Diagnosis and Treatment Modalities

**15**

proliferation.

**Chapter 2**

**Abstract**

**1. Introduction**

Molecular Diagnostics and

*Erxi Wu, Karming Fung, The Li, Ekokobe Fonkem,* 

*Jason H. Huang and A. Rao*

fluorescence in situ hybridization (FISH)

Pathology of Major Brain Tumors

*Frank Y. Shan, E. Castro, Amelia Sybenga, Sanjib Mukherjee,* 

Tumors of central nervous system (CNS) account for a small portion of tumors of human body, which include tumors occurring in the parenchyma of brain and spinal cord as well as their coverings. The following chapter covers some new development in some major brain tumors in both pediatric and adult populations, as well

as some uncommon but diagnostic and management challenging tumors.

**Keywords:** gliomas, astrocytomas, oligodendrogliomas, mixed oligoastrocytomas, WHO (World Health Organization), WHO grades, medulloblastomas (MBs), midline diffuse astrocytoma, diffuse intrinsic pontine gliomas (DIPG),

hemangioblastomas (HMBs), metastatic renal cell carcinoma (RCC), formalin-fixed, paraffin-embedded (FFPE), H3 K27M mutation, immunohistochemical (IHC) stain,

Tumors of central nervous system (CNS) include the tumors of the brain and spinal cord, as well as their covers. Those tumors are uncommon tumors, accounting for approximately 1% of all human body tumors. They can be divided into primary or secondary/metastatic tumors, benign or malignant tumors, based on the WHO classification; brain tumors are assigned into four grades, from Grade 1 very benign tumor to Grade IV highly malignant tumors (see below). By location, those tumors can be divided into extra-axial tumors (outside brain/spinal cord parenchyma), such as meningiomas, and intra-axial tumors (inside brain/spinal cord parenchyma), such as gliomas. Diagnosis of brain tumors is primarily based on the WHO Classification of Tumors of CNS; this expert consensus scheme was first completed in 1979 and then revised in 1993, 2000, and 2016. This scheme is currently the most widely utilized by neuropathologists worldwide for typing and grading the CNS tumors [1]. Neoplasms, especially those malignant ones, are biologically characterized by noncontrolled tumor cell proliferation; this uncontrolled growth is best explained by recently discovered EGFR (epidermal growth factor receptor) mutations, which mutations result in uncontrolled signal transduction downward to nuclei without ligand binding to the receptor and led to unlimited cell

#### **Chapter 2**

## Molecular Diagnostics and Pathology of Major Brain Tumors

*Frank Y. Shan, E. Castro, Amelia Sybenga, Sanjib Mukherjee, Erxi Wu, Karming Fung, The Li, Ekokobe Fonkem, Jason H. Huang and A. Rao*

#### **Abstract**

Tumors of central nervous system (CNS) account for a small portion of tumors of human body, which include tumors occurring in the parenchyma of brain and spinal cord as well as their coverings. The following chapter covers some new development in some major brain tumors in both pediatric and adult populations, as well as some uncommon but diagnostic and management challenging tumors.

**Keywords:** gliomas, astrocytomas, oligodendrogliomas, mixed oligoastrocytomas, WHO (World Health Organization), WHO grades, medulloblastomas (MBs), midline diffuse astrocytoma, diffuse intrinsic pontine gliomas (DIPG), hemangioblastomas (HMBs), metastatic renal cell carcinoma (RCC), formalin-fixed, paraffin-embedded (FFPE), H3 K27M mutation, immunohistochemical (IHC) stain, fluorescence in situ hybridization (FISH)

#### **1. Introduction**

Tumors of central nervous system (CNS) include the tumors of the brain and spinal cord, as well as their covers. Those tumors are uncommon tumors, accounting for approximately 1% of all human body tumors. They can be divided into primary or secondary/metastatic tumors, benign or malignant tumors, based on the WHO classification; brain tumors are assigned into four grades, from Grade 1 very benign tumor to Grade IV highly malignant tumors (see below). By location, those tumors can be divided into extra-axial tumors (outside brain/spinal cord parenchyma), such as meningiomas, and intra-axial tumors (inside brain/spinal cord parenchyma), such as gliomas. Diagnosis of brain tumors is primarily based on the WHO Classification of Tumors of CNS; this expert consensus scheme was first completed in 1979 and then revised in 1993, 2000, and 2016. This scheme is currently the most widely utilized by neuropathologists worldwide for typing and grading the CNS tumors [1]. Neoplasms, especially those malignant ones, are biologically characterized by noncontrolled tumor cell proliferation; this uncontrolled growth is best explained by recently discovered EGFR (epidermal growth factor receptor) mutations, which mutations result in uncontrolled signal transduction downward to nuclei without ligand binding to the receptor and led to unlimited cell proliferation.

#### *Primary Intracranial Tumors*

During the last two decades, a lot of gene mutations are identified, especially in the oncology field, which has been helpful for the development of new generation of antitumor medication focusing on the mutated gene products. As a matter of fact, those target treatments have already archived tremendous successes in the oncology field. For example, gefitinib, erlotinib, and afatinib are the current targeted medications against EGFR-mutated non-small-cell lung cancers, which already show great clinical success.

The following chapter is going to review some development in brain tumors, especially the recent understanding of adult gliomas and pediatric medulloblastomas, as well as some other uncommon tumors for their molecular diagnosis and genetic subgrouping.

#### **2. Molecular diagnosis of adult gliomas**

Glial tumors comprise approximately 25–30% of primary CNS tumors [1] and represent a spectrum ranging from low-grade, benign to the highly aggressive, malignant tumors. They are broadly classified by glial cell type of origin and determined by histology with or without the use of immunohistochemistry (IHC), which is then used to provide a WHO grade (see **Table 1**) [1]; however, histology has not been able to accurately predict response to treatment or clinical outcomes, and it is not uncommon for many of these tumors with nearly identical histologic features to have very different outcomes. As a result of these observations, and like many malignancies (lung and colorectal carcinoma for example), there has been increasing interest in attempting to further classify these tumors based on their molecular expression. With that interest there is an increase in available published data regarding these molecular alterations and a subsequent increase in the availability of myriad testing modalities; some of which are now considered well established, while others are not. In an era of test utilization awareness and rising healthcare costs, this phenomenon frequently leads to confusion regarding which tests should be utilized, how those tests should be interpreted, and how they should be reported, in order to best guide treatment and predict outcomes in this patient population [2].

**17**

*Molecular Diagnostics and Pathology of Major Brain Tumors*

We will discuss here the well-established molecular concepts, touch briefly on the evolving molecular discoveries, and provide a testing algorithm (see **Table 1**). Glioblastoma (GBM) is the most aggressive primary CNS malignancy, WHO Grade IV [1]. Despite decades of research and multiple new treatment modalities, little progress has been made in terms of substantial improvements of patients' outcomes, with the average long-term survival being measured in months rather than years [3]. However, this research has illuminated a complex series of molecular pathways and events that is improving our understanding of the pathophysiology of

Loss of heterozygosity (LOH) at chromosome 10q occurs with high frequency in both primary and secondary GBMs, occurring in both in approximately 60–80% of cases [4]. Although this is an interesting primary event in the development of GBM of either type, because of its high frequency, it is not useful in distinguishing one from the other. Instead, GBM is currently subclassified by its molecular alterations into primary and secondary GBM, based on the presence or absence of IDH1/ IDH2 and/or TP53 mutations [4]. EGFR status is also being increasingly used in this

Isocitrate dehydrogenase-1 (IDH1) is an NADP-dependent enzyme found in the cytoplasm responsible for the conversion of isocitrate to α-ketoglutarate and thereby producing NADPH, which reduces reactive oxygen species. IDH2 is similarly present in mitochondria. Their exact role in tumorigenesis is poorly understood; however, it is thought that mutations in this enzyme result in increased oxidative stress and subsequent carcinogenesis. It is therefore not surprising that this mutation is not found in primary GBMs, rather secondary GBMs that have progressed from a less aggressive tumor, and diffuse and anaplastic astrocytomas (WHO Grade II and III, respectively). IDH mutations are found with high frequency in the majority of astrocytomas, oligodendrogliomas, mixed gliomas, and secondary GBMs but not in pilocytic astrocytomas or primary GBMs. The most common mutation in IDH1 is a point mutation (arginine to histidine at codon 132), termed IDH1-R132H. IDH2 mutations (IDH2 172) represent only 3–5% of IDH mutations and are more commonly found in oligodendrogliomas. IDH mutations have also shown an association with hypoxia-inducible factor 1 alpha (HIF1), associated with upregulation of vascular endothelial growth factor (VEGF). Recent study indicates that low-grade astrocytomas with wild-type IDH1 behavior as glioblastoma clinically, suggesting again the importance of IDH1 mutation status in gliomas. IDH1 mutation can be detected by IHC, and commercially available mouse antihuman monoclonal antibody by Dianova with clone name H09 is a favored

Secondary glioblastomas confer a significantly better prognosis than those arising de novo (primary GBM), and occur in a younger age group with a history of pervious low grade gliomas [4]. Because primary and secondary GBMs cannot be distinguished morphologically, IDH1/2 mutation testing can be utilized for this task, allowing for better prognostication. IDH1/2 is commercially available as a reverse transcriptase PCR (RT-PCR) test. Although an IHC stain is available, it is not as sensitive to the less common variants of IDH1/2 mutation; however, it can be highly useful in the detection of single tumor cells in diffuse gliomas [5–7]. It is important to note that the role of IDH1/2 mutations in predicting response to therapy is still debated. It appears that

antibody to IDH1 R132H by most neuropathologists.

*DOI: http://dx.doi.org/10.5772/intechopen.80856*

this aggressive entity.

**2.1 LOH 10q**

context.

**2.2 IDH1/IDH2**

**Table 1.** *Molecular genetic map for the development of adult gliomas.*

#### *Molecular Diagnostics and Pathology of Major Brain Tumors DOI: http://dx.doi.org/10.5772/intechopen.80856*

We will discuss here the well-established molecular concepts, touch briefly on the evolving molecular discoveries, and provide a testing algorithm (see **Table 1**).

Glioblastoma (GBM) is the most aggressive primary CNS malignancy, WHO Grade IV [1]. Despite decades of research and multiple new treatment modalities, little progress has been made in terms of substantial improvements of patients' outcomes, with the average long-term survival being measured in months rather than years [3]. However, this research has illuminated a complex series of molecular pathways and events that is improving our understanding of the pathophysiology of this aggressive entity.

#### **2.1 LOH 10q**

*Primary Intracranial Tumors*

genetic subgrouping.

already show great clinical success.

**2. Molecular diagnosis of adult gliomas**

During the last two decades, a lot of gene mutations are identified, especially in the oncology field, which has been helpful for the development of new generation of antitumor medication focusing on the mutated gene products. As a matter of fact, those target treatments have already archived tremendous successes in the oncology field. For example, gefitinib, erlotinib, and afatinib are the current targeted medications against EGFR-mutated non-small-cell lung cancers, which

The following chapter is going to review some development in brain tumors, especially the recent understanding of adult gliomas and pediatric medulloblastomas, as well as some other uncommon tumors for their molecular diagnosis and

Glial tumors comprise approximately 25–30% of primary CNS tumors [1] and represent a spectrum ranging from low-grade, benign to the highly aggressive, malignant tumors. They are broadly classified by glial cell type of origin and determined by histology with or without the use of immunohistochemistry (IHC), which is then used to provide a WHO grade (see **Table 1**) [1]; however, histology has not been able to accurately predict response to treatment or clinical outcomes, and it is not uncommon for many of these tumors with nearly identical histologic features to have very different outcomes. As a result of these observations, and like many malignancies (lung and colorectal carcinoma for example), there has been increasing interest in attempting to further classify these tumors based on their molecular expression. With that interest there is an increase in available published data regarding these molecular alterations and a subsequent increase in the availability of myriad testing modalities; some of which are now considered well established, while others are not. In an era of test utilization awareness and rising healthcare costs, this phenomenon frequently leads to confusion regarding which tests should be utilized, how those tests should be interpreted, and how they should be reported, in order to best guide treatment and predict outcomes in this patient population [2].

**16**

**Table 1.**

*Molecular genetic map for the development of adult gliomas.*

Loss of heterozygosity (LOH) at chromosome 10q occurs with high frequency in both primary and secondary GBMs, occurring in both in approximately 60–80% of cases [4]. Although this is an interesting primary event in the development of GBM of either type, because of its high frequency, it is not useful in distinguishing one from the other. Instead, GBM is currently subclassified by its molecular alterations into primary and secondary GBM, based on the presence or absence of IDH1/ IDH2 and/or TP53 mutations [4]. EGFR status is also being increasingly used in this context.

#### **2.2 IDH1/IDH2**

Isocitrate dehydrogenase-1 (IDH1) is an NADP-dependent enzyme found in the cytoplasm responsible for the conversion of isocitrate to α-ketoglutarate and thereby producing NADPH, which reduces reactive oxygen species. IDH2 is similarly present in mitochondria. Their exact role in tumorigenesis is poorly understood; however, it is thought that mutations in this enzyme result in increased oxidative stress and subsequent carcinogenesis. It is therefore not surprising that this mutation is not found in primary GBMs, rather secondary GBMs that have progressed from a less aggressive tumor, and diffuse and anaplastic astrocytomas (WHO Grade II and III, respectively). IDH mutations are found with high frequency in the majority of astrocytomas, oligodendrogliomas, mixed gliomas, and secondary GBMs but not in pilocytic astrocytomas or primary GBMs. The most common mutation in IDH1 is a point mutation (arginine to histidine at codon 132), termed IDH1-R132H. IDH2 mutations (IDH2 172) represent only 3–5% of IDH mutations and are more commonly found in oligodendrogliomas. IDH mutations have also shown an association with hypoxia-inducible factor 1 alpha (HIF1), associated with upregulation of vascular endothelial growth factor (VEGF). Recent study indicates that low-grade astrocytomas with wild-type IDH1 behavior as glioblastoma clinically, suggesting again the importance of IDH1 mutation status in gliomas. IDH1 mutation can be detected by IHC, and commercially available mouse antihuman monoclonal antibody by Dianova with clone name H09 is a favored antibody to IDH1 R132H by most neuropathologists.

Secondary glioblastomas confer a significantly better prognosis than those arising de novo (primary GBM), and occur in a younger age group with a history of pervious low grade gliomas [4]. Because primary and secondary GBMs cannot be distinguished morphologically, IDH1/2 mutation testing can be utilized for this task, allowing for better prognostication. IDH1/2 is commercially available as a reverse transcriptase PCR (RT-PCR) test. Although an IHC stain is available, it is not as sensitive to the less common variants of IDH1/2 mutation; however, it can be highly useful in the detection of single tumor cells in diffuse gliomas [5–7]. It is important to note that the role of IDH1/2 mutations in predicting response to therapy is still debated. It appears that

the mutation confers an increased response to radiation therapy, while others show an increased response with chemotherapy as well. Response appears to be multifactorial, dependent not only on the type of therapy, but also on the time of that therapy in relation to surgical resection [8]. Importantly, IDH mutations serve as a surrogate marker for secondary GBMs [9]. This testing should be performed in conjunction with TP53 and Ki67 on all GBMs, and considered standard of care.

#### **2.3 P53**

P53 is a cyclin-dependent kinase responsible for tumor suppression through prevention of cell replication. Mutations in p53 in malignant tumors are well established in the literature, with greater than 50% of cancers showing p53 loss of function mutations [10]. P53 is more commonly a missense mutation that results in accumulation of the protein in the cytoplasm, resulting in diffuse, strong nuclear staining by IHC; however, alternate mutations in p53 can show complete absence of staining or cytoplasmic staining only, whereas the wild type (unmutated) p53 will show weak to moderate, patchy positivity [11].

Most tumors that express p53 mutations typically have a more aggressive course than those that do not; however, this relationship has not been established in GBMs. Currently, no statistically significant difference has been established that GBMs with p53 mutations have a worse prognosis than those that do not [12]. The utility of p53 in GBMs, similar to IDH1/2 mutation status, is as additional evidence of a secondary GBM, rather than a primary GBM, as p53 mutations are far less frequent in primary GBMs, and, when present, likely represent secondary or late events associated with increasing genetic instability [4].

#### **2.4 Epidermal growth factor receptor (EGFR)**

EGFR is a member of the transmembrane tyrosine kinase receptor family that activates MAPK and PIK3 pathways resulting in cell proliferation. EGFR testing started to gain particular popularity due in part to the development of tyrosine kinase inhibitors (TKIs) and after a 2010 study by Verhaak et al. that attempted to further subclassify GBMs based on multiple molecular markers [15]. EGFR amplification confers more aggressive behavior and poorer outcomes, autophosphorylating the PIK3 pathway, leading to increased growth, angiogenesis, metastatic potential, and reduced apoptosis [16]. In contrast to secondary GBMs, epidermal growth factor receptor (EGFR) overexpression has been demonstrated in 36% of primary GBMs, with 70% of those showing amplification. It exists most commonly as the mutation EGFR variant 3 (EGFRvIII), which has deletion of exons 2 and 7 [17]. EFGRvIII mutation testing is performed with RT-PCR [17]. Unlike other malignancies where tyrosine kinase inhibitor (TKI) immunotherapy has shown wide successes, GBMs frequently do not respond, showed only a partial response, or develop rapid resistance to TKIs. This most often attributed to PTEN loss earlier in the EGFR pathway [4]. EGFR amplification or mutation in this context can be utilized, when present, as further support of a primary GBM over a secondary GBM.

In addition to the discovery of multiple molecular alterations in MET, PDGFRA, NF1, PTEN, PIK3 and CDKN2A/B, and several others, studies have discovered alterations of several microRNAs, which are also a field of current study. Importantly, none of these have been well established in terms of either their prognostic significance or their impact on treatment response, and several studies have shown contradictory results. This likely can be attributed to the marked heterogeneity of glial tumors, particularly GBMs. It is not currently recommended to add these markers to a broader profile until their clinical significance can be better established.

**19**

*Molecular Diagnostics and Pathology of Major Brain Tumors*

their entirety is recommended due to molecular variability.

Chromosome arms 1p and 19q deletions are the most characterized genetic aberrations of oligodendrogliomas, with up to 80% of classical oligodendrogliomas (WHO Grade II) and 60% of anaplastic oligodendrogliomas (WHO Grade III) [18, 19]. Although it seems unclear what impact these deletions have on cellular function, there are two identified roles for testing these deletions: the first is as a diagnostic marker for oligodendroglial tumors and the other as an indicator of response to treatment. One study demonstrated that the presence of complete or partial codeletion of 1p and 19q conferred a significantly increased response to chemotherapy, and prolonged disease-free survival time, compared with those tumors with deletion of only one or the other chromosomal arm, regardless of histologic subtype [20], consistent with other studies, including mixed tumors. Due to the significant clinical implications for the presence of this gene, 1p/19q co-deletion testing should be performed on all glial tumors with or without oligodendroglial features since a small percentage (5%) of morphological astrocytomas are with 1p/19q co-deletion, which may confer a slightly better prognosis for the patients. Testing is available by both FISH and for LOH by real-time PCR. Evaluation of both chromosomal arms in


Methylation-specific PCR is the testing modality of choice and is widely available. An alternative is pyrosequencing, which shows high sensitivity, but is now less frequently used. Other testing modalities, such as western blot and IHC, have fallen

**Table 1** summarizes the current understanding of tumorigenesis for adult

It has been recognized for almost 20 years among pediatric neuro-oncologists that neuroimaging study defined diffuse intrinsic pontine gliomas (DIPG) had a very poor prognosis independent of histological grade (if biopsied). In that case, biopsy is most of the time considered unnecessary until recent identification of potential drug targets for individualized therapy has led to reevaluation of this approach [22]. Recent genomic analysis has demonstrated that specific genetic alterations drive distinct subsets of glial neoplasm of the central nervous system, dependent not only on tumor-type but also on the site of origin and patient age. Like this diffuse midline gliomas, with somatic mutations of the H3F3A and HIST1H3B gene encoding the histone H3 variants, H3.3 and H3.1, were recently identified in high-grade gliomas arising in the thalamus, pons, and spinal cord of

**3. Molecular diagnosis of diffuse midline gliomas with H3 K27M** 

*DOI: http://dx.doi.org/10.5772/intechopen.80856*

**2.5 1p/19q co-deletion**

**2.6 MGMT methylation**

which is not well understood [9].

into disuse due to issues with false-positive results.

O6

gliomas.

**mutation**

#### **2.5 1p/19q co-deletion**

*Primary Intracranial Tumors*

**2.3 P53**

the mutation confers an increased response to radiation therapy, while others show an increased response with chemotherapy as well. Response appears to be multifactorial, dependent not only on the type of therapy, but also on the time of that therapy in relation to surgical resection [8]. Importantly, IDH mutations serve as a surrogate marker for secondary GBMs [9]. This testing should be performed in conjunction with TP53

P53 is a cyclin-dependent kinase responsible for tumor suppression through prevention of cell replication. Mutations in p53 in malignant tumors are well established in the literature, with greater than 50% of cancers showing p53 loss of function mutations [10]. P53 is more commonly a missense mutation that results in accumulation of the protein in the cytoplasm, resulting in diffuse, strong nuclear staining by IHC; however, alternate mutations in p53 can show complete absence of staining or cytoplasmic staining only, whereas the wild type (unmutated) p53 will

Most tumors that express p53 mutations typically have a more aggressive course than those that do not; however, this relationship has not been established in GBMs. Currently, no statistically significant difference has been established that GBMs with p53 mutations have a worse prognosis than those that do not [12]. The utility of p53 in GBMs, similar to IDH1/2 mutation status, is as additional evidence of a secondary GBM, rather than a primary GBM, as p53 mutations are far less frequent in primary GBMs, and, when present, likely represent secondary or late events

EGFR is a member of the transmembrane tyrosine kinase receptor family that activates MAPK and PIK3 pathways resulting in cell proliferation. EGFR testing started to gain particular popularity due in part to the development of tyrosine kinase inhibitors (TKIs) and after a 2010 study by Verhaak et al. that attempted to further subclassify GBMs based on multiple molecular markers [15]. EGFR amplification confers more aggressive behavior and poorer outcomes, autophosphorylating the PIK3 pathway, leading to increased growth, angiogenesis, metastatic potential, and reduced apoptosis [16]. In contrast to secondary GBMs, epidermal growth factor receptor (EGFR) overexpression has been demonstrated in 36% of primary GBMs, with 70% of those showing amplification. It exists most commonly as the mutation EGFR variant 3 (EGFRvIII), which has deletion of exons 2 and 7 [17]. EFGRvIII mutation testing is performed with RT-PCR [17]. Unlike other malignancies where tyrosine kinase inhibitor (TKI) immunotherapy has shown wide successes, GBMs frequently do not respond, showed only a partial response, or develop rapid resistance to TKIs. This most often attributed to PTEN loss earlier in the EGFR pathway [4]. EGFR amplification or mutation in this context can be utilized,

when present, as further support of a primary GBM over a secondary GBM.

In addition to the discovery of multiple molecular alterations in MET, PDGFRA, NF1, PTEN, PIK3 and CDKN2A/B, and several others, studies have discovered alterations of several microRNAs, which are also a field of current study. Importantly, none of these have been well established in terms of either their prognostic significance or their impact on treatment response, and several studies have shown contradictory results. This likely can be attributed to the marked heterogeneity of glial tumors, particularly GBMs. It is not currently recommended to add these markers to a broader profile until their clinical significance can be better established.

and Ki67 on all GBMs, and considered standard of care.

show weak to moderate, patchy positivity [11].

associated with increasing genetic instability [4].

