutation, with 1p/19q codeletion and TERT mutation is characteristic of oligodendroglioma. TERT mutation in absence of IDH mutation indicates astrocytoma. In IDH wt gliomas, one with TERT mutation is associated with reduced overall survival compared to those lacking it. TERT mutation in IDH mt gliomas carries good prognosis [24].

#### **2.6 EGFR**

Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase, whose ligands include EGF and TGF-α. Most frequently amplified oncogene in astrocytic tumors. EGFR amplification is seen in nearly 40% of primary/ IDH wt glioblastoma and rarely in secondary/ IDH mt glioblastoma [1]. There are also specific EGFR mutations (the vIII mutant), which produce a truncated transmembrane receptor with constitutive activity. Both EGFR amplification and the EGFRvIII mutant are mutually exclusive with IDH mutations [25].

#### **2.7 PTEN**

Loss of large regions at 10p, 10q23 and 10q25–26 loci, or loss of an entire copy of chromosome 10 is the most frequent genetic alterations in primary glioblastoma. It is specific for astrocytic differentiation and are rare in oligodendrogliomas [25].

#### **2.8** *CDKN2A:*

Cyclin-dependent kinase inhibitor 2A (CDKN2A) is a gene located at chromosome 9, band p21.3. CDKN2A homozygous deletion is associated with poor prognosis among IDH-mutant gliomas [26, 27].

#### **2.9 MGMT methylation**

MGMT (O6-methylguanine-DNA methyltransferase) is a DNA repair enzyme and reverses the damage caused by alkylating agent temozolomide(TMZ) which adds methyl group at O6 position of guanine and this alkylation forms cross-links between adjacent strands of DNA. The MGMT protein rapidly reverses alkylation at the O6 position of guanine thereby averting the formation of lethal cross-links resulting in TMZ resistance. Promotor methylation of MGMT inactivates the gene so patients with MGMT promotor methylation are more benefitted with TMZ than patients without it [28].

MGMT promotor methylation is an essential part of molecular workup of all grade III and IV gliomas. Promoter methylation of O6-methylguanine–DNA methyltransferase (MGMT) is detected by methylation specific PCR, pyrosequencing or array based studies. MGMT determination by immunohistochemistry lacks standardization, reproducibility and, most importantly, correlation with clinical outcome so it is no longer recommended.

MGMT promotor methylation is commonly associated with IDH mutations and genome wide epigenetic changes (G-CIMP).

#### **3. Diffuse gliomas**

**Difuse astrocytoma, IDH mutant, and WHO grade II:** Diffuse astrocytoma composed of well differentiated fibrillary astrocytes in loose microcystic matrix. They show nuclear atypia in the form of variation in nuclear shape or size with accompanying hyperchromasia. All show mutation in *IDH 1* or *IDH2* supported by presence of *ATRX* characterized by gemistocytes forming nearly 20% of the tumor cells is a variant of IDH mt diffuse astrocytoma [1, 29, 30].

**Difuse astrocytoma, IDH wild type, and WHO grade II:** diffusely infiltrating astrocytoma without mutations in the IDH genes. It is extremely rare.

**Anaplastic astrocytoma, IDH mutant**: Diffusely infiltrating astrocytoma with focal or dispersed anaplasia, significant mitotic activity and mutation in *IDH 1* or *IDH.* TP53 or ATRX mutations are found in majority of tumors.

**Glioblastoma, IDH wild type/ primary glioblastoma:** They are high grade astrocytoma with nuclear atypia, cellular pleomorphism, mitosis, microvascular proliferation and necrosis. They lack IDH mutations but show TERT promotor mutations (80% cases), homozygyous deletion of CDKN2A/CDKN2B (60% cases), loss of chromosome 10p (50% cases), 10q (70%), EGFR alterations (55% cases) and PTEN (40% cases) [14, 15]. They account for nearly 90% of all glioblastoma [1].

