**4.2. Partner mutations**

DIPG trials. A recent study identified a FDA-approved epigenetic drug, Panobinostat, to be a leading therapeutic candidate by testing a plethora of promising anticancer drugs in biopsy-

A better knowledge of the genomic aberrations that are considered drivers in DIPG is essential to treat accurate animal models. Research is improving and several studies are focusing on the discovery of these important mutations and agreeing that novel combinations should be tested in genetically and histologically accurate preclinical models prior to their translation to the clinic [5,42]. This collaborative effort will elucidate many of the unanswered questions in

Recent studies and advances in DIPG and biopsy specimens available at the time of the diagnosis have permitted researchers to identify the mutations encoding for histones H3.1 (*HIST1H3B* and *HIST1H3C*), H3.2 (*HIST2H3C*), and H3.3 (*H3F3A*)—proteins known for packaging DNA into chromatin. Histone mutations are found in nearly 80% of children with DIPG, and its high frequency strongly suggests its potential as a driver mutation [45,46]. Clear evidence also indicates that the molecular pathogenesis of DIPG is distinct from non-brain‐

The *K27M* (lysine replaced by methionine at amino acid 27) or *K27I* (lysine replaced by isoleucine at amino acid 27) mutations result from a gain of function and have the potential to lower overall amounts of wild-type H3 with trimethylated lysine 27 (*H3K27me3*). This results in a loss of methylation at this site. Also, sequestration of the polycomb repressive complex 2 (*PRC2*) further results in overall histone hypomethylation. Normally, the *PRC2* complex represses gene expression through histone methylation. In the absence of *PRC2* complex member *EZH2*, genes that should be silent by methylation are expressed and transcriptional‐

Studies analyzing the differences between H3.3 and H3.1 subgroups are showing that they can have distinct cells of origin [48]. A distinct genomic expression pattern between these two subgroups, in addition to the higher frequency of H3.1 mutation in a younger age, could imply that H3.3 and H3.1 mutations target distinct progenitors. Another interesting finding is that *PDGFRA* amplification is seen mainly in combination with H3.3 mutation, while *ACVR1* is

It is known that the type of histone H3 mutation can predict the prognosis and OS of DIPG patients in a more accurate way than clinical, histological, or radiological characteristics of the tumor [29]. The discovery of the histone mutations and its importance are an incentive to the reintroduction of biopsy at the time of diagnosis, permitting to identify the genomic land‐

and autopsy-derived preclinical models of DIPG [44].

**4. Molecular basis of DIPG: major driver mutations**

ly active, leading to the mechanism of *K27M*/*K27I* tumorigenicity [47].

only seen mainly in combination with mutant H3.1 [18,48].

scape of the patient and determination of a better treatment plan.

DIPGs.

**4.1. Histone mutations**

408 Neurooncology - Newer Developments

stem HGGs [46].

Studies have shown that although about 80% DIPGs harbor histone mutation as expected, nearly all H3 mutant DIPGs also harbor partner mutations that vary across patients [23,34,46, 49]. Histone 3 mutations can be seen in combination with a variety of genomic alterations, such as *ACVR1*, *TP53*, and *PDGFRA* (**Table 1**).


**Table 1.** Specific mutations and copy number abnormalities and possible target treatments in DIPG.

Recent whole-genome sequencing studies reveal that 20–30% of DIPGs—usually patients less than 5 years old—contain mutations in the *ACVR1* gene, which encodes the type 1 bone morphogenetic protein (BMP) receptor, *ALK2* [18,50,51]. The high percentage of *ACVR1* mutation in DIPG provides strong evidence that it is an oncogenic driver in this cancer. Almost always *ACVR1* mutation occurs in combination with *HIST1H3B K27M*, encoding mutant histone H3.1, which is also associated with a younger age. When a HGG arises early in development and affect infants, usually the prognosis is better and the mutation burden is lower, suggesting that the tumor would be generated with fewer mutations [46,52].

Mutations in *ACVR1* gene activate the *ALK2* receptor, increase phosphorylation of SMAD proteins, and up-regulate genes in BMP developmental signaling pathway. These mutations are also described in patients with fibrodysplasia ossificans progressiva (FOP), although amino acid substitutions that occur in DIPGs have not been found in FOP patients [51]. Because FOP is not associated with cancer predisposition, it is likely that *ACVR1* mutations provide a selective advantage in the presence of other critical partner mutations, rather than driving tumor initiation [52].

Pathways common in a variety of tumor types occur also in DIPG. One example is *TP53* checkpoint, harmed by *TP53* mutations, which occurs in approximately 55% of patients with HGGs and is associated with *H3F3A*, *ATRX*, and *DAXX* mutations [45]. Mutant p53 proteins have an extended half-life and can be detected by immunohistochemistry (IHC) due to their protein accumulation [53]. In 9–23% of DIPGs, there are also mutually exclusive mutations in the *TP53* target gene *PPM1D*, which plays a role downstream of p53 in the DNA damage response [51,54].

Also, the association of p53 abnormalities in the context of *PDGFRA* amplification or *PI3K* mutations raises the possibility that the *PI3K* signaling pathway constitutes a major compo‐ nent of the pathogenesis of DIPG [49]. *PDGFRA* is known to be expressed in malignant gliomas and plays a role during embryonic development, suggesting an embryonic origin for DIPG, given the incidence of this disease in middle childhood [55].

Other important genomic alterations include ATRX, TERT, MYCN, and PTEN. The identifi‐ cation of driver mutations in DIPG helps more than to confirm the diagnosis at a molecular level: it provides relevant clinical and prognostic information, leading to the improvement in the genomic and epigenetic knowledge of DIPG. The combination of different mutations in a singular patient elucidates the fact that DIPG is a complex and varied pathology comprised of different molecular subgroups that share the same clinical features as well as a grim prognosis.