**2.4 Epidermal growth factor receptor (EGFR)**

**18**

Chromosome arms 1p and 19q deletions are the most characterized genetic aberrations of oligodendrogliomas, with up to 80% of classical oligodendrogliomas (WHO Grade II) and 60% of anaplastic oligodendrogliomas (WHO Grade III) [18, 19]. Although it seems unclear what impact these deletions have on cellular function, there are two identified roles for testing these deletions: the first is as a diagnostic marker for oligodendroglial tumors and the other as an indicator of response to treatment. One study demonstrated that the presence of complete or partial codeletion of 1p and 19q conferred a significantly increased response to chemotherapy, and prolonged disease-free survival time, compared with those tumors with deletion of only one or the other chromosomal arm, regardless of histologic subtype [20], consistent with other studies, including mixed tumors. Due to the significant clinical implications for the presence of this gene, 1p/19q co-deletion testing should be performed on all glial tumors with or without oligodendroglial features since a small percentage (5%) of morphological astrocytomas are with 1p/19q co-deletion, which may confer a slightly better prognosis for the patients. Testing is available by both FISH and for LOH by real-time PCR. Evaluation of both chromosomal arms in their entirety is recommended due to molecular variability.

#### **2.6 MGMT methylation**

O6 -methylguanine-DNA methyltransferase (MGMT) codes for MGMT repair protein, and methylation of this gene, which results in suppression and decreased expression of the MGMT protein, confers a significant survival benefit in patient treated with combined radiation and temozolomide therapy. MGMT methylation occurs in all types of gliomas and with frequency in primary and secondary GBMs and oligodendrogliomas (60–93%). In predicting a positive response to treatment, MGMT methylation also predicts an increased survival benefit. In lower-grade gliomas, MGMT methylation confers an increased response to radiation monotherapy, which is not well understood [9].

Methylation-specific PCR is the testing modality of choice and is widely available. An alternative is pyrosequencing, which shows high sensitivity, but is now less frequently used. Other testing modalities, such as western blot and IHC, have fallen into disuse due to issues with false-positive results.

**Table 1** summarizes the current understanding of tumorigenesis for adult gliomas.

#### **3. Molecular diagnosis of diffuse midline gliomas with H3 K27M mutation**

It has been recognized for almost 20 years among pediatric neuro-oncologists that neuroimaging study defined diffuse intrinsic pontine gliomas (DIPG) had a very poor prognosis independent of histological grade (if biopsied). In that case, biopsy is most of the time considered unnecessary until recent identification of potential drug targets for individualized therapy has led to reevaluation of this approach [22]. Recent genomic analysis has demonstrated that specific genetic alterations drive distinct subsets of glial neoplasm of the central nervous system, dependent not only on tumor-type but also on the site of origin and patient age. Like this diffuse midline gliomas, with somatic mutations of the H3F3A and HIST1H3B gene encoding the histone H3 variants, H3.3 and H3.1, were recently identified in high-grade gliomas arising in the thalamus, pons, and spinal cord of children and young adults; those tumors are named as diffuse midline gliomas with H3 K27M mutation [23].

Brainstem tumors affect primarily children and young adults. Each year, around 300–400 cases of brainstem tumors are diagnosed in the United States, and diffuse intrinsic pontine glioma (DIPG) accounts approximately 80% of these tumors [24]. DIPG has been recently categorized by WHO classification as high-grade (Grade IV) diffuse midline gliomas with H3 K27M mutation. It carries a poor prognosis, and only 1% of the patients live 5 years after diagnosis.

Clinically, diffuse midline gliomas result in brainstem dysfunction and the obstruction of cerebrospinal fluid (CSF) flow. The patients suffer from difficulty in ocular movements, weakness of facial muscles, sudden hearing problem, swallowing difficulty, muscle spasticity, clonus, and bladder dysfunction, along with multiple cranial neuropathies and ataxia.

Diagnosis of diffuse midline gliomas is initiated through imaging primarily by MRI scans indicating hypointense (T1) or hyperintense (T2) lesions, enhancing or non-enhancing after administration of contract agents. Biopsy is a standard procedure for establishing the molecular and histopathological diagnoses. This tumor shows many histopathological features of glioblastoma such as pseudopalisading necrosis and microvascular proliferation, in addition to H3 K27M positive by immunohistochemical (IHC) stain (**Figure 1**).

Unlike many other adult gliomas, debunking surgery, with gross total resection (GTR) of the tumor, is not a treatment of choice for diffuse midline gliomas, mainly due to the location of the tumors. The brainstem regulates critical bodily functions, and therefore surgical resection without damaging the vital area of the brainstem is almost impossible. Surgery is indicated only for biopsy of the tumor and to relieve the hydrocephalus that may happen in a small fraction of cases. Currently, patients are treated primarily with radiation and adjuvant chemotherapy with temozolomide.

In diffuse midline gliomas with H3 K27 mutation, lysine in 27th position of tail of Histone 3.3 is replaced by methionine. Histones are the alkaline protein that provides a scaffold, around which DNA wraps. Solomon and colleague have observed that H3K37M mutation in pontine gliomas occurred at a much younger age (median 7 years of age) than gliomas of the thalamus (median age, 24 years) or spinal cord (median age, 25 years) [23]. The H3 K27M-positive gliomas have also been reported in adult in the brainstem [25]. High-grade gliomas with H3 K27M mutation may have additional mutations (WHO, 2016). These mutations are observed to occur in the critical genes, regulating cell divisions including cell cycle checkpoints and chromatin remodeling. The most frequent additional mutation noted is tumor suppressor p53, which is noted in an estimated half of the H3 K27M

**21**

**Figure 2.**

*Molecular Diagnostics and Pathology of Major Brain Tumors*

ATRX loss (except in pontine gliomas), and monosomy 10 [23].

**4. Molecular subgroups of medulloblastomas**

**4.1 Morphologic features of medulloblastomas**

*cell/anaplastic, 15% (arrow nuclear molding, blue; wrapping, red).*

midline gliomas. Amplification of platelet-derived growth factor alpha (PDGFRA), critical for cell survival and proliferation, is observed in about 30% of the H3 K27M tumors. Cyclin-dependent kinases 4 and 6 are reported to be amplified in 20% of the tumors, and homozygous deletion of cyclin-dependent kinase inhibitor 2A/2B is noted in 5% of the H3 K27M gliomas. Mutation of activin A receptor type-1 (AVCR1) is noted in 20% of H3 K27M gliomas. A mutation of protein phosphatase 1D (PPM1D) and amplification of MYC/PVT1 may present separately, in 15% of the H3 K27M gliomas [1]. In addition, histone H3 K27M mutation is found mutually exclusive with IDH1 mutation and EGFR amplification, rarely co-occurred with BRAF-V600E mutation, and was commonly associated with p53 overexpression,

These mutations could provide us with a better understanding of the disease process and could potentially lead to the development of a better treatment strategy for this deadly disease. As a matter of fact, at least two clinical trials are underway with small molecule inhibitor of the histone demethylase, which showed some

Medulloblastoma is the most common malignant brain tumor of cerebellum in childhood, although it rarely happens in adult patients, too. It is an embryonal neuroepithelial tumor arising in the cerebellum or dorsal brainstem, which is a major cause of morbidity and mortality in pediatric brain tumor patients [27, 28], and was designed as WHO Grade IV neoplasm [1]. Histologically, medulloblastoma is a prototypical embryonal tumor, consisting of densely packed small round undifferentiated blue tumor cells with mild to moderate nuclear pleomorphism and a high mitotic count, mostly with Homer-Wright rosettes, and shows different morphological variants, such as desmoplastic/nodular, large cell, and anaplastic, etc., with predominantly neuronal differentiation and tendency to metastasize via CSF pathways [1] (**Figures 2** and **3**).

Several morphological variants of MBs are recognized, alongside the classic tumor: desmoplastic/nodular MB, MB with extensive nodularity, and large-cell/ anaplastic MB. A dominant population of undifferentiated cells with a high nuclear-to-cytoplasmic ratio and active mitotic figures is a common feature [1] (**Figures 2** and **3**). Classic MB composed of sheets of undifferentiated small blue

*Histopathology of MBs. (A) Classic type, 70%; (B) nodular, 10%; (C) extensive nodularity, 3%; and (D) large* 

*DOI: http://dx.doi.org/10.5772/intechopen.80856*

promising result [23].

**Figure 1.** *(A) Grade IV midline glioblastoma and (B) IHC + for H3 K27M mutation.*

*Molecular Diagnostics and Pathology of Major Brain Tumors DOI: http://dx.doi.org/10.5772/intechopen.80856*

*Primary Intracranial Tumors*

H3 K27M mutation [23].

temozolomide.

and only 1% of the patients live 5 years after diagnosis.

multiple cranial neuropathies and ataxia.

immunohistochemical (IHC) stain (**Figure 1**).

*(A) Grade IV midline glioblastoma and (B) IHC + for H3 K27M mutation.*

children and young adults; those tumors are named as diffuse midline gliomas with

Clinically, diffuse midline gliomas result in brainstem dysfunction and the obstruction of cerebrospinal fluid (CSF) flow. The patients suffer from difficulty in ocular movements, weakness of facial muscles, sudden hearing problem, swallowing difficulty, muscle spasticity, clonus, and bladder dysfunction, along with

Diagnosis of diffuse midline gliomas is initiated through imaging primarily by MRI scans indicating hypointense (T1) or hyperintense (T2) lesions, enhancing or non-enhancing after administration of contract agents. Biopsy is a standard procedure for establishing the molecular and histopathological diagnoses. This tumor shows many histopathological features of glioblastoma such as pseudopalisading necrosis and microvascular proliferation, in addition to H3 K27M positive by

Unlike many other adult gliomas, debunking surgery, with gross total resection (GTR) of the tumor, is not a treatment of choice for diffuse midline gliomas, mainly due to the location of the tumors. The brainstem regulates critical bodily functions, and therefore surgical resection without damaging the vital area of the brainstem is almost impossible. Surgery is indicated only for biopsy of the tumor and to relieve the hydrocephalus that may happen in a small fraction of cases. Currently, patients are treated primarily with radiation and adjuvant chemotherapy with

In diffuse midline gliomas with H3 K27 mutation, lysine in 27th position of tail of Histone 3.3 is replaced by methionine. Histones are the alkaline protein that provides a scaffold, around which DNA wraps. Solomon and colleague have observed that H3K37M mutation in pontine gliomas occurred at a much younger age (median 7 years of age) than gliomas of the thalamus (median age, 24 years) or spinal cord (median age, 25 years) [23]. The H3 K27M-positive gliomas have also been reported in adult in the brainstem [25]. High-grade gliomas with H3 K27M mutation may have additional mutations (WHO, 2016). These mutations are observed to occur in the critical genes, regulating cell divisions including cell cycle checkpoints and chromatin remodeling. The most frequent additional mutation noted is tumor suppressor p53, which is noted in an estimated half of the H3 K27M

Brainstem tumors affect primarily children and young adults. Each year, around 300–400 cases of brainstem tumors are diagnosed in the United States, and diffuse intrinsic pontine glioma (DIPG) accounts approximately 80% of these tumors [24]. DIPG has been recently categorized by WHO classification as high-grade (Grade IV) diffuse midline gliomas with H3 K27M mutation. It carries a poor prognosis,

**20**

**Figure 1.**

midline gliomas. Amplification of platelet-derived growth factor alpha (PDGFRA), critical for cell survival and proliferation, is observed in about 30% of the H3 K27M tumors. Cyclin-dependent kinases 4 and 6 are reported to be amplified in 20% of the tumors, and homozygous deletion of cyclin-dependent kinase inhibitor 2A/2B is noted in 5% of the H3 K27M gliomas. Mutation of activin A receptor type-1 (AVCR1) is noted in 20% of H3 K27M gliomas. A mutation of protein phosphatase 1D (PPM1D) and amplification of MYC/PVT1 may present separately, in 15% of the H3 K27M gliomas [1]. In addition, histone H3 K27M mutation is found mutually exclusive with IDH1 mutation and EGFR amplification, rarely co-occurred with BRAF-V600E mutation, and was commonly associated with p53 overexpression, ATRX loss (except in pontine gliomas), and monosomy 10 [23].

These mutations could provide us with a better understanding of the disease process and could potentially lead to the development of a better treatment strategy for this deadly disease. As a matter of fact, at least two clinical trials are underway with small molecule inhibitor of the histone demethylase, which showed some promising result [23].

#### **4. Molecular subgroups of medulloblastomas**

Medulloblastoma is the most common malignant brain tumor of cerebellum in childhood, although it rarely happens in adult patients, too. It is an embryonal neuroepithelial tumor arising in the cerebellum or dorsal brainstem, which is a major cause of morbidity and mortality in pediatric brain tumor patients [27, 28], and was designed as WHO Grade IV neoplasm [1]. Histologically, medulloblastoma is a prototypical embryonal tumor, consisting of densely packed small round undifferentiated blue tumor cells with mild to moderate nuclear pleomorphism and a high mitotic count, mostly with Homer-Wright rosettes, and shows different morphological variants, such as desmoplastic/nodular, large cell, and anaplastic, etc., with predominantly neuronal differentiation and tendency to metastasize via CSF pathways [1] (**Figures 2** and **3**).

#### **4.1 Morphologic features of medulloblastomas**

Several morphological variants of MBs are recognized, alongside the classic tumor: desmoplastic/nodular MB, MB with extensive nodularity, and large-cell/ anaplastic MB. A dominant population of undifferentiated cells with a high nuclear-to-cytoplasmic ratio and active mitotic figures is a common feature [1] (**Figures 2** and **3**). Classic MB composed of sheets of undifferentiated small blue

#### **Figure 2.**

*Histopathology of MBs. (A) Classic type, 70%; (B) nodular, 10%; (C) extensive nodularity, 3%; and (D) large cell/anaplastic, 15% (arrow nuclear molding, blue; wrapping, red).*

#### **Figure 3.**

*Histopathology of MBs. (A) Homer Wright rosettes and (B) nuclear wrapping (hugging), arrow, in large-cell/ anaplastic MBs.*

tumor cells with Homer-Wright rosette formations and/or palisading tumor cells forming a pseudoglandular feature, easily found mitoses and apoptosis. Other histological variants include desmoplastic MB, which contains abundant reticulin and collagen, characterized with nodular reticulin-free zones (pale islands). The nodules have reduced cellularity, a rarified fibrillar matrix and marked nuclear uniformity. A rare histologic type of MB is the so-called large-cell MB, which is composed completely or partially of cells with large, round nuclei and prominent nucleoli, commonly with large areas of necrosis. The large-cell MB sometimes resembles the rhabdoid/atypical teratoid (RT/AT) tumors of cerebellum, but its cytoplasm lacks globular hyaline inclusions and is diffusely reactive for synaptophysin, neurofilament protein, and vimentin and negative for epithelial membrane antigen, cytokeratins, and smooth-muscle actin by immunohistochemical (IHC) stains [1]. Most often associated with large-cell MB is anaplastic MB, which is characterized by angular, crowded, pleomorphic nuclei in large cells, sometimes with nuclear molding and wrapping (**Figure 3**), mitosis, and apoptosis, as well as prominent nucleoli. It has been noted for a long time that morphology of MBs was related to patient's prognosis and that those MBs with extensive nodularity are with better prognosis, while the large-cell/anaplastic MBs are usually associated with worse clinical outcomes (**Figure 1**).

Additional study indicates that poor survival outcome was significantly associated with chromosome 17p loss (loss of tumor suppressor) and high expression of oncogenes c-myc (MYCC) or N-myc (MYCN) [1].

Due to the highly heterogeneous nature of the MB, and complication caused by aggressive treatments, a more specific subgrouping of this tumor is becoming more and more important for clinical judgment.

Molecular studies from multiple groups around the world found that medulloblastoma is not a single disease but comprises a collection of clinically and molecularly diverse subgroups. Current consensus made in a 2010 meeting at Boston agrees that there are four principal subgroups of medulloblastomas [27, 28] termed as WNT, SHH, Group 3, and Group 4.

Two of these subgroups, characterized by either activated WNT or SHH signaling pathway, are thought to play prominent roles in the pathogenesis. Two other non-WNT/non-SHH groups are more closely related to each other and even produced additional different numbers of subgroups within these groups of MBs, pending additional evidence to further classify them [27, 28].

**23**

medulloblastomas.

*Molecular Diagnostics and Pathology of Major Brain Tumors*

medulloblastomas with these genetic abnormalities [27].

the nomenclature of "WNT subgroup of medulloblastomas."

activated MBs include TP53, SMARCA4, and DDX3X [1].

chromosome shattering known as chromothripsis are often present.

*4.1.2 MB, SHH subgroup with TP53 mutant*

The best known subgroup of the medulloblastoma is the WNT subgroup due to its very good long-term prognosis, compared to other subgroups. WNT indicates

More than 90% of WNT subgroup medulloblastoma patients achieved long-term survival, with those patients whose death is due to more complications of therapy or secondary tumors rather than due to recurrent WNT medulloblastomas. Germline mutations of the WNT pathway inhibit APC predispose to Turcot syndrome, which includes a proclivity to medulloblastoma; in addition, somatic mutations of CTNNB1 encoding β-catinin have been found in sporadic medulloblastomas [27]. These strong germline and somatic genetic data support an etiological role for canonical WNT signaling in the pathogenesis of this group of tumors and lead to

Almost all WNT medulloblastomas have classic histology, which often described as having CTNNB1 mutations, with nuclear labeling for β-catenin by immunohistochemical stain, and monosomy 6 (deletion of one copy of chromosome 6 in tumor cells). Medulloblastomas with large-cell/anaplastic features have also been reported in the WNT subgroup, although they appear to maintain the excellent prognosis associated with the WNT subgroup. Which of monosomy 6, nuclear staining for β-catenin, mutation of CTNNB1, immunohistochemical staining for DKK1, or a transcriptional signature that clusters with other WNT tumors should be used as a gold standard for the diagnosis of WNT medulloblastomas awaits further validation on large cohorts of well-characterized

WNT-activated MBs account for approximately 10% of all MBs, most of them present in children aged between 7 and 14 years, but they can also occur in young adults. Genetically, besides CTNNB1, genes that are recurrently mutated in WNT-

The SHH group of MBs are named after the sonic hedgehog signaling pathway. In large series of tumors, SHH-activated MBs tend to have similar transcriptome, methylome, and microRNA profiles. SHH pathway activation in TP53-mutant tumors is associated with amplification of GLI2, MYCN, or SHH. Mutations in PTCH1, SUFU, and SMO are genetically absent. Large-cell/anaplastic morphology and chromosome 17p loss are also common in SHH-activated and TP53-mutant tumors. Patterns of

WNT medulloblastomas are characterized by upregulation of the canonical WNT signaling pathway, which results in translocation of β-catenin to the nucleus. About two-thirds harbor a CTNNB1 mutation. Mutations in other pathways, such as APC and AXIN1, have been reported in the absence of a CTNNB1 mutation but are much less frequent [27]. The extent of β-catenin nuclear immunoreactivity in these WNT pathway MBs always amounts to more than a third of the total tumor area and is clearly different from the situation where very few scattered β-catenin nucleopositive cells, representing less than 2% of tumor cells, are evident. Assays for CTNNB1 mutation and monosomy 6, which occurs in nearly all WNT pathway MBs, have helped to establish the status of tumors in these immunohistochemical categories [27]. There is a close association between a WNT pathway immunophenotype and CTNNB1 mutation or monosomy 6, predicting a good outcome for

*DOI: http://dx.doi.org/10.5772/intechopen.80856*

*4.1.1 MB, WNT subgroup*

the wingless signaling pathway.

#### *4.1.1 MB, WNT subgroup*

*Primary Intracranial Tumors*

**Figure 3.**

*anaplastic MBs.*

worse clinical outcomes (**Figure 1**).

oncogenes c-myc (MYCC) or N-myc (MYCN) [1].

and more important for clinical judgment.

WNT, SHH, Group 3, and Group 4.

tumor cells with Homer-Wright rosette formations and/or palisading tumor cells forming a pseudoglandular feature, easily found mitoses and apoptosis. Other histological variants include desmoplastic MB, which contains abundant reticulin and collagen, characterized with nodular reticulin-free zones (pale islands). The nodules have reduced cellularity, a rarified fibrillar matrix and marked nuclear uniformity. A rare histologic type of MB is the so-called large-cell MB, which is composed completely or partially of cells with large, round nuclei and prominent nucleoli, commonly with large areas of necrosis. The large-cell MB sometimes resembles the rhabdoid/atypical teratoid (RT/AT) tumors of cerebellum, but its cytoplasm lacks globular hyaline inclusions and is diffusely reactive for synaptophysin, neurofilament protein, and vimentin and negative for epithelial membrane antigen, cytokeratins, and smooth-muscle actin by immunohistochemical (IHC) stains [1]. Most often associated with large-cell MB is anaplastic MB, which is characterized by angular, crowded, pleomorphic nuclei in large cells, sometimes with nuclear molding and wrapping (**Figure 3**), mitosis, and apoptosis, as well as prominent nucleoli. It has been noted for a long time that morphology of MBs was related to patient's prognosis and that those MBs with extensive nodularity are with better prognosis, while the large-cell/anaplastic MBs are usually associated with

*Histopathology of MBs. (A) Homer Wright rosettes and (B) nuclear wrapping (hugging), arrow, in large-cell/*

Additional study indicates that poor survival outcome was significantly associated with chromosome 17p loss (loss of tumor suppressor) and high expression of

Due to the highly heterogeneous nature of the MB, and complication caused by aggressive treatments, a more specific subgrouping of this tumor is becoming more

Molecular studies from multiple groups around the world found that medulloblastoma is not a single disease but comprises a collection of clinically and molecularly diverse subgroups. Current consensus made in a 2010 meeting at Boston agrees that there are four principal subgroups of medulloblastomas [27, 28] termed as

Two of these subgroups, characterized by either activated WNT or SHH signaling pathway, are thought to play prominent roles in the pathogenesis. Two other non-WNT/non-SHH groups are more closely related to each other and even produced additional different numbers of subgroups within these groups of MBs,

pending additional evidence to further classify them [27, 28].

**22**

The best known subgroup of the medulloblastoma is the WNT subgroup due to its very good long-term prognosis, compared to other subgroups. WNT indicates the wingless signaling pathway.

WNT medulloblastomas are characterized by upregulation of the canonical WNT signaling pathway, which results in translocation of β-catenin to the nucleus. About two-thirds harbor a CTNNB1 mutation. Mutations in other pathways, such as APC and AXIN1, have been reported in the absence of a CTNNB1 mutation but are much less frequent [27]. The extent of β-catenin nuclear immunoreactivity in these WNT pathway MBs always amounts to more than a third of the total tumor area and is clearly different from the situation where very few scattered β-catenin nucleopositive cells, representing less than 2% of tumor cells, are evident. Assays for CTNNB1 mutation and monosomy 6, which occurs in nearly all WNT pathway MBs, have helped to establish the status of tumors in these immunohistochemical categories [27]. There is a close association between a WNT pathway immunophenotype and CTNNB1 mutation or monosomy 6, predicting a good outcome for medulloblastomas with these genetic abnormalities [27].

More than 90% of WNT subgroup medulloblastoma patients achieved long-term survival, with those patients whose death is due to more complications of therapy or secondary tumors rather than due to recurrent WNT medulloblastomas. Germline mutations of the WNT pathway inhibit APC predispose to Turcot syndrome, which includes a proclivity to medulloblastoma; in addition, somatic mutations of CTNNB1 encoding β-catinin have been found in sporadic medulloblastomas [27]. These strong germline and somatic genetic data support an etiological role for canonical WNT signaling in the pathogenesis of this group of tumors and lead to the nomenclature of "WNT subgroup of medulloblastomas."

Almost all WNT medulloblastomas have classic histology, which often described as having CTNNB1 mutations, with nuclear labeling for β-catenin by immunohistochemical stain, and monosomy 6 (deletion of one copy of chromosome 6 in tumor cells). Medulloblastomas with large-cell/anaplastic features have also been reported in the WNT subgroup, although they appear to maintain the excellent prognosis associated with the WNT subgroup. Which of monosomy 6, nuclear staining for β-catenin, mutation of CTNNB1, immunohistochemical staining for DKK1, or a transcriptional signature that clusters with other WNT tumors should be used as a gold standard for the diagnosis of WNT medulloblastomas awaits further validation on large cohorts of well-characterized medulloblastomas.

WNT-activated MBs account for approximately 10% of all MBs, most of them present in children aged between 7 and 14 years, but they can also occur in young adults. Genetically, besides CTNNB1, genes that are recurrently mutated in WNTactivated MBs include TP53, SMARCA4, and DDX3X [1].

