**2.** *TP53* **genetic alterations in CNS tumors**

CNS tumors have historically been classified on the basis of morphological and, more recently, immunohistochemical features with less emphasis on their underlying molecular pathogene‐ sis. The past two decades, however, have seen striking advances in basic brain tumor biology, especially with regard to malignant gliomas and medulloblastomas, the most common CNS cancers of adults and children, respectively [24, 25]. Molecular signatures of tumors may play roles as diagnostic, prognostic, and predictive markers and influence the clinical decision making process. A dynamic classification of tumors is critical for the continuous integration of newly established molecular tools. This topic focuses on various genetics and epigenetics *TP53* changes in the CNS tumors which have been integrated into daily practice and gained significance for molecular diagnostic testing. Detailed discussion of neuronal and mixed neuronal-glial tumors, tumors of the pineal region, tumors of cranial and paraspinal nerves, mesenchymal tumors, lymphomas and haematopoietic neoplasms and other tumor entities is beyond the scope of this chapter, especially because there is only limited molecular information used in clinical management available for this types of tumors.

#### **2.1. Gliomas**

Gliomas are the most frequent primary brain tumors and include a variety of different histological types and malignancy grades. Although the cellular origin of gliomas is still unknown, experimental data in mice suggest an origin from neoplastically transformed neural **Figure 2.** Different approaches used in the analysis of *TP53* gene in gliomas. (A) and (B) FISH experiments using *TP53* locus specific probe (red) and 17 centromeric probe (green) in metaphase chromosomes. A normal pair of chromo‐ some 17 is showed in (A), while a heterozygous deletion of *TP53* can be observed in (B). (C) Immunopositive p53 sam‐ ple, demonstrated by immunohistochemical staining. (D) Electropherogram of an patient with the wild type sequence (CGC) in the codon 72 while (E) illustrates a base exchange mutation in this position (CCC) predicting de aminoacid

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substitution arginine → proline.

Alterations in *TP53* gene – Implications in Tumorigenesis Process and Prognosis in Central Nervous System Cancer http://dx.doi.org/10.5772/58334 131

The majority of mutations led to protein accumulation in the nucleus of the cells, which can be detected by immunohistochemistry (IHC) assays. Although some studies have shown an association between p53 positive immunostaining and poor outcomes, several studies have produced conflicting results and expectations on the use of p53 as a useful clinical biomarker failed [23]. Therefore, it seems IHC is a poor surrogate for gene mutation detection, as many mutations do not lead to protein accumulation, and because accumulation of wild-type p53 may also occur in the absence of gene mutation, producing a high rate of false negative and positive results. Hence, the use of IHC leads to an unacceptable number of misclassified cases

By contrast, the screening for *TP53* mutations by gene sequencing, precisely identifying the mutation, have produced more consistent results, at least for some types of cancers such as breast, head and neck squamous cell carcinoma (HNSCC), and leukemia, in which the presence of a *TP53* mutation is associated with poor outcomes. In other types of cancer such as brain and pancreas, mutations were also found to be associated with both poor and good prognosis, depending on the study and cancer. These results show that the type of tissue and treatment may be important determinants of the prognostic and predictive value of *TP53* mutations [1, 22]. Figure 2 illustrates the use of different techniques in the evaluation of mutational status of *TP53* and expression of p53 protein in gliomas. Fluorescence *in Situ* Hybridization (FISH),

CNS tumors have historically been classified on the basis of morphological and, more recently, immunohistochemical features with less emphasis on their underlying molecular pathogene‐ sis. The past two decades, however, have seen striking advances in basic brain tumor biology, especially with regard to malignant gliomas and medulloblastomas, the most common CNS cancers of adults and children, respectively [24, 25]. Molecular signatures of tumors may play roles as diagnostic, prognostic, and predictive markers and influence the clinical decision making process. A dynamic classification of tumors is critical for the continuous integration of newly established molecular tools. This topic focuses on various genetics and epigenetics *TP53* changes in the CNS tumors which have been integrated into daily practice and gained significance for molecular diagnostic testing. Detailed discussion of neuronal and mixed neuronal-glial tumors, tumors of the pineal region, tumors of cranial and paraspinal nerves, mesenchymal tumors, lymphomas and haematopoietic neoplasms and other tumor entities is beyond the scope of this chapter, especially because there is only limited molecular information

Gliomas are the most frequent primary brain tumors and include a variety of different histological types and malignancy grades. Although the cellular origin of gliomas is still unknown, experimental data in mice suggest an origin from neoplastically transformed neural

and to a greater inter-study variability [1, 22].

130 Tumors of the Central Nervous System – Primary and Secondary

**2.** *TP53* **genetic alterations in CNS tumors**

used in clinical management available for this types of tumors.

sequencing and IHC techniques.

**2.1. Gliomas**

**Figure 2.** Different approaches used in the analysis of *TP53* gene in gliomas. (A) and (B) FISH experiments using *TP53* locus specific probe (red) and 17 centromeric probe (green) in metaphase chromosomes. A normal pair of chromo‐ some 17 is showed in (A), while a heterozygous deletion of *TP53* can be observed in (B). (C) Immunopositive p53 sam‐ ple, demonstrated by immunohistochemical staining. (D) Electropherogram of an patient with the wild type sequence (CGC) in the codon 72 while (E) illustrates a base exchange mutation in this position (CCC) predicting de aminoacid substitution arginine → proline.

stem or progenitor cells. However, histological classification of gliomas essentially relies on morphological similarities of the tumor cells with non-neoplastic glial cells and the presence of particular architectural features; thereby, most gliomas can be classified as astrocytic, oligodendroglial, mixed oligoastrocytic or ependymal tumors according to the criteria of the WHO classification of CNS tumors [4]. Clinical experiences derived from the prospective randomized clinical trials have shown that the histomorphological criteria alone might not be sufficient to predict the clinical outcome. Moreover, lately integrated genomic studies and exome sequencing have revealed the existence of multiple distinct molecular subtypes within histologically similar looking tumors [26]. For instance, even gliomas with identical histopa‐ thological features differ considerably regarding clinical course or response to therapy.

p53 pathway is also inactivated by the amplification of *MDM4* in 4% of GBM with neither *TP53* mutation nor *MDM2* amplification [41, 42]. Additionally, the recently discovered tumor suppressor gene *CHD5* (chromodomain helicase DNA-binding domain 5), which maps to chromosome 1p36 and is therefore frequently hemizygously deleted in those human gliomas with loss of 1p, has been shown to maintain p53 levels by facilitating expression of p19 *Arf* (mouse p14ARF ortholog), and thus presents an additional mechanism for inactivation of this

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The incidence of *TP53* mutations in pilocytic astrocytomas is controversial, with some authors reporting only infrequent mutations [44-47], while more common mutations are rare [48]. Hayes *et al.* [48], were the first to find a higher rate of *TP53* mutations in an analysis of 20 pilocytic astrocytomas in children, based on a comprehensive denaturing gradient gel electrophoresis mutation detection assay of the entire coding region, including all splice site junctions of *TP53*, showed mutations considered as causative in 7 of the 20 (35%) pilocytic astrocytomas. Few Cytogenetic studies have been carried out, showing allelic losses on both 17p and 17q including the *TP53* and *NF1* loci in pilocytic astrocytomas [49]. These results suggest that *TP53* mutations may well play a role in the development of these tumors.