**Glioblastoma, IDH mutant/ secondary glioblastoma:** IDH mutations in glioblastomas are considered as a marker for glioblastoma that arise by transformation from lower-grade gliomas, regardless of clinical history. IDH mt/secondary glioblastomas differ from IDH wt/primary glioblastoma in preferential frontal location and lesser extent of necrosis. Radiologically IDHmt glioblastoma exhibited more frequent non-enhancing tumor component, larger size at diagnosis, lesser extent of edema, and increased prevalence of cystic and diffuse components [31, 32]. Median age of IDHmt glioblastoma at diagnosis is 43 years while that of primary IDH wt glioblastoma is nearly 60 years [1].

Hence in routine histopathology practice, for older patients >55 years old, glioblastoma not in midline location and no prior history of lower grade glioma, IDH wt type designation can be given solely on the basis of negative IDH R132H immunohistochemistry. Sequencing is not required as the probability of an alternate IDH mutation is <1% [1, 31, 32].

IDH mt glioblastoma manifest longer overall survival and showed more frequent promoter methylation of MGMT [6].

**Oligodendroglioma:** Diffusely infiltrating slow growing glioma composed of monomorphic cells with uniform round nuclei and variable perinuclear haloes with IDH1 or IDH2 mutation and codeletion of chromosomes arms 1p and 19q.

IDH mutant gliomas which do not show ATRX loss on IHC should be considered for 1p/19qcodeletion studies even in absence of clear cut oligodendroglial histology [33].

Rarely tumors with oligodendroglial morphology but lacking IDH mutations or 1p/19q codeletion are noted. This group belongs to pediatric type oligodendroglioma. It is important to rule out histological mimics like dysembryoplastic neuroectodermal tumor, extraventricular neurocytoma, clear cell ependymoma and pilocyctic astrocytoma before rendering diagnosis of pediatric type oligodendroglioma [34, 35]. They show FGFR1 duplications or rearrangements of MYB related MYBL1 translocation [33].

Tumors with 1p/19 q codeletion without IDH mutations are usually IDH wt high grade astrocytomas and must be evaluated for possibility of incomplete deletion of on 1p and 19q [1].

**Diffuse midline glioma:** They are Infiltrative midline high grade glioma with astrocytic differentiation and mutations in histone proteins. Pons, thalamus, spinal cord are the common locations. Median age is 5–11 years. They are always IDH wild type and are considered grade IV tumors. In humans, there are main three histone H3 proteins: H3.1 encoded by HIST1H3B and HIST1H3C, H3.2 encoded by HIST2H3C and H3.3 encoded by H3F3A and H3F3B. Most common histone mutation is H3K27M (lysine to methionine substitution in H3F3A gene) which inhibits trimethylation of H3.3 histone resulting in decrease in H3K27me3. Other less frequent mutations occur in *HIST1H3B* or *HIST1H3C*. This can be detected by Sequencing for H3F3A and HIST1H3B. However, monoclonal H3F3A K27M

#### *Molecular Classification of Diffuse Gliomas DOI: http://dx.doi.org/10.5772/intechopen.98296*

antibody is also available and intense nuclear staining in more than 80% of cells is taken as positive. Concordance between immunohistochemistry and sequencing is nearly 95%. H3 K27M mutated tumors show loss of H3K27me3 staining which can also be detected by IHC but it is not specific [1, 36].

**Not otherwise specified (NOS) designation:** It is used in tumors when either molecular testing is not available (e.g., in low-resource settings), or was performed but did not yield adequate results (assay failure), or was deliberately not done (e.g., not testing IDH status in an elderly patient with glioblastoma because of lack of implications for therapeutic management. But when molecular tests have been performed but results do not lead to a precise categorization of the tumor within the framework of the WHO 2016 classification, then term not elsewhere classified (NEC) is used [1, 26].

Final histopathological report:


Example:


#### **Key points**


### **Author details**

Kanwalpreet Kaur Department of Oncopathology, Gujarat Cancer and Research Institute, Ahmedabad, India

\*Address all correspondence to: kanwalpreet.15@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Molecular Classification of Diffuse Gliomas DOI: http://dx.doi.org/10.5772/intechopen.98296*

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Section 5