#### *4.1.2 MB, SHH subgroup with TP53 mutant*

The SHH group of MBs are named after the sonic hedgehog signaling pathway. In large series of tumors, SHH-activated MBs tend to have similar transcriptome, methylome, and microRNA profiles. SHH pathway activation in TP53-mutant tumors is associated with amplification of GLI2, MYCN, or SHH. Mutations in PTCH1, SUFU, and SMO are genetically absent. Large-cell/anaplastic morphology and chromosome 17p loss are also common in SHH-activated and TP53-mutant tumors. Patterns of chromosome shattering known as chromothripsis are often present.

SHH-activated tumors account for approximately 30% of all MBs and originate from rhombic lip-derived cerebellar granule neuron precursors. SHH-activated and TP53-mutant MBs are rare and generally found in children aged 4–17 years. Clinical outcomes in patients with SHH-activated and TP53-mutant tumors are very poor [1].

#### *4.1.3 MB, SHH subgroup, with TP53 wild type*

SHH pathway activation in TP53 wild-type tumors can be associated with germline or somatic mutation in the negative regulations PTCH1 or SUFU, as well as activating somatic mutations in SMO or (rarely) amplification of GLI2. Desmoplastic/nodular MBs and MBs with extensive nodularity are always included in the SHH-activated group, but tumors with a SHH signaling pathway can also have a classic or large-cell/anaplastic morphology, particularly in older children. Patients with SHH-activated and TP53 wild-type MBs are generally children aged <4 years, adolescents, or young adults. In addition to genetic changes activating SHH signaling, mutations in DDX3X or KMT2D and amplification of MYCN or MYCL are sometimes seen, as are deletions of chromosomal arms 9q, 10q, and 14q. Clinical outcomes in patients with SHH-activated tumors are variable [1].

#### **4.2 Epidemiology**

Research data from 1973 to 2007 suggested MB incidence rates of 0.6 cases per 1 million children aged 1–9 years and 0.6 cases per 1 million adults aged >19 years. SHH-activated MBs in general show a bimodal age distribution, being most common in infants and young adults, with a male-female ratio of approximately 1.5:1. In contrast, SHH-activated and TP53-mutant tumors are generally found in children aged 4–17 years. In one study including 133 SHH-activated MBs, 28 patients (21%) had a TP53 mutation, and the median age of these patients was approximately 15 years [1].

#### *4.2.1 Groups 3 and 4/non-WNT and non-SHH groups*

Groups 3 and 4 MBs are usually called "the non-WNT/non-SHH groups." They share some of the similarities in both clinical presentation and molecular profiling. Most tumors in these groups display classical histology. The large-cell/anaplastic and desmoplastic histologies are present but at a lower frequency. The age of onset is distributed in both groups with most patients are children; they are relatively uncommon in infants and adults. Although both groups have similar frequency of metastasis, Group 3 shows poor prognosis, while Group 4 shows intermediate prognosis. Non-WNT and non-SHH tumors account for approximately 60% of all MBs and typically have classic histopathological features [1].

One characteristic similarity between Groups 3 and 4 is both subgroups are enriched for expression of genes involved in photoreceptor differentiation, and they express high level of OTX2 and FOXG1B, well-known oncogenes of MB. However, Group 3 is distinguished by its enriched gene signatures functioning in cell cycle, protein biosynthesis, glutamate receptor signaling, and p38 mitogen-activated protein kinase (MMAPK) pathway, while Group 4 is overrepresented by genes involved in neuronal differentiation, development, cytoskeleton organization, etc.

In addition, isochromosome 17q (I17q) represents the most common structural abnormality in Groups 3 and 4. Other chromosomal alteration identified in these subgroups includes gain of 7 and 18q and loss of 8 and 11q. The major difference

**25**

represented [27].

*Molecular Diagnostics and Pathology of Major Brain Tumors*

event driving Groups 3 and 4 tumor developments.

between these two groups is the enrichment of MYC amplification in Group 3, which is very rarely observed in Group 4, as well as in WNT and SHH. Another difference is the enrichment of chromosome X loss in Group 4, found in 80% female

The signaling pathway or biological programs driving the tumorigenesis of Groups 3 and 4 still remain largely unknown, although some reports suggest that disruption of chromatin genes associated with histone methylation may be a critical

**4.3 Medulloblastoma molecular subgroups: immunophenotypes and** 

After multigroup extensive researches, the development and validation of immunohistochemical stains to define molecular subgroups of MBs finally archived, with 4 immunohistochemical staining marker identified in order to MBs subgrouping, which can be used in FFPE tissue and greatly improve the routine pathological diagnosis process for these types of tumors. Four immunostaining markers were selected for pathological subgrouping of MBs: they are β-catenin,

Combined immunoreactivities for GAB1, filamin A, and YAP1, indicating a SHH profile, were found in 31% of MBs, including all desmoplastic tumors. Desmoplastic MBs constituted 54% of SHH pathway tumors, and classic and large-cell/anaplastic tumors contributed 29 and 17%, respectively. While non-desmoplastic SHH tumor generally showed widespread and strong immunoreactivities for GAB1, YAP1, and filamin A, all three types of desmoplastic tumors displayed stronger staining for these proteins within internodular regions. Immunoreactivities for filamin A and YAP1 in SHH tumors were always strong and generally widespread. This was not always the situation for GAB1 immunoreactivity; no more than weak cytoplasmic staining for GAB1 was seen in a few non-desmoplastic SHH tumors (n = 6). These tumors were all strongly immunopositive for filamin A and YAP1, which acted to

Antibodies to β-catenin for identifying WNT tumors effective on formalin-fixed and paraffin-embedded (FFPE) tissue are well established in the diagnostic laboratory [27]. Widespread intermediate or strong cytoplasmic β-catenin immunoreactivity was a feature of nearly all MBs in the series; very few showed only patchy weak cytoplasmic staining for this antigen. WNT pathway MBs were identified by nuclear, as well as cytoplasmic, immunoreactivity for β-catenin (**Figure 4**). WNT pathway MBs defined by these types of nuclear β-catenin immunoreactivity also express filamin A. Typically, this was patchy staining and less intense than that seen in SHH tumors. Strong and widespread nuclear immunoreactivity for YAP1 was also a feature of WNT pathway tumors. This distinctive combination of β-catenin, filamin A, and YAP1 immunoreactivities robustly confirmed the status of MBs in this molecular subgroup. WNT tumors contributed 14% of all MBs in this study. Nearly all WNT pathway MBs were classic tumors. Large-cell/anaplastic tumor (n = 2, 6%) was exceptional among WNT tumors, while desmoplastic MBs were not

*DOI: http://dx.doi.org/10.5772/intechopen.80856*

**histopathological associations**

GAB1, filamin A, and YAP1 [27].

confirm the SHH phenotype [27].

*4.3.2 WNT pathway MBs*

*4.3.1 SHH pathway MBs*

MBs in Group 4.

*Molecular Diagnostics and Pathology of Major Brain Tumors DOI: http://dx.doi.org/10.5772/intechopen.80856*

between these two groups is the enrichment of MYC amplification in Group 3, which is very rarely observed in Group 4, as well as in WNT and SHH. Another difference is the enrichment of chromosome X loss in Group 4, found in 80% female MBs in Group 4.

The signaling pathway or biological programs driving the tumorigenesis of Groups 3 and 4 still remain largely unknown, although some reports suggest that disruption of chromatin genes associated with histone methylation may be a critical event driving Groups 3 and 4 tumor developments.

#### **4.3 Medulloblastoma molecular subgroups: immunophenotypes and histopathological associations**

After multigroup extensive researches, the development and validation of immunohistochemical stains to define molecular subgroups of MBs finally archived, with 4 immunohistochemical staining marker identified in order to MBs subgrouping, which can be used in FFPE tissue and greatly improve the routine pathological diagnosis process for these types of tumors. Four immunostaining markers were selected for pathological subgrouping of MBs: they are β-catenin, GAB1, filamin A, and YAP1 [27].

#### *4.3.1 SHH pathway MBs*

*Primary Intracranial Tumors*

**4.2 Epidemiology**

mately 15 years [1].

eton organization, etc.

*4.2.1 Groups 3 and 4/non-WNT and non-SHH groups*

MBs and typically have classic histopathological features [1].

*4.1.3 MB, SHH subgroup, with TP53 wild type*

SHH-activated tumors account for approximately 30% of all MBs and originate from rhombic lip-derived cerebellar granule neuron precursors. SHH-activated and TP53-mutant MBs are rare and generally found in children aged 4–17 years. Clinical outcomes in patients with SHH-activated and TP53-mutant tumors are very poor [1].

SHH pathway activation in TP53 wild-type tumors can be associated with germline or somatic mutation in the negative regulations PTCH1 or SUFU, as well as activating somatic mutations in SMO or (rarely) amplification of GLI2. Desmoplastic/nodular MBs and MBs with extensive nodularity are always included in the SHH-activated group, but tumors with a SHH signaling pathway can also have a classic or large-cell/anaplastic morphology, particularly in older children. Patients with SHH-activated and TP53 wild-type MBs are generally children aged <4 years, adolescents, or young adults. In addition to genetic changes activating SHH signaling, mutations in DDX3X or KMT2D and amplification of MYCN or MYCL are sometimes seen, as are deletions of chromosomal arms 9q, 10q, and 14q.

Clinical outcomes in patients with SHH-activated tumors are variable [1].

Research data from 1973 to 2007 suggested MB incidence rates of 0.6 cases per 1 million children aged 1–9 years and 0.6 cases per 1 million adults aged >19 years. SHH-activated MBs in general show a bimodal age distribution, being most common in infants and young adults, with a male-female ratio of approximately 1.5:1. In contrast, SHH-activated and TP53-mutant tumors are generally found in children aged 4–17 years. In one study including 133 SHH-activated MBs, 28 patients (21%) had a TP53 mutation, and the median age of these patients was approxi-

Groups 3 and 4 MBs are usually called "the non-WNT/non-SHH groups." They share some of the similarities in both clinical presentation and molecular profiling. Most tumors in these groups display classical histology. The large-cell/anaplastic and desmoplastic histologies are present but at a lower frequency. The age of onset is distributed in both groups with most patients are children; they are relatively uncommon in infants and adults. Although both groups have similar frequency of metastasis, Group 3 shows poor prognosis, while Group 4 shows intermediate prognosis. Non-WNT and non-SHH tumors account for approximately 60% of all

One characteristic similarity between Groups 3 and 4 is both subgroups are enriched for expression of genes involved in photoreceptor differentiation, and they express high level of OTX2 and FOXG1B, well-known oncogenes of MB. However, Group 3 is distinguished by its enriched gene signatures functioning in cell cycle, protein biosynthesis, glutamate receptor signaling, and p38 mitogen-activated protein kinase (MMAPK) pathway, while Group 4 is overrepresented by genes involved in neuronal differentiation, development, cytoskel-

In addition, isochromosome 17q (I17q) represents the most common structural abnormality in Groups 3 and 4. Other chromosomal alteration identified in these subgroups includes gain of 7 and 18q and loss of 8 and 11q. The major difference

**24**

Combined immunoreactivities for GAB1, filamin A, and YAP1, indicating a SHH profile, were found in 31% of MBs, including all desmoplastic tumors. Desmoplastic MBs constituted 54% of SHH pathway tumors, and classic and large-cell/anaplastic tumors contributed 29 and 17%, respectively. While non-desmoplastic SHH tumor generally showed widespread and strong immunoreactivities for GAB1, YAP1, and filamin A, all three types of desmoplastic tumors displayed stronger staining for these proteins within internodular regions. Immunoreactivities for filamin A and YAP1 in SHH tumors were always strong and generally widespread. This was not always the situation for GAB1 immunoreactivity; no more than weak cytoplasmic staining for GAB1 was seen in a few non-desmoplastic SHH tumors (n = 6). These tumors were all strongly immunopositive for filamin A and YAP1, which acted to confirm the SHH phenotype [27].

#### *4.3.2 WNT pathway MBs*

Antibodies to β-catenin for identifying WNT tumors effective on formalin-fixed and paraffin-embedded (FFPE) tissue are well established in the diagnostic laboratory [27]. Widespread intermediate or strong cytoplasmic β-catenin immunoreactivity was a feature of nearly all MBs in the series; very few showed only patchy weak cytoplasmic staining for this antigen. WNT pathway MBs were identified by nuclear, as well as cytoplasmic, immunoreactivity for β-catenin (**Figure 4**). WNT pathway MBs defined by these types of nuclear β-catenin immunoreactivity also express filamin A. Typically, this was patchy staining and less intense than that seen in SHH tumors. Strong and widespread nuclear immunoreactivity for YAP1 was also a feature of WNT pathway tumors. This distinctive combination of β-catenin, filamin A, and YAP1 immunoreactivities robustly confirmed the status of MBs in this molecular subgroup. WNT tumors contributed 14% of all MBs in this study. Nearly all WNT pathway MBs were classic tumors. Large-cell/anaplastic tumor (n = 2, 6%) was exceptional among WNT tumors, while desmoplastic MBs were not represented [27].

#### **Figure 4.**

*β-Catenin IHC stain with both nuclear and cytoplasmic positive (A and C), GAB1 stain positive (B), YAP1 (D), and filamin A (E).*

#### *4.3.3 Non-SHH/WNT MBs*

MBs (N = 130, 55%) falling outside the SHH and WNT categories displayed cytoplasmic, but not nuclear, immunoreactivity for β-catenin. Tumor cells were negative for GAB1 and YAP1. In general, tumors in this category were also immunonegative for filamin A, though very weak and patch immunoreactivity for this antigen was evident in rare non-SHH/WNT MBs (n = 9), which were classified as such on the basis of the panel of immunoreactivities. Intrinsic vascular elements were immunopositive for YAP1 and filamin A, providing an internal control. This subgroup of MBs was dominated by classic tumors (92%), including all nondesmoplastic nodular tumors and all but one MB that contained small clusters of densely packed neurocytic cells, the exception being a WNT tumor. Large-cell/ anaplastic tumors made up the remainder (n = 11) [27].

#### *4.3.4 Metastatic MBs*

Despite four subgroups, metastatic MBs exist among all subgroups although the incidence of metastatic dissemination is higher in Group 3 and 4 than WNT and

**27**

**Lindau disease**

**Table 2.**

are often present [1].

*Molecular Diagnostics and Pathology of Major Brain Tumors*

**IHC marker IHC stain for MBs MBs subgrouping**

β-Catenin N+, C+, ¼ focal C+ C+ GAB1 Neg C+ Neg Filamin A C+ C+ Neg YAP1 N+, C+ N+, C+ Neg

*N+, nuclear staining positive; C+, cytoplasmic staining positive; SHH, Sonic Hedgehog.*

SHH [33]. Metastatic MBs occur in approximately 40% of all MBs at diagnosis and are associated with poorer prognosis [34]. In 2001, McDonald et al. [35] identified potential therapeutic targets, e.g., PDGFRα PDGFR for metastatic MBs using expression array analysis. However, Gilbertson and Clifford [36] found that the probe McDonald used for PDGFRα was PDGFRβ. They further demonstrated that PDGFRβ is overexpressed in metastatic MB. Then, Kohane and his co-workers did an interesting experiment and found that genomically, human MBs were closest to mouse P (postnatal) 1-P10 cerebella, and normal human cerebella were closest to mouse P30-P60. Metastatic human MBs were highly associated with mouse P5 cerebella (non-metastatic human MB with mouse P7 cerebella). PDGFRα is highly expressed in P5; PDGFRβ in P7 [37]. However, which isoform of PDGFRs plays a role in metastatic MBs kept controversial. Ten years later, we demonstrated that PDGFRα inhibits while PDGFRβ promotes MB cell proliferation and cell survival as well as cell invasion [38], highlighting that PDGFRβ may serve as a potential therapeutic target for metastatic MBs and warrants further investigation, including clinical studies.

**WNT SHH Non-WNT/non-SHH**

**Table 2** summarizes the IHC staining for subgroups of MBs.

supply, but slightly higher-grade nuclei mostly have small nucleoli.

inactivated both in VHL-associated cases and in most sporadic cases [1].

to chromosome 3p25 and includes three highly conserved exons [31].

**5. Hemangioblastoma, metastatic renal cell carcinoma, and von Hippel-**

Hemangioblastoma (HMB) is a benign, slow-growing, WHO grade I tumor, most likely occurs in cerebellum, brainstem, and spinal cord. Most hemangioblastomas are cystic on neuroimaging with intramural nodule. Histologically, the tumor has two major components, one is tightly packed capillary small vessels, and another is so-called stromal cells with low-grade nuclei, foamy cytoplasm, and no prominent nucleoli. Mitosis and necrosis are absent. But some degenerative features

On the other hand, cerebellum is a favorite location for metastatic renal cell carcinoma (RCC). Histologically, most RCC has clear cytoplasm with rich vascular

Due to the similarity in histology and the same preference location, 70% of HMBs occur in sporadic forms, while approximately 30% of HMBs are associated with the inherited von Hippel-Lindau disease. The VHL tumor suppressor gene is

Von Hippel-Lindau disease (VHL) is a familial disorder predisposing patients to cysts and hypervascular neoplasm of multiple organs, including the CNS, eye, kidneys, adrenal medulla, pancreas, inner ear/temporal bone, and epididymis. VHL is an autosomal dominant disorder, with roughly 20% of patients presenting as sporadic cases with no family history. The VHL tumor suppressor gene maps

*DOI: http://dx.doi.org/10.5772/intechopen.80856*

*Subgrouping MBs by immunohistochemical stains.*


#### **Table 2.**

*Primary Intracranial Tumors*

*4.3.3 Non-SHH/WNT MBs*

*(D), and filamin A (E).*

**Figure 4.**

*4.3.4 Metastatic MBs*

MBs (N = 130, 55%) falling outside the SHH and WNT categories displayed cytoplasmic, but not nuclear, immunoreactivity for β-catenin. Tumor cells were negative for GAB1 and YAP1. In general, tumors in this category were also immunonegative for filamin A, though very weak and patch immunoreactivity for this antigen was evident in rare non-SHH/WNT MBs (n = 9), which were classified as such on the basis of the panel of immunoreactivities. Intrinsic vascular elements were immunopositive for YAP1 and filamin A, providing an internal control. This subgroup of MBs was dominated by classic tumors (92%), including all nondesmoplastic nodular tumors and all but one MB that contained small clusters of densely packed neurocytic cells, the exception being a WNT tumor. Large-cell/

*β-Catenin IHC stain with both nuclear and cytoplasmic positive (A and C), GAB1 stain positive (B), YAP1* 

Despite four subgroups, metastatic MBs exist among all subgroups although the incidence of metastatic dissemination is higher in Group 3 and 4 than WNT and

anaplastic tumors made up the remainder (n = 11) [27].

**26**

*Subgrouping MBs by immunohistochemical stains.*

SHH [33]. Metastatic MBs occur in approximately 40% of all MBs at diagnosis and are associated with poorer prognosis [34]. In 2001, McDonald et al. [35] identified potential therapeutic targets, e.g., PDGFRα PDGFR for metastatic MBs using expression array analysis. However, Gilbertson and Clifford [36] found that the probe McDonald used for PDGFRα was PDGFRβ. They further demonstrated that PDGFRβ is overexpressed in metastatic MB. Then, Kohane and his co-workers did an interesting experiment and found that genomically, human MBs were closest to mouse P (postnatal) 1-P10 cerebella, and normal human cerebella were closest to mouse P30-P60. Metastatic human MBs were highly associated with mouse P5 cerebella (non-metastatic human MB with mouse P7 cerebella). PDGFRα is highly expressed in P5; PDGFRβ in P7 [37]. However, which isoform of PDGFRs plays a role in metastatic MBs kept controversial. Ten years later, we demonstrated that PDGFRα inhibits while PDGFRβ promotes MB cell proliferation and cell survival as well as cell invasion [38], highlighting that PDGFRβ may serve as a potential therapeutic target for metastatic MBs and warrants further investigation, including clinical studies.

**Table 2** summarizes the IHC staining for subgroups of MBs.

#### **5. Hemangioblastoma, metastatic renal cell carcinoma, and von Hippel-Lindau disease**

Hemangioblastoma (HMB) is a benign, slow-growing, WHO grade I tumor, most likely occurs in cerebellum, brainstem, and spinal cord. Most hemangioblastomas are cystic on neuroimaging with intramural nodule. Histologically, the tumor has two major components, one is tightly packed capillary small vessels, and another is so-called stromal cells with low-grade nuclei, foamy cytoplasm, and no prominent nucleoli. Mitosis and necrosis are absent. But some degenerative features are often present [1].

On the other hand, cerebellum is a favorite location for metastatic renal cell carcinoma (RCC). Histologically, most RCC has clear cytoplasm with rich vascular supply, but slightly higher-grade nuclei mostly have small nucleoli.

Due to the similarity in histology and the same preference location, 70% of HMBs occur in sporadic forms, while approximately 30% of HMBs are associated with the inherited von Hippel-Lindau disease. The VHL tumor suppressor gene is inactivated both in VHL-associated cases and in most sporadic cases [1].

Von Hippel-Lindau disease (VHL) is a familial disorder predisposing patients to cysts and hypervascular neoplasm of multiple organs, including the CNS, eye, kidneys, adrenal medulla, pancreas, inner ear/temporal bone, and epididymis.

VHL is an autosomal dominant disorder, with roughly 20% of patients presenting as sporadic cases with no family history. The VHL tumor suppressor gene maps to chromosome 3p25 and includes three highly conserved exons [31].

VHL-associated disease includes [31] the following:


Solitary and especially multiple HMBs are diagnostic hallmarks of VHL. Roughly 75% are infratentorial, mainly involving the cerebellum. The rest of them are found

#### **Figure 5.**

*Hemangioblastoma, H&E stain ×200, with low-grade nuclei and foamy stromal cells (A); metastatic RCC with clear cytoplasm, larger nuclei, and prominent nucleoli H*&*E stain ×400 (B); and RCC is immunoreactive for CD10 (C) and cytokeratin (D).*

**29**

*Molecular Diagnostics and Pathology of Major Brain Tumors*

thology of brain tumors, please refer to Ref. [39].

in the spinal cord, brainstem, and lumbosacral nerve roots. Supratentorial HMBs are extremely rare. Of interest, only about 25–30% of cerebellar HMBs are seen in

It is may be those two tumors share the same chromosome locus of 3p25; they have some histological overlapping as well as the same preference of anatomic location (cerebellum); HMB and metastatic RCC are two tumors almost always request differentiation diagnosis, since one is benign and another is malignant, both carry different prognoses, and this two tumors become "forever differential diagnosis" for most diagnostic neuropathologists. Luckily, a simple small panel of immunohistochemical (IHC) stain would easily resolve this puzzle. HMB is negative for cytokeratin but positive for inhibin and 2D40, while RCC will be positive for

Research work in the last two decades discovered lots of genetic alterations in human brain tumors. More work will be done to further facilitate the diagnosis and classification. A recent proposal is suggested by using the epigenomics, like methylation status, to enhance brain tumor classification [32]. A new clinical trial with medication focusing on the IDH1 mutation is underway now; as more and more research data collected, we believe more effective treatment options will be developed in the near future. For a more detailed review on the molecular neuropa-

VHL patients, whereas this fraction rises to 80% in the spinal cord [1].

cytokeratin, CD10, and PAX-8 and negative for inhibin [31] (**Figure 5**).

*DOI: http://dx.doi.org/10.5772/intechopen.80856*

**6. Summary**

#### *Molecular Diagnostics and Pathology of Major Brain Tumors DOI: http://dx.doi.org/10.5772/intechopen.80856*

in the spinal cord, brainstem, and lumbosacral nerve roots. Supratentorial HMBs are extremely rare. Of interest, only about 25–30% of cerebellar HMBs are seen in VHL patients, whereas this fraction rises to 80% in the spinal cord [1].

It is may be those two tumors share the same chromosome locus of 3p25; they have some histological overlapping as well as the same preference of anatomic location (cerebellum); HMB and metastatic RCC are two tumors almost always request differentiation diagnosis, since one is benign and another is malignant, both carry different prognoses, and this two tumors become "forever differential diagnosis" for most diagnostic neuropathologists. Luckily, a simple small panel of immunohistochemical (IHC) stain would easily resolve this puzzle. HMB is negative for cytokeratin but positive for inhibin and 2D40, while RCC will be positive for cytokeratin, CD10, and PAX-8 and negative for inhibin [31] (**Figure 5**).