*TP53* mutations are a genetic hallmark of low-grade diffuse astrocytomas, for > 60% of these tumors carrying mutations in this gene [47, 50], mainly in gemistocytic astrocytomas with *TP53* mutations in up to 80% of the cases [51, 52]. In most cases, *TP53* mutation is accompanied by loss of heterozygosity (LOH) on 17p resulting in the complete absence of the wild-type *TP53* gene. Those diffuse astrocytomas with no *TP53* mutations may have altered the p53-dependent growth control by alternative mechanisms, for example, promotor methylation of the *p14 ARF* gene at 9p21. Nakamura *et al*. [53] found hypermethylation of *p14ARF* in one third of low-grade diffuse astrocytomas samples. These results suggest that aberrant *p14ARF* expression due to homozygous deletion or promoter hypermethylation is associated with the evolution of both primary and secondary GBMs, and that *p14ARF* promoter methylation is an early event in subset

Studies assessing the presence of *TP53* mutations as predictor of clinical outcome in diffuse astrocytomas have been made and the results are controversial. However most of them associated the presence of *TP53* mutations to a poor prognosis [51, 54, 55]. Peraud *et al.* [54] analyzed retrospectively timing, frequency, and prognostic impact of *TP53* mutations and p53 protein accumulation in 159 patients consecutively treated at a single neurosurgical clinic. *TP53* mutations were frequently found and univariate analysis found that gemistocytic subtype and *TP53* mutation were associated with worse prognosis, with only the gemistocytic subtype remaining an unfavourable prognostic factor on multivariate analysis. In nongemistocytic astrocytomas, a mutation in *TP53* hot spot codon 175 indicated a worse prognosis

Xanthoastrocytoma pleomorphic (PXAs) are rare astrocytic malignancies classified as grade II lesions by the WHO. Because of the relative rarity of this lesion, the molecular background is still unclear. Among the abnormalities frequently observed in astrocytic tumors, PXA shares

of astrocytomas that undergo malignant progression to secondary GBM.

in terms of time to progression and malignancy.

critical pathway [43].

*2.1.1. Astrocytic tumors*

Knowledge of the genetic alterations in the various types and malignancy grades of gliomas has drastically increased over the past years. The evolution of classical tumor molecular and cytogenetic techniques, as well as the development of newer array-based assays of compara‐ tive genomic hybridization and RNA expression, allowed subclasses of gliomas to be identified based on molecular or gene expression patterns, showing substantial genetic and geneexpression heterogeneity within and between histologic grades of different histologic types of gliomas [27]. These approaches have identified point mutations and copy number changes (deletions, amplifications, gains) in several regions; deletions and loss of heterozygosity in tumors might point to genes involved in tumor suppression, whereas amplifications and gains might point to genes involved in initiation or progression processes (e.g. oncogenes) [28].

Numerous molecular abnormalities have been associated to the underlying biology of gliomas. The p53 pathway is nearly invariably altered in sporadic gliomas: loss of p53, through either point mutations that prevent DNA binding or deletion in chromosome 17p, is a frequent and early event in the pathological progression of secondary glioblastoma (GBM) [29, 30]. The importance of p53 in gliomagenesis is also underscored by the increased incidence of gliomas in LFS, a familial cancer-predisposition syndrome associated with germline p53 mutations [31]. This genetic linkage has been reinforced by a glioma-prone condition in mice engineered with a commonly observed Li-Fraumeni p53 mutation [32] as well as in p19ARF-null mice, albeit at a low frequency [33]. In human gliomas, p53 mutations are primarily missense mutations and target the evolutionarily conserved domains in exons 5, 7, and 8, thus affecting residues that are crucial to DNA binding [30].

The finding that a second promoter drives an alternatively spliced transcript at the *CDKN2A* locus prompted the discovery of an additional tumor suppressor gene that is inactivated at this locus [34]. The second protein encoded by *CDKN2A*, p14 ARF, was subsequently shown to be an important accessory to p53 activation under conditions of oncogenic stress due to its neutralization of the p53 ubiquitin ligase, *MDM2* [35, 36], an oncogene originally discovered amplified as double minute chromosomes in a spontaneously transformed murine cell line, and then later found to be a key negative regulator of p53 during normal development and in tumorigenesis [37-39]. Concordantly, the chromosomal region containing *MDM2*, 12q14-15, is amplified in ∼10% of primary GBM, the majority of which contain intact p53 [40]. The discovery of the *MDM2*-related gene, *MDM4* (chromosome 1q32), which inhibits p53 tran‐ scription and enhances the ubiquitin ligase activity of MDM2, prompted the finding that the p53 pathway is also inactivated by the amplification of *MDM4* in 4% of GBM with neither *TP53* mutation nor *MDM2* amplification [41, 42]. Additionally, the recently discovered tumor suppressor gene *CHD5* (chromodomain helicase DNA-binding domain 5), which maps to chromosome 1p36 and is therefore frequently hemizygously deleted in those human gliomas with loss of 1p, has been shown to maintain p53 levels by facilitating expression of p19 *Arf* (mouse p14ARF ortholog), and thus presents an additional mechanism for inactivation of this critical pathway [43].

## *2.1.1. Astrocytic tumors*

stem or progenitor cells. However, histological classification of gliomas essentially relies on morphological similarities of the tumor cells with non-neoplastic glial cells and the presence of particular architectural features; thereby, most gliomas can be classified as astrocytic, oligodendroglial, mixed oligoastrocytic or ependymal tumors according to the criteria of the WHO classification of CNS tumors [4]. Clinical experiences derived from the prospective randomized clinical trials have shown that the histomorphological criteria alone might not be sufficient to predict the clinical outcome. Moreover, lately integrated genomic studies and exome sequencing have revealed the existence of multiple distinct molecular subtypes within histologically similar looking tumors [26]. For instance, even gliomas with identical histopa‐ thological features differ considerably regarding clinical course or response to therapy.

132 Tumors of the Central Nervous System – Primary and Secondary

Knowledge of the genetic alterations in the various types and malignancy grades of gliomas has drastically increased over the past years. The evolution of classical tumor molecular and cytogenetic techniques, as well as the development of newer array-based assays of compara‐ tive genomic hybridization and RNA expression, allowed subclasses of gliomas to be identified based on molecular or gene expression patterns, showing substantial genetic and geneexpression heterogeneity within and between histologic grades of different histologic types of gliomas [27]. These approaches have identified point mutations and copy number changes (deletions, amplifications, gains) in several regions; deletions and loss of heterozygosity in tumors might point to genes involved in tumor suppression, whereas amplifications and gains might point to genes involved in initiation or progression processes (e.g. oncogenes) [28].

Numerous molecular abnormalities have been associated to the underlying biology of gliomas. The p53 pathway is nearly invariably altered in sporadic gliomas: loss of p53, through either point mutations that prevent DNA binding or deletion in chromosome 17p, is a frequent and early event in the pathological progression of secondary glioblastoma (GBM) [29, 30]. The importance of p53 in gliomagenesis is also underscored by the increased incidence of gliomas in LFS, a familial cancer-predisposition syndrome associated with germline p53 mutations [31]. This genetic linkage has been reinforced by a glioma-prone condition in mice engineered with a commonly observed Li-Fraumeni p53 mutation [32] as well as in p19ARF-null mice, albeit at a low frequency [33]. In human gliomas, p53 mutations are primarily missense mutations and target the evolutionarily conserved domains in exons 5, 7, and 8, thus affecting residues that

The finding that a second promoter drives an alternatively spliced transcript at the *CDKN2A* locus prompted the discovery of an additional tumor suppressor gene that is inactivated at this locus [34]. The second protein encoded by *CDKN2A*, p14 ARF, was subsequently shown to be an important accessory to p53 activation under conditions of oncogenic stress due to its neutralization of the p53 ubiquitin ligase, *MDM2* [35, 36], an oncogene originally discovered amplified as double minute chromosomes in a spontaneously transformed murine cell line, and then later found to be a key negative regulator of p53 during normal development and in tumorigenesis [37-39]. Concordantly, the chromosomal region containing *MDM2*, 12q14-15, is amplified in ∼10% of primary GBM, the majority of which contain intact p53 [40]. The discovery of the *MDM2*-related gene, *MDM4* (chromosome 1q32), which inhibits p53 tran‐ scription and enhances the ubiquitin ligase activity of MDM2, prompted the finding that the

are crucial to DNA binding [30].