#### **6. Summary**

*Primary Intracranial Tumors*

VHL-associated disease includes [31] the following:

• Retinal hemangioblastomas (40–60%)

• CNS hemangioblastomas (60–80%)

• Endolymphatic sac tumor (2–11%)

• Pancreatic cysts or islet tumors (60–80%)

• Papillary cystadenomas of the epididymis (20–60%)

Solitary and especially multiple HMBs are diagnostic hallmarks of VHL. Roughly 75% are infratentorial, mainly involving the cerebellum. The rest of them are found

*Hemangioblastoma, H&E stain ×200, with low-grade nuclei and foamy stromal cells (A); metastatic RCC with clear cytoplasm, larger nuclei, and prominent nucleoli H*&*E stain ×400 (B); and RCC is immunoreactive* 

• Pheochromocytomas (10–25%)

• Renal cysts and RCCs (30–60%)

**28**

**Figure 5.**

*for CD10 (C) and cytokeratin (D).*

Research work in the last two decades discovered lots of genetic alterations in human brain tumors. More work will be done to further facilitate the diagnosis and classification. A recent proposal is suggested by using the epigenomics, like methylation status, to enhance brain tumor classification [32]. A new clinical trial with medication focusing on the IDH1 mutation is underway now; as more and more research data collected, we believe more effective treatment options will be developed in the near future. For a more detailed review on the molecular neuropathology of brain tumors, please refer to Ref. [39].

*Primary Intracranial Tumors*

#### **Author details**

Frank Y. Shan1,2\*, E. Castro1 , Amelia Sybenga1 , Sanjib Mukherjee3 , Erxi Wu2 , Karming Fung4 , The Li5 , Ekokobe Fonkem2 , Jason H. Huang2 and A. Rao1

1 Department of Anatomic Pathology, Scott & White Medical Center, College of Medicine, Texas A&M University, Temple, TX, USA

2 Department of Neurosurgery, Scott & White Medical Center, College of Medicine, Texas A&M University, Temple, TX, USA

3 Vasicek Cancer Center, Scott & White Medical Center, College of Medicine, Texas A&M University, Temple, TX, USA

4 Department of Pathology, Oklahoma University Medical Center, Oklahoma City, OK, USA

5 Department of Pathology, Guangdong General Hospital, Guangzhou, Guangdong, China

\*Address all correspondence to: yshan918@gmail.com

© 2018 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.

**31**

*Molecular Diagnostics and Pathology of Major Brain Tumors*

Neuroscience Reports. 2013;**13**:345. DOI: 10.1007/s11910-013-0345-4

[10] Ozaki T, Nakagawara A. Role of p53 in cell death and human cancers. Cancers. 2011;**3**:994-1013. DOI: 10.3390/

[11] Yemelyanova A, Vang R, Kshirsagar M, et al. Immunohistochemical staining patterns of p53 can serve as a surrogate marker for TP53 mutations in ovarian carcinoma: An immunohistochemical and nucleotide sequencing analysis. Modern Pathology: An Official Journal of the United States and Canadian Academy of Pathology, Inc. 2011;**24**:1248-1253. DOI: 10.1038/

0432.CCR-09-2902

cancers3010994

modpathol.2011.85

[12] England B, Huang T, Karsy M. Current understanding of the role and targeting of tumor suppressor p53 in glioblastoma multiforme. Tumour Biology: The Journal of theInternational Society for Oncodevelopmental Biology and Medicine. 2013;**34**:2063-2074. DOI:

10.1007/s13277-013-0871-3

[13] Gielen GH, Gessi M, Hammes J, et al. H3F3A K27M mutation in pediatric CNS tumors: A marker for diffuse highgrade astrocytomas. American Journal of Clinical Pathology. 2013;**139**:345-349. DOI: 10.1309/AJCPABOHBC33FVMO

[14] Venneti S, Santi M, Felicella MM,

et al. A sensitive and specific

[9] van den Bent MJ, Dubbink HJ, Marie Y, et al. IDH1 and IDH2 mutations are prognostic but not predictive for outcome in anaplastic oligodendroglial tumors: A report of the European Organization for Research and Treatment of Cancer Brain Tumor Group. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research. 2010;**16**:1597-1604. DOI: 10.1158/1078-

*DOI: http://dx.doi.org/10.5772/intechopen.80856*

[1] Louis DN, Ohgaki H, Wiestler OD, Cavenee WK. WHO Classification of Tumours of the Central Nervous System. Revised 4th ed. Lyon: International Agency for Research on

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10.1016/j.humpath.2017.05.005

[3] Lieberman F. Glioblastoma update: Molecular biology, diagnosis, treatment, response assessment, and translational clinical trials. F1000 Research. 2017;**6**:1892. DOI: 10.12688/

[4] Ohgaki H, Kleihues P. Genetic pathways to primary and secondary glioblastoma. The American Journal of Pathology. 2007;**170**:1445-1453. DOI:

[5] Capper D, Zentgraf H, Balss J, et al. Monoclonal antibody specific for IDH1 R132H mutation. Acta Neuropathologica. 2009;**118**:599-601. DOI: 10.1007/s00401-009-0595-z

[6] Capper D, Weissert S, Balss J, et al. Characterization of R132H mutationspecific IDH1 antibody binding in brain tumors. Brain Pathology (Zurich, Switzerland). 2010;**20**:245-254. DOI: 10.1111/j.1750-3639.2009.00352.x

[7] Kato Y, Jin G, Kuan C-T, et al. A monoclonal antibody IMab-1

specifically recognizes IDH1R132H, the most common glioma-derived mutation. Biochemical and Biophysical Research Communications. 2009;**390**:547-551. DOI: 10.1016/j.bbrc.2009.10.001

[8] Cohen AL, Holmen SL, Colman H. IDH1 and IDH2 mutations in gliomas. Current Neurology and

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Cancer; 2016

*Molecular Diagnostics and Pathology of Major Brain Tumors DOI: http://dx.doi.org/10.5772/intechopen.80856*

#### **References**

*Primary Intracranial Tumors*

**Author details**

Karming Fung4

Frank Y. Shan1,2\*, E. Castro1

, The Li5

A&M University, Temple, TX, USA

Oklahoma City, OK, USA

Guangdong, China

Medicine, Texas A&M University, Temple, TX, USA

Medicine, Texas A&M University, Temple, TX, USA

\*Address all correspondence to: yshan918@gmail.com

**30**

provided the original work is properly cited.

© 2018 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,

, Amelia Sybenga1

2 Department of Neurosurgery, Scott & White Medical Center, College of

1 Department of Anatomic Pathology, Scott & White Medical Center, College of

3 Vasicek Cancer Center, Scott & White Medical Center, College of Medicine, Texas

, Ekokobe Fonkem2

4 Department of Pathology, Oklahoma University Medical Center,

5 Department of Pathology, Guangdong General Hospital, Guangzhou,

, Sanjib Mukherjee3

, Jason H. Huang2

, Erxi Wu2

and A. Rao1

,

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10.1017/cjn.2016.420

**32**

[30] Thompson MC, Fuller C, Hogg TL, Dalton J, Finklestein D, Lau CC, et al. Genomic identifies medulloblastoma subgroups that are enriched for specific genetic alterations. Journal of Clinical Oncology. 2006;**24**:1924-1931

[31] Perry A, Brat DJ. Practical Surgical Neuropathology. Philadelphia, PA, USA: Churchill Livingston/Elsevier; 2010. ISBN: 978-0-443-06982-6

[32] Capper D, Jones DTW, Still M, et al. DNA methylation-based classification of central nervous system tumors. Nature. 2018, 2018;**555**:469-474. DOI: 10.1038/nature26000

[33] Zapotocky M, Mata-Mbemba D, Sumerauer D, Liby P, Lassaletta A, Zamecnik J, et al. Differential patterns of metastatic dissemination across medulloblastoma subgroups. Journal of Neurosurgery. Pediatrics. 2018;**21**(2):145-152. DOI: 10.3171/2017.8.PEDS17264. Epub: December 8, 2017

[34] Miranda Kuzan-Fischer C, Juraschka K, Taylor MD. Medulloblastoma in the Molecular Era. Journal of Korean Neurosurgical Association. 2018;**61**(3):292-301. DOI: 10.3340/jkns.2018.0028. Epub: May 1, 2018

[35] MacDonald TJ, Brown KM, LaFleur B, Peterson K, Lawlor C, Chen Y, et al. Expression profiling of medulloblastoma: PDGFRA and the RAS/MAPK pathway as therapeutic targets for metastatic disease. Nat Genet. 2001;**29**(2):143-52. Erratum. Nature Genetics. 2003;**35**(3):287

[36] Gilbertson RJ, Clifford SC. PDGFRB is overexpressed in metastatic medulloblastoma. Nature Genetics. 2003;**35**(3):197-198

[37] Kho AT, Zhao Q , Cai Z, Butte AJ, Kim JY, Pomeroy SL, et al. Conserved mechanisms across development and tumorigenesis revealed by a mouse development perspective of human cancers. Genes & Development. 2004;**18**(6):629-640

[38] Wang F, Remke M, Bhat K, Wong ET, Zhou S, Ramaswamy V, et al. A microRNA-1280/JAG2 network comprises a novel biological target in high-risk medulloblastoma. Oncotarget. 2015;**6**(5):2709-2724

[39] Velazquez VJE, Brat DJ. Incorporating advances in molecular pathology into brain tumor diagnostics. Advances in Anatomic Pathology. 2018;**25**(3):143-171

**35**

**Chapter 3**

**Abstract**

oncogenic processes.

**1. Introduction**

Astrocytoma

*Miguel Velázquez-Flores*

Potential Use of Long Noncoding

Noncoding RNAs represent a high proportion of the human genome and regulate gene expression by means of innumerable and unimaginable modes of action. Particularly, long noncoding RNAs have emerged as central regulators of gene expression and alterations on their function have been associated with many types of cancer, such as astrocytomas. Astrocytomas are the most common type of gliomas in the central nervous system, and glioblastoma multiforme is their most aggressive form. Although adult and pediatric astrocytomas exhibit certain molecular similarities, they are considered as distinct molecular entities. Since to date there is no effective treatments for these tumors, different efforts are being made to find molecular tools useful for this purpose. Studies have shown that both tumor and circulating expression of lncRNAs were altered in astrocytoma, which was useful to distinguish the patients with this neoplasia from those without cancer, as well as to determine different prognostic factors related to the disease. According to these studies, different "molecular signatures" of specific lncRNAs were established, and they have a potential use in the medical practice. From a system biological perspective, complex interaction networks, conformed by lncRNAs, microRNAs, mRNAs, and proteins, were elucidated and predicted to control many

**Keywords:** astrocytoma, biomarker, interacting network, lncRNA, microRNA

Noncoding RNAs (ncRNAs) represent a significant fraction of the human genome [1], and the great diversity and forms of action of these RNA species has put them at the center of biomedical research of diseases, such as cancer [2–4]. LncRNAs are not the exception, and many of them have been proposed as possible diagnostic and prognostic biomarkers for Ast [5]. LncRNAs are RNAs of more than >200 nucleotides in length, which have to meet certain additional criteria to be classified within this category [6]. The evolutionary conservation of lncRNAs among species is poor [7], and they are transcribed by a variety

RNAs as Biomarkers for

*Gerardo Sánchez, Griselda Ramírez and* 

*Ruth Ruiz Esparza-Garrido, Alicia Siordia-Reyes,* 

#### **Chapter 3**

## Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma

*Ruth Ruiz Esparza-Garrido, Alicia Siordia-Reyes, Gerardo Sánchez, Griselda Ramírez and Miguel Velázquez-Flores*

### **Abstract**

Noncoding RNAs represent a high proportion of the human genome and regulate gene expression by means of innumerable and unimaginable modes of action. Particularly, long noncoding RNAs have emerged as central regulators of gene expression and alterations on their function have been associated with many types of cancer, such as astrocytomas. Astrocytomas are the most common type of gliomas in the central nervous system, and glioblastoma multiforme is their most aggressive form. Although adult and pediatric astrocytomas exhibit certain molecular similarities, they are considered as distinct molecular entities. Since to date there is no effective treatments for these tumors, different efforts are being made to find molecular tools useful for this purpose. Studies have shown that both tumor and circulating expression of lncRNAs were altered in astrocytoma, which was useful to distinguish the patients with this neoplasia from those without cancer, as well as to determine different prognostic factors related to the disease. According to these studies, different "molecular signatures" of specific lncRNAs were established, and they have a potential use in the medical practice. From a system biological perspective, complex interaction networks, conformed by lncRNAs, microRNAs, mRNAs, and proteins, were elucidated and predicted to control many oncogenic processes.

**Keywords:** astrocytoma, biomarker, interacting network, lncRNA, microRNA

#### **1. Introduction**

Noncoding RNAs (ncRNAs) represent a significant fraction of the human genome [1], and the great diversity and forms of action of these RNA species has put them at the center of biomedical research of diseases, such as cancer [2–4]. LncRNAs are not the exception, and many of them have been proposed as possible diagnostic and prognostic biomarkers for Ast [5]. LncRNAs are RNAs of more than >200 nucleotides in length, which have to meet certain additional criteria to be classified within this category [6]. The evolutionary conservation of lncRNAs among species is poor [7], and they are transcribed by a variety

of transcriptional mechanisms [6]. Many cellular processes are regulated by lncRNAs and this could be at both cytoplasmic and nuclear levels, as well as distance by moving them to their target tissues through different bodily fluids, such as blood [8]. LncRNAs exert their functions by establishing interactions with other lncRNA and RNA species, as well as with proteins [9], and changes on their functioning have been associated with cancer and particularly with astrocytomas (Ast) [5]**.**

Gliomas represent 81% of the Central Nervous System (CNS) tumors of which the most common subtypes in adults are glioblastoma multiforme (GBM), anaplastic Ast (AAst), and oligodendrogliomas [10, 11]. In the pediatric counterpart, pilocytic Ast (PAst) is the most common type in pediatric age [11]. According to the new classification of the World Health Organization (WHO), Ast are now classified according to the presence or absence of *IDH1*/*IDH2* mutations, as well as by phenotypic traits and integral diagnoses [12]. Those tumors with *IDH1*/*IDH2* mutations were classified as "diffuse gliomas," a new group that includes diffuse Ast (DAst; Grade II), AAst (Grade III), GBM, and diffuse oligodendrogliomas (Grade I and II) [12]. Meanwhile, pilocytic Ast (PAst; Grade I), subependymal giant cells Ast (Grade I), and pleomorphic xanthoastrocytoma (Grade II) were excluded from the diffuse group, given that they do not have these mutations [12]. Although there are certain molecular similarities between adult and pediatric Ast (p-Ast) [13], their molecular differences are well established and based on this, they are classified as different tumor subtypes [14–17]. Although there have been advances in the Ast study—mainly on adult GBM—, to date, there are very few molecular tools useful for Ast diagnosis, prognosis, and treatment. Essentially, most studies have identified changes on the expression of lncRNAs in both tumor tissues and GBM cell lines, and according to this, some "molecular signatures" have been postulated for the diagnosis and prognosis of Ast. For instance, circulating lncRNAs have allowed the distinction of patients sensitive or resistant to treatments, specifically to temozolomide (TMZ) or radiotherapy [18, 19]. In addition, the establishment of bioinformatic algorithms identified interactome networks in which lncRNAs physically interact with other lncRNAs, as well as with messenger RNAs (mRNAs) and microRNAs (miRNAs), and proteins. These studies have shown that expression changes of lncRNAs could lead to the amplification of the aberrant signals, which in turn could lead to alterations of many signaling pathways and cellular processes [5, 20, 21]. In p-Ast, high expression levels of LINC-ROR (long intergenic nonprotein coding RNA, regulator of reprogramming) were useful to distinguish p-Ast from the control, as well as to identify the GBM from the rest of the Ast grades; this strongly suggests the involvement of LINC-ROR in p-Ast diagnosis and prognosis [5].

#### **2. Astrocytoma**

Although the new WHO classification of tumors of the CNS takes into account phenotypic traits, it also takes into account other criteria, such as the genotype and integral diagnoses of the disease [12]. According to this classification, Ast are now classified mainly by the presence or absence of *IDH1*/*IDH2* mutations and based on this, diffuse Ast (DAst; Grade II) and anaplastic Ast (AAst; Grade III), as well as the GBM, and diffuse oligodendrogliomas (Grade I and II) were classified as "diffuse gliomas" [12]. PAst, subependymal giant cells Ast, and pleomorphic xanthoastrocytoma (Grade II) were classified in a different group, because of the absence of *IDH1*/*IDH2* mutations.

**37**

**Table 1.**

*Astrocytoma classification according to the World Health Organization (2016).*

*Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma*

These tumors have a well circumscribed growth pattern, lack IDH alterations, and they frequently have *BRAF* (pilocytic Ast (Past) and pleomorphic xanthoastrocytoma) and *TSC1*/*TSC2* mutations (subependymal giant cells Ast) (**Table 1**).

PAst are the most common type of Ast in pediatric age and are characterized by their biphasic pattern: compact bipolar cells with Rosenthal fibers, microquistes, and granular bodies (**Figure 1A**). As a general rule, PAst are well-defined tumors

**2.1 Astrocytomas that lack IDH1 and IDH2 mutations**

*DOI: http://dx.doi.org/10.5772/intechopen.80561*

#### **2.1 Astrocytomas that lack IDH1 and IDH2 mutations**

*Primary Intracranial Tumors*

(Ast) [5]**.**

of transcriptional mechanisms [6]. Many cellular processes are regulated by lncRNAs and this could be at both cytoplasmic and nuclear levels, as well as distance by moving them to their target tissues through different bodily fluids, such as blood [8]. LncRNAs exert their functions by establishing interactions with other lncRNA and RNA species, as well as with proteins [9], and changes on their functioning have been associated with cancer and particularly with astrocytomas

Gliomas represent 81% of the Central Nervous System (CNS) tumors of which the most common subtypes in adults are glioblastoma multiforme (GBM), anaplastic Ast (AAst), and oligodendrogliomas [10, 11]. In the pediatric counterpart, pilocytic Ast (PAst) is the most common type in pediatric age [11]. According to the new classification of the World Health Organization (WHO), Ast are now classified according to the presence or absence of *IDH1*/*IDH2* mutations, as well as by phenotypic traits and integral diagnoses [12]. Those tumors with *IDH1*/*IDH2* mutations were classified as "diffuse gliomas," a new group that includes diffuse Ast (DAst; Grade II), AAst (Grade III), GBM, and diffuse oligodendrogliomas (Grade I and II) [12]. Meanwhile, pilocytic Ast (PAst; Grade I), subependymal giant cells Ast (Grade I), and pleomorphic xanthoastrocytoma (Grade II) were excluded from the diffuse group, given that they do not have these mutations [12]. Although there are certain molecular similarities between adult and pediatric Ast (p-Ast) [13], their molecular differences are well established and based on this, they are classified as different tumor subtypes [14–17]. Although there have been advances in the Ast study—mainly on adult GBM—, to date, there are very few molecular tools useful for Ast diagnosis, prognosis, and treatment. Essentially, most studies have identified changes on the expression of lncRNAs in both tumor tissues and GBM cell lines, and according to this, some "molecular signatures" have been postulated for the diagnosis and prognosis of Ast. For instance, circulating lncRNAs have allowed the distinction of patients sensitive or resistant to treatments, specifically to temozolomide (TMZ) or radiotherapy [18, 19]. In addition, the establishment of bioinformatic algorithms identified interactome networks in which lncRNAs physically interact with other lncRNAs, as well as with messenger RNAs (mRNAs) and microRNAs (miRNAs), and proteins. These studies have shown that expression changes of lncRNAs could lead to the amplification of the aberrant signals, which in turn could lead to alterations of many signaling pathways and cellular processes [5, 20, 21]. In p-Ast, high expression levels of LINC-ROR (long intergenic nonprotein coding RNA, regulator of reprogramming) were useful to distinguish p-Ast from the control, as well as to identify the GBM from the rest of the Ast grades; this strongly suggests the involvement of LINC-ROR in p-Ast diagnosis and

Although the new WHO classification of tumors of the CNS takes into account phenotypic traits, it also takes into account other criteria, such as the genotype and integral diagnoses of the disease [12]. According to this classification, Ast are now classified mainly by the presence or absence of *IDH1*/*IDH2* mutations and based on this, diffuse Ast (DAst; Grade II) and anaplastic Ast (AAst; Grade III), as well as the GBM, and diffuse oligodendrogliomas (Grade I and II) were classified as "diffuse gliomas" [12]. PAst, subependymal giant cells Ast, and pleomorphic xanthoastrocytoma (Grade II) were classified in a different group, because of the absence of

**36**

prognosis [5].

**2. Astrocytoma**

*IDH1*/*IDH2* mutations.

These tumors have a well circumscribed growth pattern, lack IDH alterations, and they frequently have *BRAF* (pilocytic Ast (Past) and pleomorphic xanthoastrocytoma) and *TSC1*/*TSC2* mutations (subependymal giant cells Ast) (**Table 1**).

PAst are the most common type of Ast in pediatric age and are characterized by their biphasic pattern: compact bipolar cells with Rosenthal fibers, microquistes, and granular bodies (**Figure 1A**). As a general rule, PAst are well-defined tumors

#### **Table 1.**

*Astrocytoma classification according to the World Health Organization (2016).*

#### **Figure 1.**

*(A) Pilocytic Ast. Photomicrograph that shows a glioma with astrocytes, which have an elongated cytoplasm and a pilocytic appearance, on a loose stroma (H&E, 40×). (B) Diffuse astrocytoma. Diffuse glioma with cysts and solid areas; the cells are homogenous and do not exhibit atypia (H&E, 40×). (C) Anaplastic astrocytoma. Neoplasia with hypercellularity and nuclear pleomorphism and hyperchromatism. In the upper left, a blood vessel with a glomerular pattern can be seen (H&E, 40×). (D) GBM. Hypercellular glial tumor with diffuse pleomorphism and necrosis; it is delimitated by palisaded cells, which are characteristic of GBM (H&E, 10×). Photomicrographs taken at the pediatric Pathology Service of the Children's Hospital, National Medical Center Century XXI, IMSS.*

(**Figure 2A**); so they can be surgically resected without causing damage to the adjacent tissue and they do not progress to more aggressive stages; therefore, PAst are considered as neoplasms of good prognosis.

PAst are developed along the neuroaxis, and they are preferably located in the cerebellum [22–24]. It is important to mention that there are genetic diseases such as neurofibromatosis 1 (*NF-1*), which influences the formation of PAst; approximately 15% of individuals with *NF-1* develop these type of tumors, specifically at the level of the optic nerve [25, 26].

#### **2.2 Diffuse gliomas (tumors with IDH1 and IDH2 mutations)**

In the previous WHO classification, diffuse Ast (DAst) were classified as an independent group, but now they are classified along with anaplastic Ast (Aast; Grade III) and glioblastoma (GBM; Grade IV) (**Figures 1B–D** and **2B–D**), as well as with diffuse oligodendrogliomas (Grade I and II) [12]. Although factors such as growth and tumor behavior are still taken into account, the feature that distinguishes them as diffuse gliomas are the *IDH1* and *IDH2* mutations; however, these tumors can be subclassified into the *IDH*-mutant, *IDH*-wildtype, and NOS categories [12].

*IDH*-wildtype neoplasms constitute a subgroup of uncommon tumors, which are negative for mutant R132H *IDH1* protein and genic mutations for *IDH1* (codon 132) and *IDH2* (codon 172). Importantly, DAst (WHO Grade II) and AAst (WHO Grade III) can be confused with gangliogliomas and *IDH*-wildtype GBM [27, 28].

**39**

*2.2.1 Glioblastoma*

*calcifications are rare.*

**Figure 2.**

there are different GBM variants (**Table 1**).

*Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma*

Tumors that do not have any of these molecular tests—immunohistochemistry or sequencing—are subclassified as DAst-NOS or AAst-NOS, respectively [12].

*(A and B) Pilocytic and diffuse astrocytoma. These tumors are both isodense and hypodense to the brain and show calcifications in 15–20% of cases, and they have virtually no edema. (C) Anaplastic astrocytoma is poorly defined lesions with heterogenous signal strengths. Mixed areas of isodensity to hipodensity are observed; these tumors may have hemorrhagic foci. It is common to observe a hypertensive central nucleus, which is surrounded by an intense edge with peripheral finger-like projections and secondary to a vasogenic edema. (D) GBM. Heterogenous lesion with cellular components of mixed signal, central necrosis, and hemorrhage;* 

According to the new WHO classification, the GBM was also classified into the group of diffuse gliomas and subclassified into the *IDH*-mutant or *IDH*-wildtype categories, or the NOS category. The *IDH*-wildtype form represents ~90% of cases and was associated with primary GBM (*de novo*), which are more common in patients older than 55 years of age [29]. Meanwhile, the *IDH*-mutant GBMs (~10%) are tumors that develop from low-grade diffuse glioma and are commonly present in younger patients; this type of GBM are also known as secondary GBM [29]. Similar to that described above, GBM-NOS are those tumors that do not have a full *IDH* evaluation [12]. According to phenotypic traits and the genetic background, to date,

*DOI: http://dx.doi.org/10.5772/intechopen.80561*

#### **Figure 2.**

*Primary Intracranial Tumors*

(**Figure 2A**); so they can be surgically resected without causing damage to the adjacent tissue and they do not progress to more aggressive stages; therefore, PAst

*(A) Pilocytic Ast. Photomicrograph that shows a glioma with astrocytes, which have an elongated cytoplasm and a pilocytic appearance, on a loose stroma (H&E, 40×). (B) Diffuse astrocytoma. Diffuse glioma with cysts and solid areas; the cells are homogenous and do not exhibit atypia (H&E, 40×). (C) Anaplastic astrocytoma. Neoplasia with hypercellularity and nuclear pleomorphism and hyperchromatism. In the upper left, a blood vessel with a glomerular pattern can be seen (H&E, 40×). (D) GBM. Hypercellular glial tumor with diffuse pleomorphism and necrosis; it is delimitated by palisaded cells, which are characteristic of GBM (H&E, 10×). Photomicrographs taken at the pediatric Pathology Service of the Children's Hospital, National Medical Center* 

PAst are developed along the neuroaxis, and they are preferably located in the cerebellum [22–24]. It is important to mention that there are genetic diseases such as neurofibromatosis 1 (*NF-1*), which influences the formation of PAst; approximately 15% of individuals with *NF-1* develop these type of tumors, specifically at the level

In the previous WHO classification, diffuse Ast (DAst) were classified as an independent group, but now they are classified along with anaplastic Ast (Aast; Grade III) and glioblastoma (GBM; Grade IV) (**Figures 1B–D** and **2B–D**), as well as with diffuse oligodendrogliomas (Grade I and II) [12]. Although factors such as growth and tumor behavior are still taken into account, the feature that distinguishes them as diffuse gliomas are the *IDH1* and *IDH2* mutations; however, these tumors can be subclassified into the *IDH*-mutant, *IDH*-wildtype, and NOS catego-

*IDH*-wildtype neoplasms constitute a subgroup of uncommon tumors, which are negative for mutant R132H *IDH1* protein and genic mutations for *IDH1* (codon 132) and *IDH2* (codon 172). Importantly, DAst (WHO Grade II) and AAst (WHO Grade III) can be confused with gangliogliomas and *IDH*-wildtype GBM [27, 28].

are considered as neoplasms of good prognosis.

**2.2 Diffuse gliomas (tumors with IDH1 and IDH2 mutations)**

of the optic nerve [25, 26].

**38**

ries [12].

**Figure 1.**

*Century XXI, IMSS.*

*(A and B) Pilocytic and diffuse astrocytoma. These tumors are both isodense and hypodense to the brain and show calcifications in 15–20% of cases, and they have virtually no edema. (C) Anaplastic astrocytoma is poorly defined lesions with heterogenous signal strengths. Mixed areas of isodensity to hipodensity are observed; these tumors may have hemorrhagic foci. It is common to observe a hypertensive central nucleus, which is surrounded by an intense edge with peripheral finger-like projections and secondary to a vasogenic edema. (D) GBM. Heterogenous lesion with cellular components of mixed signal, central necrosis, and hemorrhage; calcifications are rare.*

Tumors that do not have any of these molecular tests—immunohistochemistry or sequencing—are subclassified as DAst-NOS or AAst-NOS, respectively [12].

#### *2.2.1 Glioblastoma*

According to the new WHO classification, the GBM was also classified into the group of diffuse gliomas and subclassified into the *IDH*-mutant or *IDH*-wildtype categories, or the NOS category. The *IDH*-wildtype form represents ~90% of cases and was associated with primary GBM (*de novo*), which are more common in patients older than 55 years of age [29]. Meanwhile, the *IDH*-mutant GBMs (~10%) are tumors that develop from low-grade diffuse glioma and are commonly present in younger patients; this type of GBM are also known as secondary GBM [29]. Similar to that described above, GBM-NOS are those tumors that do not have a full *IDH* evaluation [12]. According to phenotypic traits and the genetic background, to date, there are different GBM variants (**Table 1**).

#### **2.3 Pediatric diffuse gliomas**

Pediatric diffuse gliomas have the K27 mutation in the gene *H3F3A* (H3 Histone Family Member 3A) and less commonly in the related gene *HIST1H3B* (Histone Cluster 1 H3 Family Member B). Although they are mainly present in children, they can also be present in adults. These tumors exhibit a diffuse growth pattern and a midline location: thalamus, brain stem, and spinal cord; therefore, they are classified as diffuse midline glioma, H3 K27 mutant, and include tumors previously known as diffuse intrinsic pontine glioma (DIPG) [12].

#### **3. LncRNAs in astrocytoma**

LncRNAs have emerged as important molecular elements in different types of cancer, and Ast are not the exception [5, 30–32]. To date, diverse studies have shown the high complexity of the lncRNA study in Ast, due to the wide variety of mechanisms by which lncRNAs exerts their biological actions and because of the high tumor heterogeneity [33–35]. Changes in the nucleotide sequence of lncRNAs, their transcription rate, the expression of specific variants, in their expression levels, among others, could lead to an aberrant amplification of cell signals [36–38]. Given that GBM is the most aggressive type of cancer that begins within the brain [39, 40], most studies have been focused on this tumor subtype and to a lesser extent in the other WHO grades of adult Ast or in all WHO grades of p-Ast. Despite the significant effort that has been made in recent years to learn more about Ast, to the best of our knowledge, to date, there are very few molecular tools really applicable to diagnose, prognose, or treatment of these tumors [41–43]. Therefore, there is great interest to establish these molecular tools for GBM and evidence indicates that lncRNAs seem to be good candidates to serve such purpose.

#### **3.1 LncRNAs as potential astrocytoma biomarkers**

Expression changes of a biomolecule are a powerful tool to establish molecular "signatures" or "fingerprints" useful to distinguish and identify subgroups of a disease with a particular clinical behavior [44–47]. In this sense, expression changes of lncRNAs have been useful to differentiate both adult and pediatric Ast from nonneoplastic tissues, and some of them have the potential to be used in the medical practice as biomarkers. The meta-analysis performed by Zhang et al. [48] demonstrated for the first time the usefulness of the lncRNAs aberrantly expressed for Ast diagnosis and prognosis. This study showed that the expression profile of lncRNAs allowed to differentiate Ast or oligodendrogliomas from nonneoplastic tissues and to associate it with Ast malignancy or with lineage distinction in gliomas (**Table 2**). Subsequently, the same group established the first "molecular signature" of lncRNAs for Ast diagnosis and prognosis, which distinguished this neoplasia from nonneoplastic tissues, as well as Ast malignancy or patient's survival (**Table 2**) [49]. Additionally, a second group of precise lncRNAs was specific for Ast, and it was functional to differentiate them from the control tissues; from this signature, two lncRNAs were also associated with Ast malignancy, since their expression distinguished Ast WHO grades (**Table 2**) [50]. However, none of the lncRNAs that were part of the first molecular signature was established in the second, which could be related to the samples included in each study—referring to age, sex, with or without treatment, radiotherapy, among others—, as well as to the bioinformatic approach used in each study. This evidence emphasizes the importance that has the

**41**

**Table 2.**

*Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma*

homogenization of patient's samples included in a study has and how crucial it is to

In addition to changes in the lncRNA expression, their promoter methylation status seems to be useful for Ast diagnosis and prognosis. Specifically, it was shown that expression and the promoter methylation pattern of *LOC285758* (Long Intergenic Non-Protein Coding RNA 1268) differentiate all Ast WHO grades and other gliomas (oligodendroglioma and oligoastrocitoma (II–III)) from the control, as well as Ast grades I–III from both primary and secondary GBM (**Table 2**) [32]. Based on this evidence, the identification of the mechanisms that lead to an aberrant expression of the lncRNAs—such as epigenetic regulation—could be part of

Specifically for GBM, different lncRNAs have also been found as potential biomarkers for its diagnosis and prognosis (**Table 3**). In this sense, Xu et al. [51] identified lncRNAs, which were associated with patient's survival; particularly, high expression of *SNHG1* (small nucleolar RNA host gene 1) was related with poor prognosis (**Table 3**). Meanwhile, *in silico* analysis showed many differentially expressed lncRNAs useful to distinguish GBM from nonneoplastic tissues and each

specify the clinic features of the included patients.

*Molecular signatures of lncRNAs in astrocytoma.*

the biomarkers package useful in the medical practice.

**3.2 LncRNAs as potential GBM biomarkers**

*DOI: http://dx.doi.org/10.5772/intechopen.80561*


#### **Table 2.**

*Primary Intracranial Tumors*

**2.3 Pediatric diffuse gliomas**

**3. LncRNAs in astrocytoma**

known as diffuse intrinsic pontine glioma (DIPG) [12].

that lncRNAs seem to be good candidates to serve such purpose.

**3.1 LncRNAs as potential astrocytoma biomarkers**

Pediatric diffuse gliomas have the K27 mutation in the gene *H3F3A* (H3 Histone Family Member 3A) and less commonly in the related gene *HIST1H3B* (Histone Cluster 1 H3 Family Member B). Although they are mainly present in children, they can also be present in adults. These tumors exhibit a diffuse growth pattern and a midline location: thalamus, brain stem, and spinal cord; therefore, they are classified as diffuse midline glioma, H3 K27 mutant, and include tumors previously

LncRNAs have emerged as important molecular elements in different types of cancer, and Ast are not the exception [5, 30–32]. To date, diverse studies have shown the high complexity of the lncRNA study in Ast, due to the wide variety of mechanisms by which lncRNAs exerts their biological actions and because of the high tumor heterogeneity [33–35]. Changes in the nucleotide sequence of lncRNAs, their transcription rate, the expression of specific variants, in their expression levels, among others, could lead to an aberrant amplification of cell signals [36–38]. Given that GBM is the most aggressive type of cancer that begins within the brain [39, 40], most studies have been focused on this tumor subtype and to a lesser extent in the other WHO grades of adult Ast or in all WHO grades of p-Ast. Despite the significant effort that has been made in recent years to learn more about Ast, to the best of our knowledge, to date, there are very few molecular tools really applicable to diagnose, prognose, or treatment of these tumors [41–43]. Therefore, there is great interest to establish these molecular tools for GBM and evidence indicates

Expression changes of a biomolecule are a powerful tool to establish molecular "signatures" or "fingerprints" useful to distinguish and identify subgroups of a disease with a particular clinical behavior [44–47]. In this sense, expression changes of lncRNAs have been useful to differentiate both adult and pediatric Ast from nonneoplastic tissues, and some of them have the potential to be used in the medical practice as biomarkers. The meta-analysis performed by Zhang et al. [48] demonstrated for the first time the usefulness of the lncRNAs aberrantly expressed for Ast diagnosis and prognosis. This study showed that the expression profile of lncRNAs allowed to differentiate Ast or oligodendrogliomas from nonneoplastic tissues and to associate it with Ast malignancy or with lineage distinction in gliomas (**Table 2**).

Subsequently, the same group established the first "molecular signature" of lncRNAs for Ast diagnosis and prognosis, which distinguished this neoplasia from nonneoplastic tissues, as well as Ast malignancy or patient's survival (**Table 2**) [49]. Additionally, a second group of precise lncRNAs was specific for Ast, and it was functional to differentiate them from the control tissues; from this signature, two lncRNAs were also associated with Ast malignancy, since their expression distinguished Ast WHO grades (**Table 2**) [50]. However, none of the lncRNAs that were part of the first molecular signature was established in the second, which could be related to the samples included in each study—referring to age, sex, with or without treatment, radiotherapy, among others—, as well as to the bioinformatic approach used in each study. This evidence emphasizes the importance that has the

**40**

*Molecular signatures of lncRNAs in astrocytoma.*

homogenization of patient's samples included in a study has and how crucial it is to specify the clinic features of the included patients.

In addition to changes in the lncRNA expression, their promoter methylation status seems to be useful for Ast diagnosis and prognosis. Specifically, it was shown that expression and the promoter methylation pattern of *LOC285758* (Long Intergenic Non-Protein Coding RNA 1268) differentiate all Ast WHO grades and other gliomas (oligodendroglioma and oligoastrocitoma (II–III)) from the control, as well as Ast grades I–III from both primary and secondary GBM (**Table 2**) [32]. Based on this evidence, the identification of the mechanisms that lead to an aberrant expression of the lncRNAs—such as epigenetic regulation—could be part of the biomarkers package useful in the medical practice.

#### **3.2 LncRNAs as potential GBM biomarkers**

Specifically for GBM, different lncRNAs have also been found as potential biomarkers for its diagnosis and prognosis (**Table 3**). In this sense, Xu et al. [51] identified lncRNAs, which were associated with patient's survival; particularly, high expression of *SNHG1* (small nucleolar RNA host gene 1) was related with poor prognosis (**Table 3**). Meanwhile, *in silico* analysis showed many differentially expressed lncRNAs useful to distinguish GBM from nonneoplastic tissues and each


#### **Table 3.**

*LncRNAs as potential GBM biomarkers.*

of the four GBM subtypes: classical, mesenchymal, neural, and proneural. The lncRNAs *CRNDE* (colorectal neoplasia differentially expressed) and *CYTOR* (cytoskeleton regulator RNA) (both upregulated) and *TUNAR* (TCL1 upstream neural differentiation-associated RNA) and *LINCO1476* (both downregulated) were those with the highest expression changes in GBM compared to the control and with a potential use for GBM diagnosis (**Table 3**) [52]. *CRNDE* overexpression has been associated with high cell proliferation, migration, and invasion, which corresponds with the promotion of tumor growth observed in *in vivo* studies [53]. In addition, the expression pattern of *RP11-334C17.6* and *BTA10* allows to group patients with a greater survival from those with worse results, as well as the prognosis of each of the four GBM subtypes [52]. Currently, the available data are promising, and based on them, specific lncRNAs have been postulated as potential biomarkers for GBM diagnosis and prognosis, which with further evidence could be used in the medical practice.

#### **3.3 Circulating lncRNAs**

It is a fact that the establishment of novel biomarkers for Ast is essential and their identification and clinical application by means of less invasive methods

**43**

[5, 75–77].

tic cells.

*Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma*

would be ideal. To date, many studies have demonstrated the usefulness of circulating lncRNAs for diagnosis and prognosis of many diseases, including GBM [46, 54, 55]. The profile expression of lncRNAs was determined in blood serum of GBM patients and high levels of *HOTAIR* (HOX transcript antisense RNA) and *GAS5* [growth arrest specific 5 (nonprotein coding)] were prognostic factors to determine patient's survival and GBM progression [56]. Overexpression of circulating *HOTAIR* has been observed in different types of cancer [57–61], but its downregulation was detected in patients with acute myocardial infarction [62]. Interestingly, the presence of high levels of circulating *HOTAIR* DNA was also detected in breast cancer (BC) patients, where it has a potential use for BC diagnosis [60]. Unlike those observed in the GBM, most studies have shown that circulating *GAS5* was downexpressed in different types of cancer, which allowed the diagnosis of both intraductal papillary mucinous neoplasms [63] and nonsmall cell lung cancer [64, 65], as well as BC prognosis [66]. By contrast, overexpression of circulating GAS5 could be used to predict treatment response in head and neck cancer [67]. According to this, the overexpression of *HOTAIR* observed in all cancer types studied to date strongly suggests a central function of this lncRNA in the establishment, maintenance, and/or progression of cancer in general. Therefore, it is very important to identify the processes that *HOTAIR* is controlling in cancer in order to postulate molecular tools to eradicate neoplas-

*MALAT1* [metastasis associated lung adenocarcinoma transcript 1 (nonprotein coding)] was another lncRNA with changes on its circulating expression levels in GBM. This lncRNA was overexpressed, and this was associated with poor overall survival and with a high GBM recurrence [19]. Overexpression of circulating *MALAT1* has been observed in many types of cancer and it seems to be useful for cancer diagnosis and prognosis [68–73]. On the contrary, Peng et al. [74] showed that *MALAT1* downregulation in blood was important for early diagnosis in nonsmall cell lung cancer. Based on the above, the presence of a biomolecule in distinct corporal fluids is a noninvasive form at the molecular level either by the presence or the absence of a disease, as well as by the patient's prognosis with an specific disease. According to the studies performed to date, the use of circulating lncRNAs

*DOI: http://dx.doi.org/10.5772/intechopen.80561*

in the medical practice seems very promising.

**3.4 Search for GBM biomarkers from a system biological perspective**

Since a biomolecule does not act alone and depends on the cellular context to carry out its biological functions, different groups of study have focused on the identification of the lncRNA interactome in GBM. Evidence indicates that lncRNAs could interact with themselves, as well as with other biomolecules, such as mRNAs, miRNAs, and proteins; changes on the lncRNA activity at distinct molecular levels could affect their interaction networks and the correct cellular functioning

Yan et al. [78] established interaction networks between lncRNAs and mRNAs aberrantly expressed in GBM, and based on this, they postulated "hub genes" which were involved in GBM pathogenesis. Similarly, under this perspective, it was found that complexes conformed by lncRNA•mRNA (*HOTAIR*-*MX11-CD58*/*PRKCE* and *HOTAIR*-*ATF5*-*NCAM1*) or lncRNA•lncRNA (*MCM3AP-AS*-MIR17HG) could be potential biomarkers for GBM prognosis [79–82]. Importantly, the *TP73-AS1*•*RFX1* complex (TP73 Antisense RNA 1 and Regulatory Factor X1, respectively) was identified as an important factor for the control of apoptosis in this type of tumor [83]. To sum up, the cancer study from a system biological perspective has allowed to identify the complex interaction networks where many biomolecules are involved

#### *Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma DOI: http://dx.doi.org/10.5772/intechopen.80561*

*Primary Intracranial Tumors*

of the four GBM subtypes: classical, mesenchymal, neural, and proneural. The lncRNAs *CRNDE* (colorectal neoplasia differentially expressed) and *CYTOR* (cytoskeleton regulator RNA) (both upregulated) and *TUNAR* (TCL1 upstream neural differentiation-associated RNA) and *LINCO1476* (both downregulated) were those with the highest expression changes in GBM compared to the control and with a potential use for GBM diagnosis (**Table 3**) [52]. *CRNDE* overexpression has been associated with high cell proliferation, migration, and invasion, which corresponds with the promotion of tumor growth observed in *in vivo* studies [53]. In addition, the expression pattern of *RP11-334C17.6* and *BTA10* allows to group patients with a greater survival from those with worse results, as well as the prognosis of each of the four GBM subtypes [52]. Currently, the available data are promising, and based on them, specific lncRNAs have been postulated as potential biomarkers for GBM diagnosis and prognosis, which with further evidence could be used in the medical

It is a fact that the establishment of novel biomarkers for Ast is essential and their identification and clinical application by means of less invasive methods

**42**

practice.

**Table 3.**

**3.3 Circulating lncRNAs**

*LncRNAs as potential GBM biomarkers.*

would be ideal. To date, many studies have demonstrated the usefulness of circulating lncRNAs for diagnosis and prognosis of many diseases, including GBM [46, 54, 55]. The profile expression of lncRNAs was determined in blood serum of GBM patients and high levels of *HOTAIR* (HOX transcript antisense RNA) and *GAS5* [growth arrest specific 5 (nonprotein coding)] were prognostic factors to determine patient's survival and GBM progression [56]. Overexpression of circulating *HOTAIR* has been observed in different types of cancer [57–61], but its downregulation was detected in patients with acute myocardial infarction [62]. Interestingly, the presence of high levels of circulating *HOTAIR* DNA was also detected in breast cancer (BC) patients, where it has a potential use for BC diagnosis [60]. Unlike those observed in the GBM, most studies have shown that circulating *GAS5* was downexpressed in different types of cancer, which allowed the diagnosis of both intraductal papillary mucinous neoplasms [63] and nonsmall cell lung cancer [64, 65], as well as BC prognosis [66]. By contrast, overexpression of circulating GAS5 could be used to predict treatment response in head and neck cancer [67]. According to this, the overexpression of *HOTAIR* observed in all cancer types studied to date strongly suggests a central function of this lncRNA in the establishment, maintenance, and/or progression of cancer in general. Therefore, it is very important to identify the processes that *HOTAIR* is controlling in cancer in order to postulate molecular tools to eradicate neoplastic cells.

*MALAT1* [metastasis associated lung adenocarcinoma transcript 1 (nonprotein coding)] was another lncRNA with changes on its circulating expression levels in GBM. This lncRNA was overexpressed, and this was associated with poor overall survival and with a high GBM recurrence [19]. Overexpression of circulating *MALAT1* has been observed in many types of cancer and it seems to be useful for cancer diagnosis and prognosis [68–73]. On the contrary, Peng et al. [74] showed that *MALAT1* downregulation in blood was important for early diagnosis in nonsmall cell lung cancer. Based on the above, the presence of a biomolecule in distinct corporal fluids is a noninvasive form at the molecular level either by the presence or the absence of a disease, as well as by the patient's prognosis with an specific disease. According to the studies performed to date, the use of circulating lncRNAs in the medical practice seems very promising.

#### **3.4 Search for GBM biomarkers from a system biological perspective**

Since a biomolecule does not act alone and depends on the cellular context to carry out its biological functions, different groups of study have focused on the identification of the lncRNA interactome in GBM. Evidence indicates that lncRNAs could interact with themselves, as well as with other biomolecules, such as mRNAs, miRNAs, and proteins; changes on the lncRNA activity at distinct molecular levels could affect their interaction networks and the correct cellular functioning [5, 75–77].

Yan et al. [78] established interaction networks between lncRNAs and mRNAs aberrantly expressed in GBM, and based on this, they postulated "hub genes" which were involved in GBM pathogenesis. Similarly, under this perspective, it was found that complexes conformed by lncRNA•mRNA (*HOTAIR*-*MX11-CD58*/*PRKCE* and *HOTAIR*-*ATF5*-*NCAM1*) or lncRNA•lncRNA (*MCM3AP-AS*-MIR17HG) could be potential biomarkers for GBM prognosis [79–82]. Importantly, the *TP73-AS1*•*RFX1* complex (TP73 Antisense RNA 1 and Regulatory Factor X1, respectively) was identified as an important factor for the control of apoptosis in this type of tumor [83]. To sum up, the cancer study from a system biological perspective has allowed to identify the complex interaction networks where many biomolecules are involved

#### *Primary Intracranial Tumors*

to regulate specific cellular processes; alterations in the operation of any of these components will affect the correct functioning of the cell. Specifically, lncRNA changes could lead to an amplification of the aberrant signals and this could be more significant if the lncRNAs interact with other ncRNAs, given that they have many targets of regulation.

#### **3.5 Radio and chemoresistance**

A major clinical problem is the resistance to chemotherapy and radiotherapy; therefore, identification of "molecular tools" that can predict and in the best-case scenario, improve the cellular response to these treatments would be ideal. Wang et al. [80] established a prediction model for radiosensitivity by detecting differential expressed lncRNAs and mRNAs after irradiation. Interestingly, the algorithm differentiated those patients that were radiosensitive and with a greater survival, from the patients with radioresistance; unfortunately, as far as we know, this is the only study focused on GBM radioresistance.