The incidence of *TP53* mutations in pilocytic astrocytomas is controversial, with some authors reporting only infrequent mutations [44-47], while more common mutations are rare [48]. Hayes *et al.* [48], were the first to find a higher rate of *TP53* mutations in an analysis of 20 pilocytic astrocytomas in children, based on a comprehensive denaturing gradient gel electrophoresis mutation detection assay of the entire coding region, including all splice site junctions of *TP53*, showed mutations considered as causative in 7 of the 20 (35%) pilocytic astrocytomas. Few Cytogenetic studies have been carried out, showing allelic losses on both 17p and 17q including the *TP53* and *NF1* loci in pilocytic astrocytomas [49]. These results suggest that *TP53* mutations may well play a role in the development of these tumors.

*TP53* mutations are a genetic hallmark of low-grade diffuse astrocytomas, for > 60% of these tumors carrying mutations in this gene [47, 50], mainly in gemistocytic astrocytomas with *TP53* mutations in up to 80% of the cases [51, 52]. In most cases, *TP53* mutation is accompanied by loss of heterozygosity (LOH) on 17p resulting in the complete absence of the wild-type *TP53* gene. Those diffuse astrocytomas with no *TP53* mutations may have altered the p53-dependent growth control by alternative mechanisms, for example, promotor methylation of the *p14 ARF* gene at 9p21. Nakamura *et al*. [53] found hypermethylation of *p14ARF* in one third of low-grade diffuse astrocytomas samples. These results suggest that aberrant *p14ARF* expression due to homozygous deletion or promoter hypermethylation is associated with the evolution of both primary and secondary GBMs, and that *p14ARF* promoter methylation is an early event in subset of astrocytomas that undergo malignant progression to secondary GBM.

Studies assessing the presence of *TP53* mutations as predictor of clinical outcome in diffuse astrocytomas have been made and the results are controversial. However most of them associated the presence of *TP53* mutations to a poor prognosis [51, 54, 55]. Peraud *et al.* [54] analyzed retrospectively timing, frequency, and prognostic impact of *TP53* mutations and p53 protein accumulation in 159 patients consecutively treated at a single neurosurgical clinic. *TP53* mutations were frequently found and univariate analysis found that gemistocytic subtype and *TP53* mutation were associated with worse prognosis, with only the gemistocytic subtype remaining an unfavourable prognostic factor on multivariate analysis. In nongemistocytic astrocytomas, a mutation in *TP53* hot spot codon 175 indicated a worse prognosis in terms of time to progression and malignancy.

Xanthoastrocytoma pleomorphic (PXAs) are rare astrocytic malignancies classified as grade II lesions by the WHO. Because of the relative rarity of this lesion, the molecular background is still unclear. Among the abnormalities frequently observed in astrocytic tumors, PXA shares only *TP53* mutations, and, although *TP53* mutations in anaplastic PXA have previously been reported, the significance of this alteration for tumor malignant progression is not clear [56, 57].The high frequency of *TP53* mutations in low-grade astrocytomas raises the question of whether these alterations play an important role in the tumorigenesis of PXA. Paulus and coworkers [58] reported the highest frequency of TP53 mutations, around 25%. However, in contrast, Giannini *et al.* [59] identified mutation in only 1 of 47 samples, all of which were nonrecurrent lesions and all lacked anaplastic transformation, while Bettegowda *et al.* [60] sequenced the exomes of 12 PXAs and identified mutation in only 2 cases.

frequently in GBMs (76%), and correlated with homozygous deletion or promoter methylation of the p14 ARF gene [53]. Comparing the overall frequency of p14 ARF alterations between primary and secondary GBMs, no significant difference was observed, while p14 ARF promoter methylation was more frequent in secondary than primary GBMs [53]. The analysis of multiple biopsies from the same patients revealed p14 ARF methylation already in one-third of low-grade

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But, have all these data any prognostic value for GBMs? Although there is some discordance among different studies, promising data have already been gained. Hence, some studies showed no association between *TP53* status and outcome of GBM patients [73, 74] or between p53 score analyzed by IHC and patient survival [75]. Schmidt *et al.* [76] analyzed 97 GBM cases and found that the presence of *TP53* mutations was a favorable prognostic factor. In the same way, Ohgaki *et al.* [67] showed that the presence of *TP53* mutations was a favorable prognostic factor, and at the population level, univariate analysis revealed that the presence of these mutations was predictive of longer survival; however, age-adjusted multivariate analysis revealed no difference in survival between patients with and without *TP53* alterations.

El Hallani *et al.* [77] showed that the Pro/Pro genotype (a functional single nucleotide poly‐ morphism at codon 72 of *TP53* gene results in the presence of either proline (Pro) or arginine (Arg) in the amino acid sequence) is significantly over-represented in young patients with GBM (<45 years) (7 of 43 cases, 16.3%) compared to older patients (>45 years) (14 of 217, 6.5%) (*P*=0.05), whereas no difference of frequencies for Arg/Arg versus Arg/Pro between the two groups were observed. These data suggest a recessive effect of the Pro allele on the oncogenesis of GBM in young patients. This result is in line with previous reports showing consistent associations between the codon 72 polymorphism with age of onset in oral cancer, head and neck carcinomas, hereditary nonpolyposis colorectal cancer, and prostate cancer [78, 79]. The polymorphism described by El Hallani *et al*. [77] at *TP53* codon 72 is associated with age at onset of glioblastoma. In a study in 2009, Zawlik et al. (66) revealed that *TP53* codon 72 Pro allele was significantly associated with shorter survival among patients with GBMs carrying a *TP53* mutation (Arg/Pro or Pro/Pro), and among those treated with surgery plus radiother‐

Considering the association between mutations and treatments, recent studies have shown that the status of the *TP53* gene interferes with the effectiveness of treatment by DNA alkylating agent temozolomide (TMZ), the most effective chemotherapeutic for GBM. Blough *et al.* [80] related that GBM cell lines that did not express a functional p53 were significantly more sensitive to TMZ than cell lines with functionally intact wild-type p53 expression, while altered p53 expression or function had only minor effects on TMZ sensitivity in brain tumor initiating

In contrast to diffuse astrocytomas, loss of 17p and *TP53* mutations are rare in oligodendroglial tumors (~10%) and mutually exclusive to 1p/19q deletion, a hallmark alteration in oligoden‐ drogliomas (~ 70%), while oligoastrocytomas frequently carry either *TP53* mutations (~40%)

astrocytomas [53].

apy (Arg/Pro).

cells and tended to decrease sensitivity to TMZ.

*2.1.2. Oligodendroglial and oligoastrocytic tumors*

Anaplastic astrocytomas, from a clinical, morphologic and genetic point of view, represents an intermediate stage on the route of progression to GBM. They exhibit high *TP53* mutation rate (40-70%) similar to diffuse astrocytomas and high frequency of LOH at 17p [61, 62]. In a study of almost 200 astrocytomas of grades II-IV, 72% of anaplastic astrocytomas were found to have a disruption in the p53 pathway [63].

An important clue to pathways involved in gliomagenesis may lie in the two GBM subtypes that have been clinically identified [50]. Primary GBM is typically present in older patients as aggressive, highly invasive tumor, usually without any evidence of prior clinical disease. Secondary GBM have a very different clinical history, being usually observed in younger patients who initially presented low-grade astrocytoma that transformed in GBM within 5–10 years of the initial diagnosis, regardless of prior therapy. The cataloging of genetic lesions in these GBM subtypes has identified differences in their genetic profiles, predominantly in the penetrance of specific genetic mutations. As a result, it has been proposed that primary and secondary GBMs represent two distinct clinical entities, each developing along distinct genetic pathways [50].