In addition, the involvement of lncRNAs in chemoresistance has been widely studied. LncRNAs *RP11-838 N2.4* [84] and *MALAT1* [19, 55] were shown to be associated with TMZ resistance (**Table 3**). Hiseq sequencing identified the profile expression of lncRNAs, which was specific and differentiated patient resistant to TMZ from those sensitive to this drug. This analysis showed that overexpression of *MALAT1* and its circulating form was related to a lower response to chemotherapy and to a shorter survival time of patients with GBM by controlling the miR-203 and *TYMS* (thymidylate synthase) levels, which was tested in TMZ resistant GBM cells [19]. Another fact worthy of mention is that other components of the *MALAT1* interactome have been elucidated to be important for TMZ resistance. *MALAT1* overexpression maintained high levels of expression of specific genes, such as *ABCB1* (ATP binding cassette subfamily B member 1), *ABCC5* (ATP binding cassette subfamily C member 5), *LRP1* (LDL receptor related protein 1), and *ZEB1* (zinc finger E-box binding homeobox). Notably, forced decrease of *MALAT1* resulted in TMZ sensitization by decreasing the levels of *ZEB1* [55]. Meanwhile, alterations in the axis RP11-838 N2.4•miR-10•EphA8 (EPH Receptor A8) were also involved in GBM cell resistance to TMZ [21]. All these facts supported the importance of the study of lncRNAs for clinical purposes and specifically gain knowledge regarding the prognosis of patients to radiotherapy or chemotherapy.

#### **3.6 LncRNAs in stem cells**

Many lines of evidence have shown the involvement of lncRNAs in the control of many cellular processes in cancer stem cells (CSCs) [85–87], but their participation in Ast has been very poorly studied. These cells are able to self-renew and differentiate into diverse cancer cell lineages to form tumors, so CSCs have been proposed as potential targets for cancer treatment. To further understand this, Balci et al. [88] determined the profile expression of lncRNAs in GBM stem cells (GSCs) relative to control stem cells. From these differentially expressed lncRNAs, *PCAT-1* (prostate cancer associated transcript 1 (nonprotein coding)), *MEG3* (maternally expressed 3 (nonprotein coding)), and *HOTAIR* functioned as tumor suppressors in GBM. This was related to alterations in gene expression. Interestingly, another study identified that even identical GSCs showed variations in their expression profile of lncRNAs, as well as in the variants produced by specific subgroups of cells. Despite this, the authors could establish a stem cell

**45**

evidence.

**4.1 Sponge lncRNAs**

*Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma*

signature of 31 lncRNAs according to their expression levels [57]. Meanwhile, in hypoxic conditions, the expression of the lncRNA *HIF1A-AS2* (hypoxia inducible factor 1 alpha-antisense RNA 2) was induced and this led to positive control of the growth, self-renewal, and molecular reprograming of the GSCs [20]. Significantly, the control of these cellular processes was possible by regulating an interaction

Although many studies have focused on studying the changes on the expression of lncRNAs, very few have attempted to determine the mechanisms underlying this deregulation. In this sense, Zhang et al. [89] showed a feedback loop which controlled the expression of the lncRNA *FOXM1-AS* (Forkhead box M1 antisense) and it proved to be important for GSC tumorigenesis. *ALKBH5* (AlkB homolog 5, RNA demethylase) is a demethylase highly expressed in GBM GSCs, which was associated with an enhanced self-renewal and tumorigenesis of these cells. These malignant cell processes were controlled by *FOXM1* (Forkhead box M1) and *FOXM1-AS*, which increased their expression levels by a greater demethylation of the immature transcripts of *FOXM1*. In this pathway of regulation, *FOXM1-AS* was important to facilitate the action of *ALKBH5* on the nascent transcripts of *FOXM1*; therefore, a therapy in which the action of this lncRNA was reduced or blocked could be important to prevent GBM tumorigenesis. Taken together, these studies showed that although expression changes of lncRNAs were useful for GBM diagnosis and prognosis, they necessarily not represent the entire tumor, but rather this seems to associate with certain subgroups of cells that predominate over others and express particular lncRNAs. Therefore, the applicability of a differentially expressed biomolecule in the medical practice—particularly lncRNAs—must be done with caution and with all the required

In addition to expression changes, it is necessary for the elucidation of the action mechanisms by which lncRNAs are acting. Evidence showed that lncRNAs act at both cytoplasmic and nuclear levels and that this is done directly and/or by their interaction with protein complexes and/or with other lncRNAs or different RNA species, such as mRNAs and miRNAs [5, 75–77]. Also, lncRNAs can regulate many signaling pathways by controlling the cytoplasmic disposal of mRNAs and miRNAs

This class of lncRNAs regulates miRNA disposal in the cell cytoplasm by capturing them and blocking their action [90, 91]. To date, all lncRNAs identified as "sponges" in the GBM acting as suppressors and involved in lncRNA upregulation and miRNA attenuation were associated with GBM **Table 4**). LncRNAs *H19* (imprinted maternally expressed transcript (nonprotein coding)) and *NEAT1* (nuclear paraspeckle assembly transcript 1 (nonprotein coding)) controlled the action of the miRNA let-7e, whose levels were downregulated in GBM due to the overexpression of these lncRNAs [92, 93]. Specifically, the axis *H19*•let-7e was involved in maintaining the phenotype of stem cells, which was associated with tumor malignancy and TMZ chemoresistance [93]. Similarly, a low disposal of let-7e by *NEAT1* overexpression, resulted in a higher activity of its mRNA target *NRAS* (NRAS proto-oncogene, GTPase), which leads to GBM malignancy [92].

*DOI: http://dx.doi.org/10.5772/intechopen.80561*

network, which will be described later.

**4. Action mechanisms of lncRNAs in GBM**

and even by producing small RNA species, such as miRNAs [89].

*Primary Intracranial Tumors*

many targets of regulation.

**3.5 Radio and chemoresistance**

only study focused on GBM radioresistance.

to regulate specific cellular processes; alterations in the operation of any of these components will affect the correct functioning of the cell. Specifically, lncRNA changes could lead to an amplification of the aberrant signals and this could be more significant if the lncRNAs interact with other ncRNAs, given that they have

A major clinical problem is the resistance to chemotherapy and radiotherapy; therefore, identification of "molecular tools" that can predict and in the best-case scenario, improve the cellular response to these treatments would be ideal. Wang et al. [80] established a prediction model for radiosensitivity by detecting differential expressed lncRNAs and mRNAs after irradiation. Interestingly, the algorithm differentiated those patients that were radiosensitive and with a greater survival, from the patients with radioresistance; unfortunately, as far as we know, this is the

In addition, the involvement of lncRNAs in chemoresistance has been widely studied. LncRNAs *RP11-838 N2.4* [84] and *MALAT1* [19, 55] were shown to be associated with TMZ resistance (**Table 3**). Hiseq sequencing identified the profile expression of lncRNAs, which was specific and differentiated patient resistant to TMZ from those sensitive to this drug. This analysis showed that overexpression of *MALAT1* and its circulating form was related to a lower response to chemotherapy and to a shorter survival time of patients with GBM by controlling the miR-203 and *TYMS* (thymidylate synthase) levels, which was tested in TMZ resistant GBM cells [19]. Another fact worthy of mention is that other components of the *MALAT1* interactome have been elucidated to be important for TMZ resistance. *MALAT1* overexpression maintained high levels of expression of specific genes, such as *ABCB1* (ATP binding cassette subfamily B member 1), *ABCC5* (ATP binding cassette subfamily C member 5), *LRP1* (LDL receptor related protein 1), and *ZEB1* (zinc finger E-box binding homeobox). Notably, forced decrease of *MALAT1* resulted in TMZ sensitization by decreasing the levels of *ZEB1* [55]. Meanwhile, alterations in the axis RP11-838 N2.4•miR-10•EphA8 (EPH Receptor A8) were also involved in GBM cell resistance to TMZ [21]. All these facts supported the importance of the study of lncRNAs for clinical purposes and specifically gain knowledge regarding the prognosis of patients to radiotherapy or

Many lines of evidence have shown the involvement of lncRNAs in the control

of many cellular processes in cancer stem cells (CSCs) [85–87], but their participation in Ast has been very poorly studied. These cells are able to self-renew and differentiate into diverse cancer cell lineages to form tumors, so CSCs have been proposed as potential targets for cancer treatment. To further understand this, Balci et al. [88] determined the profile expression of lncRNAs in GBM stem cells (GSCs) relative to control stem cells. From these differentially expressed lncRNAs, *PCAT-1* (prostate cancer associated transcript 1 (nonprotein coding)), *MEG3* (maternally expressed 3 (nonprotein coding)), and *HOTAIR* functioned as tumor suppressors in GBM. This was related to alterations in gene expression. Interestingly, another study identified that even identical GSCs showed variations in their expression profile of lncRNAs, as well as in the variants produced by specific subgroups of cells. Despite this, the authors could establish a stem cell

**44**

chemotherapy.

**3.6 LncRNAs in stem cells**

signature of 31 lncRNAs according to their expression levels [57]. Meanwhile, in hypoxic conditions, the expression of the lncRNA *HIF1A-AS2* (hypoxia inducible factor 1 alpha-antisense RNA 2) was induced and this led to positive control of the growth, self-renewal, and molecular reprograming of the GSCs [20]. Significantly, the control of these cellular processes was possible by regulating an interaction network, which will be described later.

Although many studies have focused on studying the changes on the expression of lncRNAs, very few have attempted to determine the mechanisms underlying this deregulation. In this sense, Zhang et al. [89] showed a feedback loop which controlled the expression of the lncRNA *FOXM1-AS* (Forkhead box M1 antisense) and it proved to be important for GSC tumorigenesis. *ALKBH5* (AlkB homolog 5, RNA demethylase) is a demethylase highly expressed in GBM GSCs, which was associated with an enhanced self-renewal and tumorigenesis of these cells. These malignant cell processes were controlled by *FOXM1* (Forkhead box M1) and *FOXM1-AS*, which increased their expression levels by a greater demethylation of the immature transcripts of *FOXM1*. In this pathway of regulation, *FOXM1-AS* was important to facilitate the action of *ALKBH5* on the nascent transcripts of *FOXM1*; therefore, a therapy in which the action of this lncRNA was reduced or blocked could be important to prevent GBM tumorigenesis. Taken together, these studies showed that although expression changes of lncRNAs were useful for GBM diagnosis and prognosis, they necessarily not represent the entire tumor, but rather this seems to associate with certain subgroups of cells that predominate over others and express particular lncRNAs. Therefore, the applicability of a differentially expressed biomolecule in the medical practice—particularly lncRNAs—must be done with caution and with all the required evidence.

#### **4. Action mechanisms of lncRNAs in GBM**

In addition to expression changes, it is necessary for the elucidation of the action mechanisms by which lncRNAs are acting. Evidence showed that lncRNAs act at both cytoplasmic and nuclear levels and that this is done directly and/or by their interaction with protein complexes and/or with other lncRNAs or different RNA species, such as mRNAs and miRNAs [5, 75–77]. Also, lncRNAs can regulate many signaling pathways by controlling the cytoplasmic disposal of mRNAs and miRNAs and even by producing small RNA species, such as miRNAs [89].

#### **4.1 Sponge lncRNAs**

This class of lncRNAs regulates miRNA disposal in the cell cytoplasm by capturing them and blocking their action [90, 91]. To date, all lncRNAs identified as "sponges" in the GBM acting as suppressors and involved in lncRNA upregulation and miRNA attenuation were associated with GBM **Table 4**). LncRNAs *H19* (imprinted maternally expressed transcript (nonprotein coding)) and *NEAT1* (nuclear paraspeckle assembly transcript 1 (nonprotein coding)) controlled the action of the miRNA let-7e, whose levels were downregulated in GBM due to the overexpression of these lncRNAs [92, 93]. Specifically, the axis *H19*•let-7e was involved in maintaining the phenotype of stem cells, which was associated with tumor malignancy and TMZ chemoresistance [93]. Similarly, a low disposal of let-7e by *NEAT1* overexpression, resulted in a higher activity of its mRNA target *NRAS* (NRAS proto-oncogene, GTPase), which leads to GBM malignancy [92].


#### **Table 4.**

*LncRNAs as sponges in adult GBM.*

Other lncRNAs that function as sponges in GBM were related to tumor malignancy. For example, the upregulation of *XIST* (X inactive specific transcript (nonprotein coding)) was related to GSC malignancy, tumor growth, and poor mice survival by controlling the action of miR-152 [94]. Meanwhile, the attenuation of the miR-299 disposal was controlled by the overexpression of *TUG1* (lncRNA taurine upregulated 1), which was related to tumor malignancy by the overactivation of *VEGFA* (vascular endothelial growth factor A) [95] and apoptosis evasion [93].

Similarly, GBM malignancy was mediated by the overexpression of *RP11- 838N2.4* and *SNHG7* (small nucleolar RNA host gene 7), which regulated the function of miR-10 and miR-5095, respectively. In the first case, the attenuation of the action of miR-10 was associated with apoptosis evasion, and the reestablishment of the axis *RP11-838 N2.4*•miR-10•*EphA8* (EPH receptor A8) induced this programmed cell death [21]. Meanwhile, reestablishment of the *SNGH7*•miR-5095•*CTNNB1* (catenin beta 1) axis arrested tumor growth and decreased metastasis by decreasing the expression of *CTNNB1*, which is involved in the Wnt/β-catenin pathway [96]. Finally, it was observed that GBM proliferation, migration, and invasion were also promoted by the overexpression of *CRNDE* and the consequent attenuation of the miR-136-5p expression; all these led to the overactivation of BCL2 and WNT2, which are target genes of this miRNA [97]. According to the LNCipedia compendium, there are many variants reported for these lncRNAs; therefore, it would be very interesting and important to identify which lncRNA

**47**

**Figure 3.**

*targets.*

*Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma*

regulate the action of let-7e in a synergistic manner.

variants are expressed in GBM and which of them have the binding sites for trapping these miRNAs. Also, further studies are necessary to know if *H19* and *NEAT1*

A very interesting case was that of the lncRNA *CASC2c* (cancer susceptibility candidate 2; formerly C10orf5). Besides its interaction with miR-101, this lncRNA was involved in the processing of the pre-miR-101 into mature miR-101 and competed with this miRNA for the mRNA *CPEB1* (cytoplasmic polyadenylation element binding protein 1). High levels of *CASC2c* and consequently a reduced activity of the axis miR-101•CPEB1 were associated with a high cell proliferation and tumorigenesis. Therefore, a decrease in *CASC2c* expression and an increased disposal of miR-101 were related to better patient's prognosis [98]. This evidence is an indication of all biological functions that an lncRNA can play in the cell and how the system ensures the regulation of gene expression by regulating at different levels the biogenesis of miRNAs (**Figure 3**). In consequence, if something modifies the processing of the pre-miR-101 or affects the regulation of its mature form, *CASC2c* would try to compensate the miRNA action by competing for its target genes. Evidently, other mechanisms must be involved in the biogenesis of this miRNA.

Besides the lncRNA interaction with miRNAs, there is evidence indicating that lncRNAs can carry out their biological functions when they interact with mRNAs and/or proteins [5, 75–77]. As mentioned above, *HIF1A-AS2* was involved in the GSC malignancy under hypoxia conditions. The action of this lncRNA was performed in part by directly interacting with *IGF2BP2* (insulin-like growth factor 2 MRNA binding protein 2) and *DHX9* (DExH-box helicase 9), which finally controlled the action of *HMGA1* (high mobility group AT-Hook 1) [20]. According to this, elucidation of all the components that formed the interactome network of HIF1A-AS2 in the GSCs would be crucial to establish molecular tools for GBM

*The lncRNA CASC2 regulated the function of the miR-101 at different molecular levels. CASC2 was involved in the processing of the pre-miR-101 and also interact with its mature form to regulate the function of this miRNA. If any of these mechanisms fail, CASC2 ensures the miR-101 regulation by interacting with its mRNA* 

*DOI: http://dx.doi.org/10.5772/intechopen.80561*

**4.2 By interacting with mRNAs**

treatment.

#### *Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma DOI: http://dx.doi.org/10.5772/intechopen.80561*

variants are expressed in GBM and which of them have the binding sites for trapping these miRNAs. Also, further studies are necessary to know if *H19* and *NEAT1* regulate the action of let-7e in a synergistic manner.

A very interesting case was that of the lncRNA *CASC2c* (cancer susceptibility candidate 2; formerly C10orf5). Besides its interaction with miR-101, this lncRNA was involved in the processing of the pre-miR-101 into mature miR-101 and competed with this miRNA for the mRNA *CPEB1* (cytoplasmic polyadenylation element binding protein 1). High levels of *CASC2c* and consequently a reduced activity of the axis miR-101•CPEB1 were associated with a high cell proliferation and tumorigenesis. Therefore, a decrease in *CASC2c* expression and an increased disposal of miR-101 were related to better patient's prognosis [98]. This evidence is an indication of all biological functions that an lncRNA can play in the cell and how the system ensures the regulation of gene expression by regulating at different levels the biogenesis of miRNAs (**Figure 3**). In consequence, if something modifies the processing of the pre-miR-101 or affects the regulation of its mature form, *CASC2c* would try to compensate the miRNA action by competing for its target genes. Evidently, other mechanisms must be involved in the biogenesis of this miRNA.

#### **4.2 By interacting with mRNAs**

*Primary Intracranial Tumors*

**"Sponges" LncRNAs**

H19 NEAT1

**Table 4.**

*LncRNAs as sponges in adult GBM.*

*Tumor suppressors*

Other lncRNAs that function as sponges in GBM were related to tumor malignancy. For example, the upregulation of *XIST* (X inactive specific transcript (nonprotein coding)) was related to GSC malignancy, tumor growth, and poor mice survival by controlling the action of miR-152 [94]. Meanwhile, the attenuation of the miR-299 disposal was controlled by the overexpression of *TUG1* (lncRNA taurine upregulated 1), which was related to tumor malignancy by the overactivation of *VEGFA*

Polyadenylation Element Binding Protein 1)

**microRNA mRNA target Cellular process altered Signaling** 

H19: stem cells phenotype

invasion, apoptosis evasion, tumor growth and poor mice survival

Angiogenesis induction

Proliferation, migration, invasion, apoptosis

Low chemotherapy response

Cell proliferation Tumorigenesis

Shorter survival time of

Apoptosis evasion Wnt

Apoptosis evasion Apoptosis

NEAT1:

evasion

patients

Let-7e NRAS (NRAS Proto-

A8)

1)

Synthetase)

Member A2) BCL2 (BCL2 Apoptosis

Regulator)

TUG1 miR-299 VEGFA (Vascular

RP11-838N2.4 miR-10 EphA8 (EPH Receptor

SNHG7 miR-5095 CTNNB1 (Catenin Beta

MALAT1 miR-203 TYMS (Thymidylate

CRNDE miR-136-5p Wnt2 (Wnt Family

CASC2 miR-101 CPEB1 (Cytoplasmic

Oncogene, GTPase)

XIST miR-152 Proliferation, migration,

Endothelial Growth Factor A)

**pathway**

Wnt/β catenin

Apoptosis

(vascular endothelial growth factor A) [95] and apoptosis evasion [93].

Similarly, GBM malignancy was mediated by the overexpression of *RP11- 838N2.4* and *SNHG7* (small nucleolar RNA host gene 7), which regulated the function of miR-10 and miR-5095, respectively. In the first case, the attenuation of the action of miR-10 was associated with apoptosis evasion, and the reestablishment of the axis *RP11-838 N2.4*•miR-10•*EphA8* (EPH receptor A8) induced this programmed cell death [21]. Meanwhile, reestablishment of the *SNGH7*•miR-5095•*CTNNB1* (catenin beta 1) axis arrested tumor growth and decreased metastasis by decreasing the expression of *CTNNB1*, which is involved in the Wnt/β-catenin pathway [96]. Finally, it was observed that GBM proliferation, migration, and invasion were also promoted by the overexpression of *CRNDE* and the consequent attenuation of the miR-136-5p expression; all these led to the overactivation of BCL2 and WNT2, which are target genes of this miRNA [97]. According to the LNCipedia compendium, there are many variants reported for these lncRNAs; therefore, it would be very interesting and important to identify which lncRNA

**46**

Besides the lncRNA interaction with miRNAs, there is evidence indicating that lncRNAs can carry out their biological functions when they interact with mRNAs and/or proteins [5, 75–77]. As mentioned above, *HIF1A-AS2* was involved in the GSC malignancy under hypoxia conditions. The action of this lncRNA was performed in part by directly interacting with *IGF2BP2* (insulin-like growth factor 2 MRNA binding protein 2) and *DHX9* (DExH-box helicase 9), which finally controlled the action of *HMGA1* (high mobility group AT-Hook 1) [20]. According to this, elucidation of all the components that formed the interactome network of HIF1A-AS2 in the GSCs would be crucial to establish molecular tools for GBM treatment.

#### **Figure 3.**

*The lncRNA CASC2 regulated the function of the miR-101 at different molecular levels. CASC2 was involved in the processing of the pre-miR-101 and also interact with its mature form to regulate the function of this miRNA. If any of these mechanisms fail, CASC2 ensures the miR-101 regulation by interacting with its mRNA targets.*

#### **5. Pediatric Ast**

Adult and p-Ast are distinct molecular entities and are classified into different groups; therefore, studies in pediatric Ast are imperative. The first study performed in p-Ast was the one where the overexpression *HOTAIR* and *HOX* was detected in different pediatric brain tumors, including juvenile pediatric Ast (JPA); however, the biological meaning of this was not further studied [99].

We identified in the laboratory the expression profile of lncRNAs in p-Ast of WHO grades I–IV, given that the function of lncRNAs in p-Ast has been poorly studied. Similar to that observed for adult Ast, p-Ast showed many lncRNAs with expression changes relative to the control tissues, among histological grades or even in the same histological grade [5]. In addition, it was identified that the interaction of many differentially expressed lncRNAs with mRNAs and/or miRNAs aberrantly expressed was identified. As explained above, these interactions could lead to the amplification of the aberrant signals and to the modification of many signaling pathways. According to this, there were several hub lncRNAs in p-Ast that in relation to their interactions with mRNAs could be altering pathways such as FOXO, chemokine, hedgehog, MAPK, and others (**Figure 4**). Additionally, hub lncRNAs potentially useful to distinguish GBM from the other histopathological WHO grades were predicted to control diverse metabolic pathways and signaling pathways such as Ras, hippo, apellin, etc. (**Figure 4**).

The interaction of differentially expressed lncRNAs and miRNAs was shown to be a complex network that could be involved in modifications on proteoglycans in cancer, fatty acid metabolism, cell cycle, and spliceosome. Notably, data analysis revealed the presence of circular lncRNAs (circRNAs) with expression changes in p-Ast (**Figure 5**). According to the interactions of circRNAs with miRNAs, this type

#### **Figure 4.**

*Predicted interaction networks between lncRNAs and mRNAs were predicted to be involved in the control of signaling pathways. Data showed hub mRNAs that were analyzed with the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. Hub mRNAs were those mRNAs with the highest number of interactions with lncRNAs. Data were taken from [5] and analyzed with KEGG.*

**49**

**Figure 5.**

*Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma*

of lncRNAs was predicted to be involved in regulating cellular growth, survival,

*Circular lncRNAs in pediatric astrocytoma. Predictions showed differentially expressed circRNAs in pediatric* 

The integration of proteome and mirnome, as well as transcriptome data showed a convergence of all these biomolecules in the control of common signaling pathways, which gave an overview of the action of complex networks in cancer, particularly p-Ast [5, 47]. For example, although it is widely known that the MAPK pathway is altered in ~88% of gliomas, these data showed novel molecular components involved in this signaling pathway in p-Ast, which also allow to differentiate GBM from the other histological grades. The lncRNA *GRPEL1-1:1* was aberrantly expressed in all p-Ast grades when compared to the control tissues, but it was downregulated in WHO grades I–III relative to GBM. It is noteworthy to add, this lncRNA was predicted to interact with miR-15b-5p, and its expression levels were inversely correlated to those of *lnc-GRPEL1-1:1* (**Figure 6**). Other lncRNAs such as *TIMM22-1:1*, *Noc4L-1:1*, and *LINC-ROR* were predicted to be involved in the MAPK pathway, as well as in the Wnt pathway and extracellular matrix interactions [5]. In pediatric GBM, the overexpression of linc-Ror could lead to the downregulation of miR-145, since there is evidence indicating that linc-Ror sponges to miR-145, which was associated with cancer malignancy [100, 101]. According to our model, the linc-Ror•miR-145 axis could be increasing the expression of *IGFR1* (insulin-like growth factor 1 receptor), *c-Myc* (MYC proto-oncogene, BHLH transcription factor), and *STAT1* (signal transducer and activator of transcription 1), which causes a sustained angiogenesis and increased cell proliferation; however, this must be tested (**Figure 6**). In patients with glioma, linc-Ror was downregulated and this correlated positively and negatively with the expression of *SOX11* (SRY-box 11) and *KFL4* (Kruppel-like factor 4), respectively [101]. In the GBM cell line U87, *in vitro* assays showed the involvement of this lncRNA in the induction of cell proliferation, *CD133* expression, and in the formation of neurospheres [101], which are the factors of tumor malignancy. Similarly, linc-Ror was downregulated in p-Ast grades I–III, but it was upregulated in GBM relative to control tissues and other p-Ast grades [5]. Therefore, linc-Ror seems to be a

migration, invasion, adhesion, among others [5] (**Table 5**).

*astrocytoma, which have many binding sites for miRNAs.*

candidate to function as a biomarker for p-Ast diagnosis and prognosis.

*DOI: http://dx.doi.org/10.5772/intechopen.80561*

*Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma DOI: http://dx.doi.org/10.5772/intechopen.80561*

#### **Figure 5.**

*Primary Intracranial Tumors*

Adult and p-Ast are distinct molecular entities and are classified into different groups; therefore, studies in pediatric Ast are imperative. The first study performed in p-Ast was the one where the overexpression *HOTAIR* and *HOX* was detected in different pediatric brain tumors, including juvenile pediatric Ast (JPA); however,

We identified in the laboratory the expression profile of lncRNAs in p-Ast of WHO grades I–IV, given that the function of lncRNAs in p-Ast has been poorly studied. Similar to that observed for adult Ast, p-Ast showed many lncRNAs with expression changes relative to the control tissues, among histological grades or even in the same histological grade [5]. In addition, it was identified that the interaction of many differentially expressed lncRNAs with mRNAs and/or miRNAs aberrantly expressed was identified. As explained above, these interactions could lead to the amplification of the aberrant signals and to the modification of many signaling pathways. According to this, there were several hub lncRNAs in p-Ast that in relation to their interactions with mRNAs could be altering pathways such as FOXO, chemokine, hedgehog, MAPK, and others (**Figure 4**). Additionally, hub lncRNAs potentially useful to distinguish GBM from the other histopathological WHO grades were predicted to control diverse metabolic pathways and signaling

The interaction of differentially expressed lncRNAs and miRNAs was shown to be a complex network that could be involved in modifications on proteoglycans in cancer, fatty acid metabolism, cell cycle, and spliceosome. Notably, data analysis revealed the presence of circular lncRNAs (circRNAs) with expression changes in p-Ast (**Figure 5**). According to the interactions of circRNAs with miRNAs, this type

*Predicted interaction networks between lncRNAs and mRNAs were predicted to be involved in the control of signaling pathways. Data showed hub mRNAs that were analyzed with the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. Hub mRNAs were those mRNAs with the highest number of interactions with* 

*lncRNAs. Data were taken from [5] and analyzed with KEGG.*

the biological meaning of this was not further studied [99].

pathways such as Ras, hippo, apellin, etc. (**Figure 4**).

**5. Pediatric Ast**

**48**

**Figure 4.**

*Circular lncRNAs in pediatric astrocytoma. Predictions showed differentially expressed circRNAs in pediatric astrocytoma, which have many binding sites for miRNAs.*

of lncRNAs was predicted to be involved in regulating cellular growth, survival, migration, invasion, adhesion, among others [5] (**Table 5**).

The integration of proteome and mirnome, as well as transcriptome data showed a convergence of all these biomolecules in the control of common signaling pathways, which gave an overview of the action of complex networks in cancer, particularly p-Ast [5, 47]. For example, although it is widely known that the MAPK pathway is altered in ~88% of gliomas, these data showed novel molecular components involved in this signaling pathway in p-Ast, which also allow to differentiate GBM from the other histological grades. The lncRNA *GRPEL1-1:1* was aberrantly expressed in all p-Ast grades when compared to the control tissues, but it was downregulated in WHO grades I–III relative to GBM. It is noteworthy to add, this lncRNA was predicted to interact with miR-15b-5p, and its expression levels were inversely correlated to those of *lnc-GRPEL1-1:1* (**Figure 6**). Other lncRNAs such as *TIMM22-1:1*, *Noc4L-1:1*, and *LINC-ROR* were predicted to be involved in the MAPK pathway, as well as in the Wnt pathway and extracellular matrix interactions [5]. In pediatric GBM, the overexpression of linc-Ror could lead to the downregulation of miR-145, since there is evidence indicating that linc-Ror sponges to miR-145, which was associated with cancer malignancy [100, 101]. According to our model, the linc-Ror•miR-145 axis could be increasing the expression of *IGFR1* (insulin-like growth factor 1 receptor), *c-Myc* (MYC proto-oncogene, BHLH transcription factor), and *STAT1* (signal transducer and activator of transcription 1), which causes a sustained angiogenesis and increased cell proliferation; however, this must be tested (**Figure 6**). In patients with glioma, linc-Ror was downregulated and this correlated positively and negatively with the expression of *SOX11* (SRY-box 11) and *KFL4* (Kruppel-like factor 4), respectively [101]. In the GBM cell line U87, *in vitro* assays showed the involvement of this lncRNA in the induction of cell proliferation, *CD133* expression, and in the formation of neurospheres [101], which are the factors of tumor malignancy. Similarly, linc-Ror was downregulated in p-Ast grades I–III, but it was upregulated in GBM relative to control tissues and other p-Ast grades [5]. Therefore, linc-Ror seems to be a candidate to function as a biomarker for p-Ast diagnosis and prognosis.


#### **Table 5.**

*Pathways potentially regulated by differentially expressed super sponges in pediatric astrocytoma; DIANA MirPath V 3.0 analysis.*

**51**

*Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma*

The lncRNA study in Ast has demonstrated an aberrant expression of this type of RNAs in both tumors and blood, which was useful to distinguish Ast from its nonneoplastic counterpart. The elucidation of molecular signatures from circulating lncRNAs is very promising due to their potential use as noninvasive tools for the diagnosis and prognosis of Ast. From another approach, it could be relevant the identification of complete interaction networks in which lncRNAs, other RNA species, and proteins were involved, since this would give a "panoramic vision" of how the aberrant system functions in astrocytic tumors. This could be crucial for

*LncRNAs were predicted to be involved in the control of signaling pathways. Differentially expressed lncRNAs were involved in controlling many signaling pathways by interacting with both mRNAs and miRNAs. Further* 

This work was partially supported by the grant FIS/IMSS/PROT/G17 from The Mexican Institute of Social Security (IMSS). The English edition was carried out by

Areli Ruiz Esparza, translator in-chief at SINTAGMA TRANSLATIONS.

All the authors declare that there is no conflict of interest.

*DOI: http://dx.doi.org/10.5772/intechopen.80561*

the creation of molecular tools for their treatment.

**6. Conclusions**

*experimental validation is necessary.*

**Figure 6.**

**Acknowledgements**

**Conflict of interest**

*Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma DOI: http://dx.doi.org/10.5772/intechopen.80561*

#### **Figure 6.**

*Primary Intracranial Tumors*

Proteoglycans in cancer

Protein processing in the endoplasmic reticulum

Hippo signaling pathway

TGF-beta signaling

pathway

**KEGG pathway p-value Number** 

**of genes**

Fatty acid metabolism 9.64e−09 28 12 Fatty acid metabolism Adherens junction 3.85e−08 49 12 Actin polymerization

Cell cycle 3.85e−08 85 14 Ubiquitin mediated proteolysis

Fatty acid elongation 1.78e-06 13 7 Fatty acid degradation

p53 signaling pathway 2.12e−06 50 14 Cell cycle arrest

Prion diseases 9.44e−06 15 9 Neuronal apoptosis

*Pathways potentially regulated by differentially expressed super sponges in pediatric astrocytoma; DIANA* 

2.59e−07 101 14 Proteasome

2.29e-06 77 14 Pro-apoptotic genes

2.32e−06 48 12 Differentiation, neurogenesis,

**Number of miRNAs**

8.91e−11 120 14 Cellular growth and survival

**Potential cellular processes altered**

Cell migration and invasion Cell adhesion Apoptosis Angiogenesis Vascular permeability

Cell growth and differentiation Gene expression

DNA biosynthesis Origin recognition complex Mini-Chromosome maintenance

Apoptosis

Fatty acid biosynthesis

Apoptosis Inhibition of angiogenesis and metastasis DNA repair and damage prevention Inhibition of IGF-1/mTOR pathway Exosome mediated secretion p53 negative feedback Cellular senescence

> Anti-apoptotic genes Pro-proliferation genes Cell contact inhibition Organ size control Adherens junctions

ventral mesoderm specification Angiogenesis, extracellular matrix neogenesis, immunosuppression, apoptosis induction. G1 arrest Gonadal growth, embryo differentiation, placenta formation Left-right axis determination

> Autophagy Oxidative stress Proliferation of astrocytes

**50**

**Table 5.**

*MirPath V 3.0 analysis.*

*LncRNAs were predicted to be involved in the control of signaling pathways. Differentially expressed lncRNAs were involved in controlling many signaling pathways by interacting with both mRNAs and miRNAs. Further experimental validation is necessary.*

#### **6. Conclusions**

The lncRNA study in Ast has demonstrated an aberrant expression of this type of RNAs in both tumors and blood, which was useful to distinguish Ast from its nonneoplastic counterpart. The elucidation of molecular signatures from circulating lncRNAs is very promising due to their potential use as noninvasive tools for the diagnosis and prognosis of Ast. From another approach, it could be relevant the identification of complete interaction networks in which lncRNAs, other RNA species, and proteins were involved, since this would give a "panoramic vision" of how the aberrant system functions in astrocytic tumors. This could be crucial for the creation of molecular tools for their treatment.

#### **Acknowledgements**

This work was partially supported by the grant FIS/IMSS/PROT/G17 from The Mexican Institute of Social Security (IMSS). The English edition was carried out by Areli Ruiz Esparza, translator in-chief at SINTAGMA TRANSLATIONS.

#### **Conflict of interest**

All the authors declare that there is no conflict of interest.

*Primary Intracranial Tumors*

#### **Author details**

Ruth Ruiz Esparza-Garrido1 , Alicia Siordia-Reyes2 , Gerardo Sánchez<sup>3</sup> , Griselda Ramírez3 and Miguel Velázquez-Flores1 \*

1 Functional Genomics Laboratory, Unit of Medical Research on Human Genomics, Children's Hospital "Silvestre Frenk Freund", National Medical Center Century XXI, Institute of Social Security (IMSS), CDMX, Mexico

2 Pediatric Pathology Service, Children's Hospital "Silvestre Frenk Freund", National Medical Center Century XXI, Institute of Social Security (IMSS), CDMX, Mexico

3 Pediatric Neurosurgery Service, Children's Hospital "Silvestre Frenk Freund", National Medical Center Century XXI, Institute of Social Security (IMSS), CDMX, Mexico

\*Address all correspondence to: dr.velazquez.imss@gmail.com

© 2018 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.

**53**

*Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma*

The evolution of lncRNA repertoires and expression patterns in tetrapods. Nature. 2014;**505**:635-640. DOI:

[8] Botti G, Marra L, Malzone MG, Anniciello A, Botti C, Franco R, et al. LncRNA HOTAIR as prognostic circulating marker and potential therapeutic target in patients with tumor diseases. Current Drug Targets.

[9] Noh JH, Kim KM, McClusky WG,

Abdelmohsen K, Gorospe M. Cytoplasmic functions of long noncoding RNAs. Wiley Interdisciplinary Reviews: RNA. 2018;**9**:e1471. DOI: 10.1002/wrna.1471

[10] Ostrom QT, Gittleman H, Stetson L, Virk S, Barnholtz-Sloan JS. Epidemiology of intracranial gliomas. Progress in Neurological Surgery. 2018;**30**:1-11. DOI:

[11] Ostrom QT, Gittleman H, Liao P, Rouse C, Chen Y, Dowling J, et al. CBTRUS statistical report: Primary brain and central nervous system tumors diagnosed in the United States in 2007-2011. Neuro-Oncology.

[12] Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, et al. The 2016 World Health Organization classification of tumors of the central nervous system: A summary. Acta Neuropathologica. 2016;**131**:803-820. DOI: 10.1007/

[13] Kim W, Liau LM. IDH mutations in human glioma. Neurosurgery Clinics of North America. 2012;**23**:471-480. DOI:

[14] Pathak P, Jha P, Purkait S, Sharma V, Suri V, Sharma MC, et al. Altered

10.1159/000464374

2014;**16**(Suppl 4):iv1-iv63

s00401-016-1545-1

10.1016/j.nec.2012.04.009

10.1038/nature12943

2017;**18**:27-34

*DOI: http://dx.doi.org/10.5772/intechopen.80561*

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[2] Zhang C, Zhao LM, Wu H, Tian G, Dai SL, Zhao RY, et al. C/D-Box Snord105b promotes tumorigenesis in gastric cancer via ALDOA/C-Myc pathway. Cellular Physiology and Biochemistry. 2018;**45**:2471-2482. DOI:

[3] López-Aguilar JE, Velázquez-Flores MA, Simón-Martínez LA, Ávila-Miranda R, Rodríguez-Florido MA, Ruiz-Esparza Garrido R. Circulating microRNAs as biomarkers for pediatric astrocytomas. Archives of

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#### **References**

*Primary Intracranial Tumors*

**Author details**

Griselda Ramírez3

Mexico

Mexico

Ruth Ruiz Esparza-Garrido1

**52**

provided the original work is properly cited.

© 2018 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,

, Alicia Siordia-Reyes2

2 Pediatric Pathology Service, Children's Hospital "Silvestre Frenk Freund",

1 Functional Genomics Laboratory, Unit of Medical Research on Human Genomics, Children's Hospital "Silvestre Frenk Freund", National Medical Center Century XXI,

National Medical Center Century XXI, Institute of Social Security (IMSS), CDMX,

3 Pediatric Neurosurgery Service, Children's Hospital "Silvestre Frenk Freund", National Medical Center Century XXI, Institute of Social Security (IMSS), CDMX,

\*

and Miguel Velázquez-Flores1

\*Address all correspondence to: dr.velazquez.imss@gmail.com

Institute of Social Security (IMSS), CDMX, Mexico

, Gerardo Sánchez<sup>3</sup>

,

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10.4161/rna.28725

10.3390/ncrna4010006

s13058-018-0959-1

120-122. DOI: 10.1016/j. gene.2016.06.024

*DOI: http://dx.doi.org/10.5772/intechopen.80561*

[80] Cao Y, Wang P, Ning S, Xiao W, Xiao B, Li X. Identification of prognostic biomarkers in glioblastoma using a long non-coding RNA-mediated, competitive endogenous RNA network. Oncotarget. 2016;**7**:41737-41747. DOI: 10.18632/

[81] Li Q, Jia H, Li H, Dong C, Wang Y, Zou Z. LncRNA and mRNA expression profiles of glioblastoma multiforme (GBM) reveal the potential roles of lncRNAs in GBM pathogenesis. Tumour

[82] Zhang K, Li Q, Kang X, Wang Y, Wang S. Identification and functional characterization of lncRNAs acting as ceRNA involved in the malignant

[83] Wang WA, Lai LC, Tsai MH, Lu TP, Chuang EY. Development of a prediction model for radiosensitivity using the expression values of genes and long non-coding RNAs. Oncotarget. 2016;**7**:26739-26750. DOI: 10.18632/

Biology. 2016;**37**:14537-14552

progression of glioblastoma multiforme. Oncology Reports. 2016;**36**:2911-2925. DOI: 10.3892/

or.2016.5070

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[84] Izuogu OG, Alhasan AA,

[85] Ruan ZB, Chen GC, Ren Y, Zhu L. Expression profile of long non-coding RNAs during the differentiation of human umbilical cord derived mesenchymal stem cells into cardiomyocyte-like cells. Cytotechnology. 2018;**70**:1247-1260. DOI: 10.1007/s10616-018-0217-5

[86] Balci T, Yilmaz Susluer S, Kayabasi C, Ozmen Yelken B, Biray Avci C, Gunduz C. Analysis of dysregulated

Mellough C, Collin J, Gallon R, Hyslop J, et al. Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular. BMC Genomics. 2018;**19**:276. DOI: 10.1186/

oncotarget.9569

#### *Potential Use of Long Noncoding RNAs as Biomarkers for Astrocytoma DOI: http://dx.doi.org/10.5772/intechopen.80561*

[80] Cao Y, Wang P, Ning S, Xiao W, Xiao B, Li X. Identification of prognostic biomarkers in glioblastoma using a long non-coding RNA-mediated, competitive endogenous RNA network. Oncotarget. 2016;**7**:41737-41747. DOI: 10.18632/ oncotarget.9569

*Primary Intracranial Tumors*

circulating long non-coding RNAs (lncRNAs) (LincRNA-p21, GAS 5, HOTAIR) predict the treatment

[68] Zidan HE, Karam RA, El-Seifi OS, Abd Elrahman TM. Circulating long non-coding RNA MALAT1 expression as molecular biomarker in Egyptian patients with breast cancer. Cancer Genetics. 2018;**220**:32-37. DOI: 10.1016/j.cancergen.2017.11.005

[69] Zhang R, Xia Y, Wang Z, Zheng J, Chen Y, Li X, et al. Serum long non coding RNA MALAT-1 protected by exosomes is up-regulated and promotes cell proliferation and migration in nonsmall cell lung cancer. Biochemical and Biophysical Research Communications. 2017;**490**(2):406-414. DOI: 10.1016/j.

[70] He B, Zeng J, Chao W, Chen X, Huang Y, Deng K, et al. Serum long noncoding RNAs MALAT1, AFAP1-AS1 and AL359062 as diagnostic and prognostic

biomarkers for nasopharyngeal carcinoma. Oncotarget. 2017;**8**:41166- 41177. DOI: 10.18632/oncotarget.17083

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target for castration resistant prostate cancer. The Journal of Urology. 2013;**190**:2278-2287. DOI: 10.1016/j.

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[75] Jalali S, Gandhi S, Scaria V. Distinct and modular organization of protein interacting sites in long non-coding RNAs. Frontiers in Molecular Biosciences. 2018;**5**:27. DOI: 10.3389/

[76] Li G, Liu K, Du X. Long non-coding RNA TUG1 promotes proliferation and inhibits apoptosis of osteosarcoma cells by sponging miR-132-3p and upregulating SOX4 expression. Yonsei Medical Journal. 2018;**59**:226-235. DOI:

[77] Sun XJ, Wang Q, Guo B, Liu XY, Wang B. Identification of skin-related lncRNAs as potential biomarkers that involved in Wnt pathways in keloids. Oncotarget. 2017;**8**:34236-34244. DOI:

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and Clinical Oncology. 2015;**141**:

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827-838. DOI: 10.1007/ s00432-014-1861-6

**58**

[81] Li Q, Jia H, Li H, Dong C, Wang Y, Zou Z. LncRNA and mRNA expression profiles of glioblastoma multiforme (GBM) reveal the potential roles of lncRNAs in GBM pathogenesis. Tumour Biology. 2016;**37**:14537-14552

[82] Zhang K, Li Q, Kang X, Wang Y, Wang S. Identification and functional characterization of lncRNAs acting as ceRNA involved in the malignant progression of glioblastoma multiforme. Oncology Reports. 2016;**36**:2911-2925. DOI: 10.3892/ or.2016.5070

[83] Wang WA, Lai LC, Tsai MH, Lu TP, Chuang EY. Development of a prediction model for radiosensitivity using the expression values of genes and long non-coding RNAs. Oncotarget. 2016;**7**:26739-26750. DOI: 10.18632/ oncotarget.8496

[84] Izuogu OG, Alhasan AA, Mellough C, Collin J, Gallon R, Hyslop J, et al. Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular. BMC Genomics. 2018;**19**:276. DOI: 10.1186/ s12864-018-4660-7

[85] Ruan ZB, Chen GC, Ren Y, Zhu L. Expression profile of long non-coding RNAs during the differentiation of human umbilical cord derived mesenchymal stem cells into cardiomyocyte-like cells. Cytotechnology. 2018;**70**:1247-1260. DOI: 10.1007/s10616-018-0217-5

[86] Balci T, Yilmaz Susluer S, Kayabasi C, Ozmen Yelken B, Biray Avci C, Gunduz C. Analysis of dysregulated

long non-coding RNA expressions in glioblastoma cells. Gene. 2016;**590**: 120-122. DOI: 10.1016/j. gene.2016.06.024

[87] Zhang S, Zhao BS, Zhou A, Lin K, Zheng S, Lu Z, et al. m6A demethylase ALKBH5 maintains tumorigenicity of glioblastoma stem-like cells by sustaining FOXM1 expression and cell proliferation program. Cancer Cell. 2017;**31**:591-606. e6. DOI: 10.1016/j.ccell.2017.02.013

[88] Ma X, Shao C, Jin Y, Wang H, Meng Y. Long non-coding RNAs: A novel endogenous source for the generation of Dicer-like 1-dependent small RNAs in Arabidopsis thaliana. RNA Biology. 2014;**11**:373-390. DOI: 10.4161/rna.28725

[89] Gaiti F, Hatleberg WL, Tanurdžić M, Degnan BM. Sponge long noncoding RNAs are expressed in specific cell types and conserved networks. Noncoding RNA. 2018;**4**:E6. DOI: 10.3390/ncrna4010006

[90] Zhou Y, Meng X, Chen S, Li W, Li D, Singer R, et al. IMP1 regulates UCA1-mediated cell invasion through facilitating UCA1 decay and decreasing the sponge effect of UCA1 for miR-122-5p. Breast Cancer Research. 2018;**20**:32. DOI: 10.1186/ s13058-018-0959-1

[91] Gong W, Zheng J, Liu X, Ma J, Liu Y, Xue Y. Knockdown of NEAT1 restrained the malignant progression of glioma stem cells by activating microRNA let-7e. Oncotarget. 2016;**7**:62208-62223. DOI: 10.18632/oncotarget.11403

[92] Li W, Jiang P, Sun X, Xu S, Ma X, Zhan R. Suppressing H19 modulates tumorigenicity and stemness in U251 and U87MG glioma cells. Cellular and Molecular Neurobiology. 2016;**36**(8):1219-1227

[93] Yao Y, Ma J, Xue Y, Wang P, Li Z, Liu J, et al. Knockdown of long non-coding

RNA XIST exerts tumor-suppressive functions in human glioblastoma stem cells by up-regulating miR-152. Cancer Letters. 2015;**359**:75-86. DOI: 10.1016/j. canlet.2014.12.051

[94] Cai H, Liu X, Zheng J, Xue Y, Ma J, Li Z, et al. Long non-coding RNA taurine upregulated 1 enhances tumorinduced angiogenesis through inhibiting microRNA-299 in human glioblastoma. Oncogene. 2017;**36**:318-331. DOI: 10.1038/onc.2016.212

[95] Ren J, Yang Y, Xue J, Xi Z, Hu L, Pan SJ, et al. Long noncoding RNA SNHG7 promotes the progression and growth of glioblastoma via inhibition of miR-5095. Biochemical and Biophysical Research Communications. 2018;**496**:712-718. DOI: 10.1016/j.bbrc.2018.01.109

[96] Li DX, Fei XR, Dong YF, Cheng CD, Yang Y, Deng XF, et al. The long noncoding RNA CRNDE acts as a ceRNA and promotes glioma malignancy by preventing miR-136-5p-mediated downregulation of Bcl-2 and Wnt2. Oncotarget. 2017;**8**:88163-88178. DOI: 10.18632/oncotarget.21513

[97] Liu C, Sun Y, She X, Tu C, Cheng X, Wang L, et al. CASC2c as an unfavorable prognosis factor interacts with miR-101 to mediate astrocytoma tumorigenesis. Cell Death & Disease. 2017;**8**:e2639. DOI: 10.1038/cddis.2017.11

[98] Chakravadhanula M, Ozols VV, Hampton CN, Zhou L, Catchpoole D, Bhardwaj RD. Expression of the HOX genes and HOTAIR in a typical teratoid rhabdoid tumors and other pediatric brain tumors. Cancer Genetics. 2014;**207**:425-428. DOI: 10.1016/j. cancergen.2014.05.014

[99] Yan ZY, Sun XC. LincRNA-ROR functions as a ceRNA to regulate Oct4, Sox2, and Nanog expression by sponging miR-145 and its effect on biologic characteristics of colonic cancer stem cells. Zhonghua Bing Li Xue Za

Zhi. 2018;**47**:284-290. DOI: 10.3760/ cma.j.issn.0529-5807.2018.04.011

[100] Li C, Lu L, Feng B, Zhang K, Han S, Hou D, et al. The lincRNA-ROR/ miR-145 axis promotes invasion and metastasis in hepatocellular carcinoma via induction of epithelial-mesenchymal transition by targeting ZEB2. Scientific Reports. 2017;**7**:4637. DOI: 10.1038/ s41598-017-04113-w

[101] Feng S, Yao J, Chen Y, Geng P, Zhang H, Ma X, et al. Expression and functional role of reprogrammingrelated long noncoding RNA (lincRNA-ROR) in glioma. Journal of Molecular Neuroscience. 2015;**56**:623-630. DOI: 10.1007/s12031-014-0488-z

**61**

**Chapter 4**

**Abstract**

ing safety and effectiveness.

can bring catastrophic consequences.