*TP53* mutations are a genetic hallmark of secondary GBM, because these tumors have a high incidence of mutations in this gene (>65%), suggesting that p53 pathway plays a crucial role in their development tumors [62, 64-66]. *TP53* mutations are the first detectable genetic alteration in > 60% of precursor low-grade diffuse astrocytomas or in anaplastic astrocytomas in a similar frequency, and secondary GMBs derived thereof [64, 67]. *TP53* mutations also is present in primary GMBs, but with significantly lesser frequency (25-30% of cases) [47, 67]. Giant cell glioblastoma, a histological variant of GBM, carry *TP53* mutations in high frequency (75 – 90%) [68, 69], while gliosarcoma, another GBM variant characterized by a biphasic tissue pattern, has a lower *TP53* mutation rate (23–24%) [70, 71], and identical *TP53* mutations in both gliomatous and sarcomatous components [70].

In secondary GBMs, 57% of mutations have been reported to be located in the two hotspot codons 248 and 273; however, in primary GBMs, mutations were more equally distributed through all exons, with only 17% occurring in codons 248 and 273 [67]. Furthermore, G:C > A:T transitions at CpG sites were significantly more frequent in secondary than in primary GBMs [67]. The less specific pattern of *TP53* mutations in primary GBMs suggests a different molecular mechanism underlying the acquisition of *TP53* mutations in these subtypes.

Amplification of *MDM2* is present in < 10% of GBMs, and this event appears to be associated to primary GBMs with no *TP53* mutations [72]. Loss of p14 ARF expression has been observed frequently in GBMs (76%), and correlated with homozygous deletion or promoter methylation of the p14 ARF gene [53]. Comparing the overall frequency of p14 ARF alterations between primary and secondary GBMs, no significant difference was observed, while p14 ARF promoter methylation was more frequent in secondary than primary GBMs [53]. The analysis of multiple biopsies from the same patients revealed p14 ARF methylation already in one-third of low-grade astrocytomas [53].

But, have all these data any prognostic value for GBMs? Although there is some discordance among different studies, promising data have already been gained. Hence, some studies showed no association between *TP53* status and outcome of GBM patients [73, 74] or between p53 score analyzed by IHC and patient survival [75]. Schmidt *et al.* [76] analyzed 97 GBM cases and found that the presence of *TP53* mutations was a favorable prognostic factor. In the same way, Ohgaki *et al.* [67] showed that the presence of *TP53* mutations was a favorable prognostic factor, and at the population level, univariate analysis revealed that the presence of these mutations was predictive of longer survival; however, age-adjusted multivariate analysis revealed no difference in survival between patients with and without *TP53* alterations.

El Hallani *et al.* [77] showed that the Pro/Pro genotype (a functional single nucleotide poly‐ morphism at codon 72 of *TP53* gene results in the presence of either proline (Pro) or arginine (Arg) in the amino acid sequence) is significantly over-represented in young patients with GBM (<45 years) (7 of 43 cases, 16.3%) compared to older patients (>45 years) (14 of 217, 6.5%) (*P*=0.05), whereas no difference of frequencies for Arg/Arg versus Arg/Pro between the two groups were observed. These data suggest a recessive effect of the Pro allele on the oncogenesis of GBM in young patients. This result is in line with previous reports showing consistent associations between the codon 72 polymorphism with age of onset in oral cancer, head and neck carcinomas, hereditary nonpolyposis colorectal cancer, and prostate cancer [78, 79]. The polymorphism described by El Hallani *et al*. [77] at *TP53* codon 72 is associated with age at onset of glioblastoma. In a study in 2009, Zawlik et al. (66) revealed that *TP53* codon 72 Pro allele was significantly associated with shorter survival among patients with GBMs carrying a *TP53* mutation (Arg/Pro or Pro/Pro), and among those treated with surgery plus radiother‐ apy (Arg/Pro).

Considering the association between mutations and treatments, recent studies have shown that the status of the *TP53* gene interferes with the effectiveness of treatment by DNA alkylating agent temozolomide (TMZ), the most effective chemotherapeutic for GBM. Blough *et al.* [80] related that GBM cell lines that did not express a functional p53 were significantly more sensitive to TMZ than cell lines with functionally intact wild-type p53 expression, while altered p53 expression or function had only minor effects on TMZ sensitivity in brain tumor initiating cells and tended to decrease sensitivity to TMZ.

#### *2.1.2. Oligodendroglial and oligoastrocytic tumors*

only *TP53* mutations, and, although *TP53* mutations in anaplastic PXA have previously been reported, the significance of this alteration for tumor malignant progression is not clear [56, 57].The high frequency of *TP53* mutations in low-grade astrocytomas raises the question of whether these alterations play an important role in the tumorigenesis of PXA. Paulus and coworkers [58] reported the highest frequency of TP53 mutations, around 25%. However, in contrast, Giannini *et al.* [59] identified mutation in only 1 of 47 samples, all of which were nonrecurrent lesions and all lacked anaplastic transformation, while Bettegowda *et al.* [60]

Anaplastic astrocytomas, from a clinical, morphologic and genetic point of view, represents an intermediate stage on the route of progression to GBM. They exhibit high *TP53* mutation rate (40-70%) similar to diffuse astrocytomas and high frequency of LOH at 17p [61, 62]. In a study of almost 200 astrocytomas of grades II-IV, 72% of anaplastic astrocytomas were found

An important clue to pathways involved in gliomagenesis may lie in the two GBM subtypes that have been clinically identified [50]. Primary GBM is typically present in older patients as aggressive, highly invasive tumor, usually without any evidence of prior clinical disease. Secondary GBM have a very different clinical history, being usually observed in younger patients who initially presented low-grade astrocytoma that transformed in GBM within 5–10 years of the initial diagnosis, regardless of prior therapy. The cataloging of genetic lesions in these GBM subtypes has identified differences in their genetic profiles, predominantly in the penetrance of specific genetic mutations. As a result, it has been proposed that primary and secondary GBMs represent two distinct clinical entities, each developing along distinct genetic

*TP53* mutations are a genetic hallmark of secondary GBM, because these tumors have a high incidence of mutations in this gene (>65%), suggesting that p53 pathway plays a crucial role in their development tumors [62, 64-66]. *TP53* mutations are the first detectable genetic alteration in > 60% of precursor low-grade diffuse astrocytomas or in anaplastic astrocytomas in a similar frequency, and secondary GMBs derived thereof [64, 67]. *TP53* mutations also is present in primary GMBs, but with significantly lesser frequency (25-30% of cases) [47, 67]. Giant cell glioblastoma, a histological variant of GBM, carry *TP53* mutations in high frequency (75 – 90%) [68, 69], while gliosarcoma, another GBM variant characterized by a biphasic tissue pattern, has a lower *TP53* mutation rate (23–24%) [70, 71], and identical *TP53* mutations in both

In secondary GBMs, 57% of mutations have been reported to be located in the two hotspot codons 248 and 273; however, in primary GBMs, mutations were more equally distributed through all exons, with only 17% occurring in codons 248 and 273 [67]. Furthermore, G:C > A:T transitions at CpG sites were significantly more frequent in secondary than in primary GBMs [67]. The less specific pattern of *TP53* mutations in primary GBMs suggests a different molecular mechanism underlying the acquisition of *TP53* mutations in these subtypes.

Amplification of *MDM2* is present in < 10% of GBMs, and this event appears to be associated to primary GBMs with no *TP53* mutations [72]. Loss of p14 ARF expression has been observed

sequenced the exomes of 12 PXAs and identified mutation in only 2 cases.

to have a disruption in the p53 pathway [63].

134 Tumors of the Central Nervous System – Primary and Secondary

gliomatous and sarcomatous components [70].

pathways [50].

In contrast to diffuse astrocytomas, loss of 17p and *TP53* mutations are rare in oligodendroglial tumors (~10%) and mutually exclusive to 1p/19q deletion, a hallmark alteration in oligoden‐ drogliomas (~ 70%), while oligoastrocytomas frequently carry either *TP53* mutations (~40%) or loss of 1p/19q (~45%), indicating that oligoastrocytomas are genetically monoclonal, and carry genetic alterations similar to either diffuse astrocytomas or oligodendrogliomas. Furthermore, G:C > A:T transitions at CpG sites are the most frequent *TP53* mutations in these tumors [81-83]. According to Muller *et al.* [84] oligoastrocytomas in the temporal lobe showed LOH on 1p and 19q less frequently (33%) than *TP53* mutations (45%). In contrast, oligoastro‐ cytomas arising outside the temporal lobe demonstrated LOH on 1p and 19q in nearly 75% of the cases while *TP53* mutations were found in less than 20% [85].

(< 1.0%), while eighteen of 48 grade III tumors (37.5%) showed expression of p53 and mean positivity was 5.5%. Manasa *et al.* [95] reported 66% p53 positivity, performing p53 immuno‐ histochemical analysis in 54 samples of different grades and subtypes of ependymomas and observed that p53 indices were higher in grade II and grade III tumors (26.27 % and 26.08% respectively) as compared to subependymomas (grade I) (7.25%). However, p53 index of myxopapillary ependymoma (grade I) (26%) was similar to grade II and grade III tumors. But these values did not show statistical significance (P=0.2). Papillary ependymoma (grade II)

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Some authors have advocated that p53 immunolabeling are important prognostic markers in ependymomas. Zamecnik *et al.* [96] found that p53 immunopositivity is the strongest indicator of aggressive tumor behavior and poor prognosis. Gaspar *et al.,* [88] studied the p53 pathway in primary intracranial childhood ependymomas and p53-mediated response to DNA-damage in two newly described ependymoma xenograft models. Their findings do not suggest a role of p53 genetic/epigenetic alterations in the tumorigenesis or progression of childhood epen‐ dymomas; however, radioresistance of these tumors might be due to alterations in p53 mediated growth arrest. Despite the lack of *TP53* mutations, immunocytochemical accumulation of p53 occurs, particularly in tumors with poor outcome. Moreover, the data concerning immunoexpression of the p53 protein indicate its usefulness in identification of

The genetic and genomic understanding of medulloblastoma (MB) has evolved dramatically in the past few years, but the role of p53 in MB pathogenesis has only initiated to be elucidated. Patients with LFS, caused by a germline mutation in p53, develop MB at a higher incidence than the general population [97, 98]. Similarly, p53 deficiency in mice in combination with mutations in other genes, including poly (ADP-ribose) polymerase (PARP), the cell cycle regulatory protein retinoblastoma (Rb), or the Sonic hedgehog (Shh) receptor Patched1 (Ptch1), greatly increases tumor incidence [99, 100], indicating that loss of p53 can promote MB tumorigenesis. However, in contrast with the high incidence of p53 mutations in most human tumors, the *TP53* gene is altered in <10% of sporadic human MB. Chromosome 17p, where *TP53* is located, is lost in 40% to 50% of sporadic MB tumors. However, it has been found that

New support for a role for p53 in MB tumorigenesis came from a better understanding of heterogeneity underlying MB tumors. Recently, several groups were able to demonstrate that although morphologically similar, MBs could be divided into several subgroups on the basis of expression profiling [103, 104]. A consensus meeting resulted in the current molecular subclassification of MB into four subgroups: wingless (WNT), sonic hedgehog (SHH), group 3, and group 4 [105]. Hopefully, in the near future, this subclassification will be used to select

targeted therapies and improve understanding of the behavior of this disease.

more aggressive clones in ependymomas and its superior predictive value [94].

losses of 17p and p53 status are unrelated in MB [101, 102].

showed p53 expression in 24% cells.

**2.2. Embryonal tumors**

*2.2.1. Medulloblastoma*

Watanabe *et al.* [86] reported that genetic alterations in the p53 pathway are more frequent in anaplastic oligodendroglioma (50%) than in oligodendroglioma WHO grade II (21%), and showed that simultaneous disruption of the *RB1/CDK4/p16 INK4a /p15 INK4b* and the *TP53/p14 ARF /MDM2* pathways occurs in 45% (9/20) of anaplastic oligodendrogliomas, suggesting that these phenomena contribute to their malignant phenotype. Anaplastic oligoastrocytoma typically exhibits the type and distribution of molecular lesions observed in oligoastrocytoma: loss of 1p/19q or *TP53* mutations [84].

A number of genetic alterations have been correlated with poorer response to chemotherapy or worse overall survival in anaplastic oligodendrogliomas. Ino *et al.* [87] suggested that a variety of relatively infrequent genetic alterations (*EGFR*gene amplification, 10q loss, *CDKN2A* homozygous deletion, *PTEN* mutation, and *TP53* mutation) were associated with worse prognosis. Interestingly, *TP53* mutation was associated with an improved likelihood of chemotherapeutic response but with a poor overall prognosis, since responses were not durable in the setting of *TP53* mutation.

Kim *et al.* [83] evaluated 413 tumors confirmed as low-grade diffuse gliomas WHO grade II (206 diffuse astrocytomas, 73 oligoastrocytomas, and 134 oligodendrogliomas) and observed that the median survival of patients with *TP53* mutation combined with *IDH1/2* mutation was significantly shorter than the observed in patients with 1p/19q loss combined with *IDH1/2* mutation (51.8 months vs. 58.7 months, respectively; P=0.0037). A Multivariate analysis with adjustment for age and treatment confirmed these results (P=0.0087) and also revealed that *TP53* mutation is a significant prognostic marker for shorter survival (P=0.0005) and 1p/19q loss for longer survival (P=0.0002).

#### *2.1.3. Ependymal tumors*

*TP53* mutations were rarely reported in ependymal tumors by molecular analysis [88, 89]. However, p53 protein is identified in about 60% of ependymal tumors [90]. Shuangshoti *et al.* [91] suggested that the discrepancy may be due to expression of wild type p53 gene in tumor cells, alternative mechanisms of p53 gene inactivation or simply a cross-reaction of the antigenantibody complex.

A number of studies have documented a correlation between p53 expression and tumor grade in ependymomas [90, 92, 93]. Sharma *et al.* [94] analyzed p53 protein expression in 119 ependymomas tumors (17 cases were of grade I, 54 of grade II and 48 of grade III) and observed its expression in only two cases of grade I tumors (11.5% and 6.4%). Five cases of grade II tumors showed p53 protein expression and this percentage of nuclear positivity was very low (< 1.0%), while eighteen of 48 grade III tumors (37.5%) showed expression of p53 and mean positivity was 5.5%. Manasa *et al.* [95] reported 66% p53 positivity, performing p53 immuno‐ histochemical analysis in 54 samples of different grades and subtypes of ependymomas and observed that p53 indices were higher in grade II and grade III tumors (26.27 % and 26.08% respectively) as compared to subependymomas (grade I) (7.25%). However, p53 index of myxopapillary ependymoma (grade I) (26%) was similar to grade II and grade III tumors. But these values did not show statistical significance (P=0.2). Papillary ependymoma (grade II) showed p53 expression in 24% cells.

Some authors have advocated that p53 immunolabeling are important prognostic markers in ependymomas. Zamecnik *et al.* [96] found that p53 immunopositivity is the strongest indicator of aggressive tumor behavior and poor prognosis. Gaspar *et al.,* [88] studied the p53 pathway in primary intracranial childhood ependymomas and p53-mediated response to DNA-damage in two newly described ependymoma xenograft models. Their findings do not suggest a role of p53 genetic/epigenetic alterations in the tumorigenesis or progression of childhood epen‐ dymomas; however, radioresistance of these tumors might be due to alterations in p53 mediated growth arrest. Despite the lack of *TP53* mutations, immunocytochemical accumulation of p53 occurs, particularly in tumors with poor outcome. Moreover, the data concerning immunoexpression of the p53 protein indicate its usefulness in identification of more aggressive clones in ependymomas and its superior predictive value [94].

#### **2.2. Embryonal tumors**

or loss of 1p/19q (~45%), indicating that oligoastrocytomas are genetically monoclonal, and carry genetic alterations similar to either diffuse astrocytomas or oligodendrogliomas. Furthermore, G:C > A:T transitions at CpG sites are the most frequent *TP53* mutations in these tumors [81-83]. According to Muller *et al.* [84] oligoastrocytomas in the temporal lobe showed LOH on 1p and 19q less frequently (33%) than *TP53* mutations (45%). In contrast, oligoastro‐ cytomas arising outside the temporal lobe demonstrated LOH on 1p and 19q in nearly 75% of

Watanabe *et al.* [86] reported that genetic alterations in the p53 pathway are more frequent in anaplastic oligodendroglioma (50%) than in oligodendroglioma WHO grade II (21%), and showed that simultaneous disruption of the *RB1/CDK4/p16 INK4a /p15 INK4b* and the *TP53/p14 ARF /MDM2* pathways occurs in 45% (9/20) of anaplastic oligodendrogliomas, suggesting that these phenomena contribute to their malignant phenotype. Anaplastic oligoastrocytoma typically exhibits the type and distribution of molecular lesions observed in oligoastrocytoma:

A number of genetic alterations have been correlated with poorer response to chemotherapy or worse overall survival in anaplastic oligodendrogliomas. Ino *et al.* [87] suggested that a variety of relatively infrequent genetic alterations (*EGFR*gene amplification, 10q loss, *CDKN2A* homozygous deletion, *PTEN* mutation, and *TP53* mutation) were associated with worse prognosis. Interestingly, *TP53* mutation was associated with an improved likelihood of chemotherapeutic response but with a poor overall prognosis, since responses were not

Kim *et al.* [83] evaluated 413 tumors confirmed as low-grade diffuse gliomas WHO grade II (206 diffuse astrocytomas, 73 oligoastrocytomas, and 134 oligodendrogliomas) and observed that the median survival of patients with *TP53* mutation combined with *IDH1/2* mutation was significantly shorter than the observed in patients with 1p/19q loss combined with *IDH1/2* mutation (51.8 months vs. 58.7 months, respectively; P=0.0037). A Multivariate analysis with adjustment for age and treatment confirmed these results (P=0.0087) and also revealed that *TP53* mutation is a significant prognostic marker for shorter survival (P=0.0005) and 1p/19q

*TP53* mutations were rarely reported in ependymal tumors by molecular analysis [88, 89]. However, p53 protein is identified in about 60% of ependymal tumors [90]. Shuangshoti *et al.* [91] suggested that the discrepancy may be due to expression of wild type p53 gene in tumor cells, alternative mechanisms of p53 gene inactivation or simply a cross-reaction of the antigen-

A number of studies have documented a correlation between p53 expression and tumor grade in ependymomas [90, 92, 93]. Sharma *et al.* [94] analyzed p53 protein expression in 119 ependymomas tumors (17 cases were of grade I, 54 of grade II and 48 of grade III) and observed its expression in only two cases of grade I tumors (11.5% and 6.4%). Five cases of grade II tumors showed p53 protein expression and this percentage of nuclear positivity was very low

the cases while *TP53* mutations were found in less than 20% [85].

loss of 1p/19q or *TP53* mutations [84].

136 Tumors of the Central Nervous System – Primary and Secondary

durable in the setting of *TP53* mutation.

loss for longer survival (P=0.0002).

*2.1.3. Ependymal tumors*

antibody complex.

#### *2.2.1. Medulloblastoma*

The genetic and genomic understanding of medulloblastoma (MB) has evolved dramatically in the past few years, but the role of p53 in MB pathogenesis has only initiated to be elucidated. Patients with LFS, caused by a germline mutation in p53, develop MB at a higher incidence than the general population [97, 98]. Similarly, p53 deficiency in mice in combination with mutations in other genes, including poly (ADP-ribose) polymerase (PARP), the cell cycle regulatory protein retinoblastoma (Rb), or the Sonic hedgehog (Shh) receptor Patched1 (Ptch1), greatly increases tumor incidence [99, 100], indicating that loss of p53 can promote MB tumorigenesis. However, in contrast with the high incidence of p53 mutations in most human tumors, the *TP53* gene is altered in <10% of sporadic human MB. Chromosome 17p, where *TP53* is located, is lost in 40% to 50% of sporadic MB tumors. However, it has been found that losses of 17p and p53 status are unrelated in MB [101, 102].

New support for a role for p53 in MB tumorigenesis came from a better understanding of heterogeneity underlying MB tumors. Recently, several groups were able to demonstrate that although morphologically similar, MBs could be divided into several subgroups on the basis of expression profiling [103, 104]. A consensus meeting resulted in the current molecular subclassification of MB into four subgroups: wingless (WNT), sonic hedgehog (SHH), group 3, and group 4 [105]. Hopefully, in the near future, this subclassification will be used to select targeted therapies and improve understanding of the behavior of this disease.

As observed for other CNS, reports detailing the prognostic impact of *TP53* mutations in MB offer conflicting conclusions. Pfaff *et al.* [106] reported that *TP53* mutations occur at low frequency in MBs but are overrepresented in the prognostically favorable subgroup featuring alterations in the Wnt pathway. In addition, because no correlation between *TP53* mutation status and patient outcome was observed in more than 300 patients, these authors concluded that *TP53* mutation is not a universal prognostic marker for MB. These results were supported by Lindsey *et al.* [107] in an independent and representative series of all major established clinical and molecular subtypes of MBs. Nevertheless, Gessi *et al.* [108] reported that *TP53* expression is associated with rapid disease progression and poor prognosis in patients with metastatic MB, with a statistically significant inverse correlation between *TP53* expression and patient survival.

Although the presence of *TP53* mutations seems to mainly occur in adult s-PNETs, nuclear accumulation of p53 has been described to be frequent not only in adults but also in pediatric CNS-PNETs [118]. This observation led to the hypothesis that the p53 pathway is pivotal in

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Immunohistochemical staining for the p53 gene product is a good predictor of poor outcome in PNETs. Robert *et al.* [119] observed stained intensely for the p53 protein in 10 patients (n=40) with PNETs and 11 had weakly staining nuclei, while 19 specimens had no staining. The patients with specimens that stained intensely had a statistically significant decreased disease free survival (P=0. 03). Intense p53 immunostaining may predict a poor prognosis in PNETs of childhood [120], however, the significance of p53 in recurrent CNS PNETs is unknown.

The role of p53 in atypical teratoid/ rhabdoid tumor (AT/RT) is also poorly understood. Cell lines established from malignant rhabdoid tumor (MRT) show overexpression of p53, without associated *TP53* gene mutations [120]. On the other hand, missense mutations in *TP53* were reported in 3/6 cases of non-CNS MRT [121]. Knockdown of *SMARCB1* in cell lines and animal models results in activation of p53 [122, 123]. Intriguingly, combined inactivation of *Smarcb1* and *TP53*, but not *Rb* or *p16ink4a*, leads to accelerated development of MRT in mouse models [122, 124]. These data have led to the hypothesis that two successive hits involving *SMARCB1* and *TP53* may contribute to malignant transformation and tumor development. Venneti *et al.* [125] studied the expression of p53 and determined *TP53* mutational status in 36 AT/RT and 16 non-CNS MRT patients. They also studied the relationship of p53 expression with its regulators *p14ARF/MDM2* in AT/RT and non-CNS MRT. p14 ARF expression was seen in many cases, which correlated positively with p53 and inversely with Mdm2 immunostaining in AT/ RT, while *TP53* mutational analysis in 19/25 AT/RT and 8 in 11 non-CNS MRT cases showed point mutations in only 3 AT/RT cases, suggesting that p53 expression was driven mainly by

Choroid plexus tumors (CPT) are rare tumors, often occurring during childhood. Previous studies have shown high frequencies of germline *TP53* mutations in patients with CPT (44– 100%) irrespective of family history [126, 127]. According to the latest clinical criteria for LFS, it is suggested that patients with CPT should be considered for *TP53* testing [128, 129]. In addition, somatic mutations of the *TP53* gene and subsequent accumulation of p53 protein

The prognostic role of p53 in choroid plexus carcinomas has been recently demonstrated. Tabori *et al.* [131] studied 54 patients with CPTs, including CPC (n=36) and choroid plexus papilloma (CPP) (n=18), and demonstrated that patients with CPC who have low tumor total structural variation and absence of *TP53* dysfunction had a favorable prognosis and could be successfully treated without radiation therapy. Krzyzankova *et al.,* [132], investigated the role of p53 in the growth-inhibitory potential of a variety of anticancer agents in the immortalized

have been described in up to 50 % of choroid plexus carcinomas (CPC) [130, 131].

CNS-PNET biology and can also be activated by mechanisms other than mutation.

*2.2.3. Atypical teratoid/ Rhabdoid tumor*

p14 ARF.

**2.3. Choroid plexus tumors**

A large whole-genome and exome sequencing efforts recently published by different groups revealed an additional, albeit small number, of *TP53* mutations in MB [109, 110]. These independent groups found *TP53* mutations enriched in the SHH group and associated with poor survival. Zhukova *et al.* [111] evaluated the association of *TP53* mutations, molecular groups, and survival in MBs patients and confirmed that *TP53* mutations are enriched among SHH MBs, in which they portend poor outcome and account for a large proportion of treatment failures in these patients.

Carvalho *et al.*[112] were the first to investigate the role of the *TP53* Arg72Pro SNP as a potential risk factor and/or prognostic marker of MB by performing a case–control analysis using a polymerase chain reaction-restriction fragment length polymorphism approach. The date suggested that, although there is no association between the *TP53* Arg72Pro SNP and MB risk, the Pro/Pro genotype is associated with shorter overall survival of patients submitted to adjuvant therapy.

Some researchers justify the p53 inactivation in MB tumors lacking *TP53* gene mutations through alternative mechanisms. Mendrysa *et al.* [113] supported MDM2 as an important contributor to the inhibition of p53 in SHH-driven MB tumorigenesis. In cerebellar develop‐ ment, *MDM2* is required to inhibit p53-mediated apoptosis in granular neuronal precursors, the presumed cell of origin for MB tumors of the Shh subgroup, and *MDM2* deficiency potently restricts cerebellar tumorigenesis in Ptch1+/− mice, a model of human Shh-induced MB.

#### *2.2.2. CNS primitive neuroectodermal tumors*

The presence of *TP53* mutations have been identified in CNS primitive neuroectodermal tumors (PNETs), mainly in adult patients [114]. However, *TP53* mutations in PNET have also been occasionally reported in children [115, 116], but the overall incidence of somatic *TP53* mutation in pediatric CNS-PNET seems to be very low [115]. Gessi *et al.* [117] analyzed the clinicopathologic and molecular features of 12 cases of PNETs in adult patients. The p53 staining showed strong nuclear positivity (>20% of stained nuclei) in 9 cases, evidencing the presence of *TP53* mutations in these tumors. The use of single strand conformation polymor‐ phism (SSCP) followed by sequencing of the *TP53* gene showed point mutations of this gene in 4 of these 9 cases, identifying 5 mutations in exons 4, 5, 7, and 8.

Although the presence of *TP53* mutations seems to mainly occur in adult s-PNETs, nuclear accumulation of p53 has been described to be frequent not only in adults but also in pediatric CNS-PNETs [118]. This observation led to the hypothesis that the p53 pathway is pivotal in CNS-PNET biology and can also be activated by mechanisms other than mutation.

Immunohistochemical staining for the p53 gene product is a good predictor of poor outcome in PNETs. Robert *et al.* [119] observed stained intensely for the p53 protein in 10 patients (n=40) with PNETs and 11 had weakly staining nuclei, while 19 specimens had no staining. The patients with specimens that stained intensely had a statistically significant decreased disease free survival (P=0. 03). Intense p53 immunostaining may predict a poor prognosis in PNETs of childhood [120], however, the significance of p53 in recurrent CNS PNETs is unknown.

#### *2.2.3. Atypical teratoid/ Rhabdoid tumor*

As observed for other CNS, reports detailing the prognostic impact of *TP53* mutations in MB offer conflicting conclusions. Pfaff *et al.* [106] reported that *TP53* mutations occur at low frequency in MBs but are overrepresented in the prognostically favorable subgroup featuring alterations in the Wnt pathway. In addition, because no correlation between *TP53* mutation status and patient outcome was observed in more than 300 patients, these authors concluded that *TP53* mutation is not a universal prognostic marker for MB. These results were supported by Lindsey *et al.* [107] in an independent and representative series of all major established clinical and molecular subtypes of MBs. Nevertheless, Gessi *et al.* [108] reported that *TP53* expression is associated with rapid disease progression and poor prognosis in patients with metastatic MB, with a statistically significant inverse correlation between *TP53* expression and

A large whole-genome and exome sequencing efforts recently published by different groups revealed an additional, albeit small number, of *TP53* mutations in MB [109, 110]. These independent groups found *TP53* mutations enriched in the SHH group and associated with poor survival. Zhukova *et al.* [111] evaluated the association of *TP53* mutations, molecular groups, and survival in MBs patients and confirmed that *TP53* mutations are enriched among SHH MBs, in which they portend poor outcome and account for a large proportion of treatment

Carvalho *et al.*[112] were the first to investigate the role of the *TP53* Arg72Pro SNP as a potential risk factor and/or prognostic marker of MB by performing a case–control analysis using a polymerase chain reaction-restriction fragment length polymorphism approach. The date suggested that, although there is no association between the *TP53* Arg72Pro SNP and MB risk, the Pro/Pro genotype is associated with shorter overall survival of patients submitted to

Some researchers justify the p53 inactivation in MB tumors lacking *TP53* gene mutations through alternative mechanisms. Mendrysa *et al.* [113] supported MDM2 as an important contributor to the inhibition of p53 in SHH-driven MB tumorigenesis. In cerebellar develop‐ ment, *MDM2* is required to inhibit p53-mediated apoptosis in granular neuronal precursors, the presumed cell of origin for MB tumors of the Shh subgroup, and *MDM2* deficiency potently restricts cerebellar tumorigenesis in Ptch1+/− mice, a model of human Shh-induced MB.

The presence of *TP53* mutations have been identified in CNS primitive neuroectodermal tumors (PNETs), mainly in adult patients [114]. However, *TP53* mutations in PNET have also been occasionally reported in children [115, 116], but the overall incidence of somatic *TP53* mutation in pediatric CNS-PNET seems to be very low [115]. Gessi *et al.* [117] analyzed the clinicopathologic and molecular features of 12 cases of PNETs in adult patients. The p53 staining showed strong nuclear positivity (>20% of stained nuclei) in 9 cases, evidencing the presence of *TP53* mutations in these tumors. The use of single strand conformation polymor‐ phism (SSCP) followed by sequencing of the *TP53* gene showed point mutations of this gene

in 4 of these 9 cases, identifying 5 mutations in exons 4, 5, 7, and 8.

patient survival.

138 Tumors of the Central Nervous System – Primary and Secondary

failures in these patients.

adjuvant therapy.

*2.2.2. CNS primitive neuroectodermal tumors*

The role of p53 in atypical teratoid/ rhabdoid tumor (AT/RT) is also poorly understood. Cell lines established from malignant rhabdoid tumor (MRT) show overexpression of p53, without associated *TP53* gene mutations [120]. On the other hand, missense mutations in *TP53* were reported in 3/6 cases of non-CNS MRT [121]. Knockdown of *SMARCB1* in cell lines and animal models results in activation of p53 [122, 123]. Intriguingly, combined inactivation of *Smarcb1* and *TP53*, but not *Rb* or *p16ink4a*, leads to accelerated development of MRT in mouse models [122, 124]. These data have led to the hypothesis that two successive hits involving *SMARCB1* and *TP53* may contribute to malignant transformation and tumor development. Venneti *et al.* [125] studied the expression of p53 and determined *TP53* mutational status in 36 AT/RT and 16 non-CNS MRT patients. They also studied the relationship of p53 expression with its regulators *p14ARF/MDM2* in AT/RT and non-CNS MRT. p14 ARF expression was seen in many cases, which correlated positively with p53 and inversely with Mdm2 immunostaining in AT/ RT, while *TP53* mutational analysis in 19/25 AT/RT and 8 in 11 non-CNS MRT cases showed point mutations in only 3 AT/RT cases, suggesting that p53 expression was driven mainly by p14 ARF.

#### **2.3. Choroid plexus tumors**

Choroid plexus tumors (CPT) are rare tumors, often occurring during childhood. Previous studies have shown high frequencies of germline *TP53* mutations in patients with CPT (44– 100%) irrespective of family history [126, 127]. According to the latest clinical criteria for LFS, it is suggested that patients with CPT should be considered for *TP53* testing [128, 129]. In addition, somatic mutations of the *TP53* gene and subsequent accumulation of p53 protein have been described in up to 50 % of choroid plexus carcinomas (CPC) [130, 131].

The prognostic role of p53 in choroid plexus carcinomas has been recently demonstrated. Tabori *et al.* [131] studied 54 patients with CPTs, including CPC (n=36) and choroid plexus papilloma (CPP) (n=18), and demonstrated that patients with CPC who have low tumor total structural variation and absence of *TP53* dysfunction had a favorable prognosis and could be successfully treated without radiation therapy. Krzyzankova *et al.,* [132], investigated the role of p53 in the growth-inhibitory potential of a variety of anticancer agents in the immortalized rodent choroid plexus epithelial cell line Z310 and observed that growth-inhibitory activity of vincristine, doxorubicin, carboplatin, etoposide, and TMZ was significantly impaired by silencing of *TP53*, showing the potential predictive role of p53 in choroid plexus carcinomas.

**3. Epigenetic mechanisms in CNS tumors**

genetic alterations and transformation [150].

nomics of CNS tumors is needed [151].

**3.1. DNA methylation of gene** *TP53*

Epigenetics is defined as mitotically heritable changes in gene expression that are not due to changes in the primary DNA sequence. The coordinated interaction of these changes regulates gene expression activity and several types of epigenetic marks work in concert to drive appropriate gene expression, like DNA methylation at CpG dinucleotides, covalent modifications of histone proteins, non-coding RNAs, and other complementary mechanisms controlling higher order chromatin organization within the cell nucleus. Epigenetic alterations have been recognized as important mechanisms in neoplastic transformation, malignant progression of cancer, and although epigenetic changes are somatically inheritable, they are

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reversible and hence may represent actionable targets for novel therapies [148, 149]

Epigenetic changes are often observed at the earliest stages of neoplasia within the altered tissue stem and progenitor cells. These observations have led to the epigenetic progenitor model [149]. This model explains that transformation to a malignant state occurs in three steps. First, there is an expansion of an epigenetically permissive population due to an essential early epigenetic disruption of stem/progenitor cells. Second, an initiating genetic alteration in an oncogene or tumor suppressor gene occurs. Finally, genetic and epigenetic plasticity resulting in an enhanced ability to stably evolve the phenotype is observed. An important difference to the clonal genetic model is that the epigenetic 'hits' occur early, and are necessary to create an appropriate expansion of a polyclonal population, that is the cellular substrate for subsequent

To better understand the multiple cellular pathways involved in their development, estab‐ lishment markers of resistance to traditional therapies, and contribution to the development of targeted therapies, a comprehensive appreciation of the integrated genomics and epige‐

Hypermethylation of promoters usually occurs at CpG islands. Methylation of *TP53* was reported as a mechanism for its inactivation in neoplasias, such as acute lymphoblastic leukemia, multiple myeloma, malignant glioma cells, and brain metastases of solid tumors [152]. Since the promoter region of *TP53* does not contain a classic CpG island, methylation of one or two sites may produce a proportionately greater effect in downregulation of transcrip‐ tion compared to a tumor suppressor gene with a classic CpG island in the promoter [153]. The *TP53* promoter region has been sequenced and basal promoter activity localized to an 85 bp region (nucleotide 760–844) that is indispensable for full promoter activity and the *TP53* promoter has putative binding sites for transcriptional factors [154]. Schroeder and Mass [155] have shown that methylation in the promoter region of the p53 gene reduces reporter gene activity. They found down-regulation of p53 in cultured cells transfected with a plasmid incorporating a *TP53* promoter containing methylated CpG dinucleotides. Furthermore, this

region has been shown to be methylated in several cancers [156].

#### **2.4. Meningiomas**

Few studies have examined the *TP53* gene directly for mutations in meningiomas, [133-135], and these studies typically have not observed mutations in this gene, although rare mutants have been described, mainly associated with malignant histology [134, 136]. One group working on specimens from Korean patients has documented a rate of nearly 40% of p53 overexpressing meningiomas as having mutations, and observed that the mutation rate was associated with both histological grade and recurrence [137].

In contrast the low frequency of *TP53* point mutations, expression of p53 was found in 10% to 90% of meningiomas [138], but their role in pathogenesis is still uncertain. Studies suggested the involvement of the p53 pathway in meningioma development: the correlation of p53 protein expression with histological tumor grade and meningioma recurrence [139]; methyl‐ ation of the *p14 ARF* gene in 8.6% of benign, 20% of atypical and in 50% of anaplastic meningi‐ omas and loss of detectable Mdm2 protein in high grade meningiomas [140]; defective p53 response to gamma ray stress in meningioma cells [141]. In addition, the *NF2* protein product was reported to increase p53 stability through downregulation of Mdm2 levels in mouse fibroblast [142]. It follows that loss of NF2 may increase the likelihood of p53 suppression, thus decreasing tumor suppression activity and providing a possible mechanism for the involve‐ ment of the p53 pathway in meningiomas.

Many studies have examined benign and atypical/malignant meningiomas for over-expres‐ sion of the p53 protein with diverse results: p53 over-expression has been reported in 0–10% of benign, 50–72.7% of atypical and 77–88.9% of anaplastic or malignant meningiomas [143]. Despite the differing rates, all of the studies are consistent, with atypical/malignant tumors showing higher rates of over-expression than benign meningiomas. However, studies on the biological significance of p53 over-expression are highly contradictory. While over-expression of p53 has been associated with recurrence in some studies [139, 144], no association has been found in others [133, 145]; and still other studies have suggested that expression of high levels of p53 may be protective against recurrence [146].

Terzi *et al.* [147] analyzed the immunohistochemical expression of Ki-67, p53, p21, p16, and *PTEN* proteins in 130 meningiomas (64 benign, 39 atypical, and 27 malignant meningiomas) using tissue microarray and demonstrated that Histological grade, p53, Ki-67 labeling indices, and overexpression of p16 were strongly associated with decreased event-free survival in univariate analysis and Ki-67 and p53 labeling indices are useful additional tools in discrimi‐ nating atypical from benign or anaplastic meningiomas.