**1. Introduction**

Neurosurgical Tools to Improve

Safety and Survival in Patients

Neuronavigation, MRI, and 5-ALA

*Guilherme Augusto de Souza Machado and Ricardo Ramina*

This chapter describes the usefulness of surgical technologies such as intraoperative MRI, 5-ALA fluorescence-guided surgery, and neuronavigation as tools to make brain tumor resections safer and more effective. The focuses are practical aspects and the relevant literature regarding the impact of their use in avoidance of complications, improvement in survival rates, and some tips and tricks acquired in the experience of our department. All three strategies have an important role in neuro-oncological surgery. The future probably will prove that the combination of these tools, selected case by case, is the best way to achieve the best results regard-

**Keywords:** neurosurgical procedures, brain neoplasms, neuronavigation,

In all areas of science and knowledge, technology development is thought to bring solutions that optimize process, reduce costs, and make things safer. Brain tumor resection is a routine procedure in neurosurgical practice. In most of the cases, complete surgical resection remains as the gold standard of treatment. But some cases are real challenges to the neurosurgical team. Deep-seated tumors demand planned pathways to achieve it considering functions of each area of the brain, including white fiber tracts to avoid injury related to the approach. Besides eloquent area involvement, in some cases, despite simple or complex approaches, some aspects of the lesion turns them more difficult to resect such as its consistency, adherence to neighboring structures, and the presence of a well-defined cleavage plan. Neurosurgery has this cardinal aspect that every structure matters and injuries

Depending on the aggressiveness of the tumor, the tolerance to incomplete resection changes. But, for example, in benign tumors with incomplete resection, remnants can be followed by the "watch-and-wait" policy. Only in case of progression, a new decision should be done: reoperation, complementary treatment such

fluorescence-guided surgery, magnetic resonance imaging

with Intracranial Tumors:

*Luis Fernando Moura da Silva,* 

#### **Chapter 4**

*Primary Intracranial Tumors*

canlet.2014.12.051

10.1038/onc.2016.212

RNA XIST exerts tumor-suppressive functions in human glioblastoma stem cells by up-regulating miR-152. Cancer Letters. 2015;**359**:75-86. DOI: 10.1016/j.

Zhi. 2018;**47**:284-290. DOI: 10.3760/ cma.j.issn.0529-5807.2018.04.011

[100] Li C, Lu L, Feng B, Zhang K, Han S, Hou D, et al. The lincRNA-ROR/ miR-145 axis promotes invasion and metastasis in hepatocellular carcinoma via induction of epithelial-mesenchymal transition by targeting ZEB2. Scientific Reports. 2017;**7**:4637. DOI: 10.1038/

[101] Feng S, Yao J, Chen Y, Geng P, Zhang H, Ma X, et al. Expression and functional role of reprogrammingrelated long noncoding RNA (lincRNA-ROR) in glioma. Journal of Molecular Neuroscience. 2015;**56**:623-630. DOI:

10.1007/s12031-014-0488-z

s41598-017-04113-w

[94] Cai H, Liu X, Zheng J, Xue Y, Ma J, Li Z, et al. Long non-coding RNA taurine upregulated 1 enhances tumorinduced angiogenesis through inhibiting microRNA-299 in human glioblastoma. Oncogene. 2017;**36**:318-331. DOI:

[95] Ren J, Yang Y, Xue J, Xi Z, Hu L, Pan SJ, et al. Long noncoding RNA SNHG7 promotes the progression and growth of glioblastoma via inhibition of miR-5095. Biochemical and Biophysical Research Communications. 2018;**496**:712-718. DOI: 10.1016/j.bbrc.2018.01.109

[96] Li DX, Fei XR, Dong YF, Cheng CD, Yang Y, Deng XF, et al. The long noncoding RNA CRNDE acts as a ceRNA and promotes glioma malignancy by preventing miR-136-5p-mediated downregulation of Bcl-2 and Wnt2. Oncotarget. 2017;**8**:88163-88178. DOI:

[97] Liu C, Sun Y, She X, Tu C, Cheng X, Wang L, et al. CASC2c as an unfavorable prognosis factor interacts with miR-101 to mediate astrocytoma tumorigenesis. Cell Death & Disease. 2017;**8**:e2639.

10.18632/oncotarget.21513

DOI: 10.1038/cddis.2017.11

cancergen.2014.05.014

[98] Chakravadhanula M, Ozols VV, Hampton CN, Zhou L, Catchpoole D, Bhardwaj RD. Expression of the HOX genes and HOTAIR in a typical teratoid rhabdoid tumors and other pediatric brain tumors. Cancer Genetics. 2014;**207**:425-428. DOI: 10.1016/j.

[99] Yan ZY, Sun XC. LincRNA-ROR functions as a ceRNA to regulate Oct4, Sox2, and Nanog expression by sponging miR-145 and its effect on biologic characteristics of colonic cancer stem cells. Zhonghua Bing Li Xue Za

**60**

## Neurosurgical Tools to Improve Safety and Survival in Patients with Intracranial Tumors: Neuronavigation, MRI, and 5-ALA

*Luis Fernando Moura da Silva, Guilherme Augusto de Souza Machado and Ricardo Ramina*

#### **Abstract**

This chapter describes the usefulness of surgical technologies such as intraoperative MRI, 5-ALA fluorescence-guided surgery, and neuronavigation as tools to make brain tumor resections safer and more effective. The focuses are practical aspects and the relevant literature regarding the impact of their use in avoidance of complications, improvement in survival rates, and some tips and tricks acquired in the experience of our department. All three strategies have an important role in neuro-oncological surgery. The future probably will prove that the combination of these tools, selected case by case, is the best way to achieve the best results regarding safety and effectiveness.

**Keywords:** neurosurgical procedures, brain neoplasms, neuronavigation, fluorescence-guided surgery, magnetic resonance imaging

#### **1. Introduction**

In all areas of science and knowledge, technology development is thought to bring solutions that optimize process, reduce costs, and make things safer. Brain tumor resection is a routine procedure in neurosurgical practice. In most of the cases, complete surgical resection remains as the gold standard of treatment. But some cases are real challenges to the neurosurgical team. Deep-seated tumors demand planned pathways to achieve it considering functions of each area of the brain, including white fiber tracts to avoid injury related to the approach. Besides eloquent area involvement, in some cases, despite simple or complex approaches, some aspects of the lesion turns them more difficult to resect such as its consistency, adherence to neighboring structures, and the presence of a well-defined cleavage plan. Neurosurgery has this cardinal aspect that every structure matters and injuries can bring catastrophic consequences.

Depending on the aggressiveness of the tumor, the tolerance to incomplete resection changes. But, for example, in benign tumors with incomplete resection, remnants can be followed by the "watch-and-wait" policy. Only in case of progression, a new decision should be done: reoperation, complementary treatment such

as radiosurgery, radiotherapy, chemotherapy, or immunotherapy (depending on its characteristics). In cases of malignant tumors such as gliomas and metastasis, the extent of resection (EOR) is directly related to recurrence and survival. Incomplete resection for these patients should be only discussed if the risk of neurological injury is high. Obviously, *not to harm* is always the most important principle. Increase in survival only makes sense if accompanied by quality.

Even with intense microsurgical training, the multidisciplinary treatment challenge remains. Some strategies such as intraoperative monitoring, awake surgery, and intraoperative histology (margin biopsy) can be used to improve the goal. In this chapter neuronavigation, intraoperative magnet resonance imaging (ioMRI), and 5-aminolevulinic acid (5-ALA) are discussed as tools to improve the safety and efficacy of intracranial tumor resection.

#### **2. Neuronavigation**

Neuronavigation has a fundamental role in contemporary neurosurgery. This tool allowed surgeons to better individualize treatment tailoring craniotomies and localizing structures or lesions intraoperatively. It consists of a frameless stereotactic system of localization based on pre- or intraoperative image data. The data used can be a fusion of different techniques like CT, MRI anatomical or functional sequences, US, or PET-CT.

The most important indications of the use of navigation are planning of craniotomy, intraoperative localization of lesions or structures, and guided biopsies.

#### **2.1 Craniotomy planning**

Using metastasis as an example, neurosurgeons increasingly attempt to resect as much tumor tissue as possible to impact disease control and survival. If a patient has four metastases of 4 centimeters that can be completely resected, this procedure should be indicated. Even if multiple craniotomies are needed, this should not dissuade the surgeon to indicate it [1]. In these special cases, considering that these metastases are in different places of the brain, neuronavigation makes a real difference with a tailored and focused approach to each lesion.

Neuronavigation allows direct access to the lesions, even if small, reducing the size of craniotomy, dural opening, unwanted manipulation of the brain, duration of surgery, blood loss, volume of the tissue to be healed, length of stay in the hospital, recurrence rate, time to be available for complementary treatment if needed, and costs and improving recurrence-free survival (RFS) and performance status [2, 3].

In cases of ventricular endoscopic approach, neuronavigation can also be very useful. Some patients with pineal or third ventricle-located tumors with noncommunicating hydrocephalus, for example, need third ventriculostomy and biopsy. In order to offer a direct straightforward approach, avoiding lesions of related structures, two different trepanations/small craniotomies can be performed guided by neuronavigation (**Figure 1**).

Besides defining the position of the craniotomy, still regarding surgical approach, neuronavigation can help in many ways to improve safety of neurosurgical procedures. Identification of sinus position in retrosigmoid craniotomy has been demonstrated successfully avoiding unnecessary sinus exposition reducing complications [4]. Also, superficial vein identification before dural opening was demonstrated, eliminating the need to use indocyanine to make a transdural analysis, for

**63**

tumor type) [6].

**Figure 1.**

*achieved their targets.*

brain. Loss of 20 cm3

*Neurosurgical Tools to Improve Safety and Survival in Patients with Intracranial Tumors…*

example [5]. These strategies reduce also the risk of bleeding and venous closure,

*Patient with indication of a third ventricle lesion and third ventriculostomy. Neuronavigation plan of two different craniotomies to straightforward approach avoiding critical structures. The yellow trajectory with direct approach to Monro's foramen and Liliequist membrane. The green target to direct approach of a third ventricle lesion. Approaches point distant 7 cm. Two small craniotomies were preferred and successfully* 

When used to localize superficial lesions/anatomical structures and tailor surgical approach, neuronavigation has high accuracy, being a very reliable tool, because the intracranial compartment remains untouched. However, the main drawback of

The accuracy between preoperative images and real intraoperative anatomy is influenced during many surgical steps that result in dislocation of structures, called brain shift. Several surgical aspects are not related to wrong landmark selection, hardware movement, or software algorithm influence on brain shift. The causes are classified as physical (hardware movement, patient position, and gravity), surgical (fluid loss, tissue loss, and surgical equipment), and biological (mannitol and

The effect of gravity is an important physical factor of brain shift. It interacts with two surgical causes: fluid loss and tissue loss. After tumor resection or relevant CSF drainage, adjacent healthy tissue becomes unsupported with sagging of the

strated to result in the shift of the anterior commissure by approximately 2 mm [7]. Mannitol administration during surgery also can influence, especially in cases

of CSF in deep brain stimulation (DBS) surgery was demon-

which can have a negative impact on surgical outcome.

**2.2 Intraoperative localization of structures/lesions**

neuronavigation is that it is not a real-time evaluation.

*DOI: http://dx.doi.org/10.5772/intechopen.81211*

*Neurosurgical Tools to Improve Safety and Survival in Patients with Intracranial Tumors… DOI: http://dx.doi.org/10.5772/intechopen.81211*

#### **Figure 1.**

*Primary Intracranial Tumors*

as radiosurgery, radiotherapy, chemotherapy, or immunotherapy (depending on its characteristics). In cases of malignant tumors such as gliomas and metastasis, the extent of resection (EOR) is directly related to recurrence and survival. Incomplete resection for these patients should be only discussed if the risk of neurological injury is high. Obviously, *not to harm* is always the most important principle.

Even with intense microsurgical training, the multidisciplinary treatment challenge remains. Some strategies such as intraoperative monitoring, awake surgery, and intraoperative histology (margin biopsy) can be used to improve the goal. In this chapter neuronavigation, intraoperative magnet resonance imaging (ioMRI), and 5-aminolevulinic acid (5-ALA) are discussed as tools to improve the safety and

Neuronavigation has a fundamental role in contemporary neurosurgery. This tool allowed surgeons to better individualize treatment tailoring craniotomies and localizing structures or lesions intraoperatively. It consists of a frameless stereotactic system of localization based on pre- or intraoperative image data. The data used can be a fusion of different techniques like CT, MRI anatomical or functional

The most important indications of the use of navigation are planning of craniotomy, intraoperative localization of lesions or structures, and guided biopsies.

Using metastasis as an example, neurosurgeons increasingly attempt to resect as much tumor tissue as possible to impact disease control and survival. If a patient has four metastases of 4 centimeters that can be completely resected, this procedure should be indicated. Even if multiple craniotomies are needed, this should not dissuade the surgeon to indicate it [1]. In these special cases, considering that these metastases are in different places of the brain, neuronavigation makes a real differ-

Neuronavigation allows direct access to the lesions, even if small, reducing the size of craniotomy, dural opening, unwanted manipulation of the brain, duration of surgery, blood loss, volume of the tissue to be healed, length of stay in the hospital, recurrence rate, time to be available for complementary treatment if needed, and costs and improving recurrence-free survival (RFS) and performance

In cases of ventricular endoscopic approach, neuronavigation can also be very useful. Some patients with pineal or third ventricle-located tumors with noncommunicating hydrocephalus, for example, need third ventriculostomy and biopsy. In order to offer a direct straightforward approach, avoiding lesions of related structures, two different trepanations/small craniotomies can be performed guided by

Besides defining the position of the craniotomy, still regarding surgical approach, neuronavigation can help in many ways to improve safety of neurosurgical procedures. Identification of sinus position in retrosigmoid craniotomy has been demonstrated successfully avoiding unnecessary sinus exposition reducing complications [4]. Also, superficial vein identification before dural opening was demonstrated, eliminating the need to use indocyanine to make a transdural analysis, for

ence with a tailored and focused approach to each lesion.

Increase in survival only makes sense if accompanied by quality.

efficacy of intracranial tumor resection.

**2. Neuronavigation**

sequences, US, or PET-CT.

**2.1 Craniotomy planning**

**62**

status [2, 3].

neuronavigation (**Figure 1**).

*Patient with indication of a third ventricle lesion and third ventriculostomy. Neuronavigation plan of two different craniotomies to straightforward approach avoiding critical structures. The yellow trajectory with direct approach to Monro's foramen and Liliequist membrane. The green target to direct approach of a third ventricle lesion. Approaches point distant 7 cm. Two small craniotomies were preferred and successfully achieved their targets.*

example [5]. These strategies reduce also the risk of bleeding and venous closure, which can have a negative impact on surgical outcome.

#### **2.2 Intraoperative localization of structures/lesions**

When used to localize superficial lesions/anatomical structures and tailor surgical approach, neuronavigation has high accuracy, being a very reliable tool, because the intracranial compartment remains untouched. However, the main drawback of neuronavigation is that it is not a real-time evaluation.

The accuracy between preoperative images and real intraoperative anatomy is influenced during many surgical steps that result in dislocation of structures, called brain shift. Several surgical aspects are not related to wrong landmark selection, hardware movement, or software algorithm influence on brain shift. The causes are classified as physical (hardware movement, patient position, and gravity), surgical (fluid loss, tissue loss, and surgical equipment), and biological (mannitol and tumor type) [6].

The effect of gravity is an important physical factor of brain shift. It interacts with two surgical causes: fluid loss and tissue loss. After tumor resection or relevant CSF drainage, adjacent healthy tissue becomes unsupported with sagging of the brain. Loss of 20 cm3 of CSF in deep brain stimulation (DBS) surgery was demonstrated to result in the shift of the anterior commissure by approximately 2 mm [7]. Mannitol administration during surgery also can influence, especially in cases

#### *Primary Intracranial Tumors*

where high intracranial pressure levels or large edema are present. Neuronavigation does not contraindicate the administration of mannitol. But its use should be used judiciously, not routinely. Regarding biological causes, some authors observed an association between tumor biology and unique patterns of the shift. But the reasons are not well understood, and more studies should analyze this before generalization can be made [6].

Previously, many attempts to identify intra-axial tumor margins using neuronavigation were performed, but it could be done with reliable results due to brain shift. Other options such as fluorescence and ioMRI have superior results. Otherwise, targets located in fixed structures like the bone, brainstem, and skull base meninges tolerate better intracranial manipulation. The dural implantation of a skull base meningioma, for example, can be checked with navigation during the procedure, because it will suffer few the effect of brain shift. But as accuracy should be low, the shift needs to be weighted in every procedure. In brainstem biopsies, the passage of the biopsy needle through the parenchyma does not change target position significantly; but if the trajectory accidentally passes through the ventricle with CSF drainage, the brain shift can have significant influence hindering correct target achievement.

Correction of brain shift can be done using intraoperative MRI to update the navigation; or other real-time exams, where ioMRI is not available, can be performed to compare and adjust it such as ultrasound (US) [2, 8].

Ultrasound is a fast, cheap, real-time, and commonly available exam. Although its image quality is not comparable to MRI, it plays an important role in brain tumor surgery. After craniotomy, for example, brain shift can occur even if brain deformation is still not present. Placing the probe directly on the dura and superimposing identifiable structures on both techniques can confirm if neuronavigation is still adequate. The main concept of using intraoperative US is that the focus is not on diagnosis but on localization. Undoubtedly, MRI is the gold standard exam to analyze brain lesions and define diagnosis. But to locate lesions and some structures, US is sometimes enough with the advantage of being easily and real-time performed. ioUS can affect the decision of further resection in 59% of cases [9]. Association of these two techniques offers the possibility to overcome the limitations of each one separately improving the safety of the procedure (**Figure 2**).

Another important intraoperative use of navigation is in the association with other tools such as awake surgery and transcranial magnetic stimulation (TMS). Navigated TMS-based DTI-fiber tracking in awake surgery has been demonstrated

#### **Figure 2.**

*MRI of a hemorrhagic tumor with ioUS view. Easy identification of both limits and differences of cystic and solid components.*

**65**

**Figure 3.**

*Neurosurgical Tools to Improve Safety and Survival in Patients with Intracranial Tumors…*

as a useful tool in the treatment of highly eloquent gliomas with results considering craniotomy size, EOR, duration of surgery, postoperative deficits, Karnofsky Performance Scale (KPS), and length of stay in the hospital [10]. Association of image-guided resection of glioblastoma in eloquent brain areas facilitated by laser surface thermal therapy was also demonstrated with favorable long-term results. This strategy allowed the higher rates of complete resection and improved overall survival without the negative effect on postoperative functional status [11].

Biopsy of intracranial lesion is an important diagnostic tool in neurosurgery. With the progression of genetic and molecular characterization of tumors, biopsy becomes even more important in deep-seated lesions with difficult access such as in

Frame-based intracranial biopsy has been the gold standard technique for intracranial biopsy for a long time. The stereotactic system provides excellent precision of target achievement. After development of neuronavigation, the frameless intracranial biopsy, guided by neurosurgery, has evolved a lot. Both methods have similar effectiveness to histological diagnosis. But a frameless system has become increasingly the first choice among neurosurgeons due to reduced equipment size; reduced work of calculations to define targets, entry point, and trajectory; patient's comfort; reduced surgical time with navigation; and the absence of the need to redo

The use of real-time ioMRI-guided biopsy has also been compared to framebased and frameless neuronavigation-guided biopsy with comparable diagnostic yield in patients with no prior treatment. ioMRI-guided biopsy was associated with short hospital stay [12]. But ioMRI is not available in many places, and navigation-

The most common complications of deep biopsies are brain shift, hemorrhage, and failure in representativeness of samples. Brain shift was discussed before in Section 2.2. Hemorrhage can be directly related to biopsy (intratumoral) or to the trajectory

guided frameless biopsy continues as the first option in most departments. In pineal tumors, as some patients have hydrocephalus, endoscopic biopsy

associated with third ventriculostomy is a feasible option, as cited before.

*MRI of a frameless-based biopsy (neuronavigation guided) of a deep-seated lesion. Trajectory planning* 

*without any passage through ventricular system to avoid CSF drainage and brain shift.*

image examination after placement of the frame (**Figure 3**) [12].

*DOI: http://dx.doi.org/10.5772/intechopen.81211*

the thalamus, brainstem, and pineal gland.

**2.3 Guided biopsy**

*Neurosurgical Tools to Improve Safety and Survival in Patients with Intracranial Tumors… DOI: http://dx.doi.org/10.5772/intechopen.81211*

as a useful tool in the treatment of highly eloquent gliomas with results considering craniotomy size, EOR, duration of surgery, postoperative deficits, Karnofsky Performance Scale (KPS), and length of stay in the hospital [10]. Association of image-guided resection of glioblastoma in eloquent brain areas facilitated by laser surface thermal therapy was also demonstrated with favorable long-term results. This strategy allowed the higher rates of complete resection and improved overall survival without the negative effect on postoperative functional status [11].

#### **2.3 Guided biopsy**

*Primary Intracranial Tumors*

can be made [6].

target achievement.

where high intracranial pressure levels or large edema are present. Neuronavigation does not contraindicate the administration of mannitol. But its use should be used judiciously, not routinely. Regarding biological causes, some authors observed an association between tumor biology and unique patterns of the shift. But the reasons are not well understood, and more studies should analyze this before generalization

Previously, many attempts to identify intra-axial tumor margins using neuronavigation were performed, but it could be done with reliable results due to brain shift. Other options such as fluorescence and ioMRI have superior results. Otherwise, targets located in fixed structures like the bone, brainstem, and skull base meninges tolerate better intracranial manipulation. The dural implantation of a skull base meningioma, for example, can be checked with navigation during the procedure, because it will suffer few the effect of brain shift. But as accuracy should be low, the shift needs to be weighted in every procedure. In brainstem biopsies, the passage of the biopsy needle through the parenchyma does not change target position significantly; but if the trajectory accidentally passes through the ventricle with CSF drainage, the brain shift can have significant influence hindering correct

Correction of brain shift can be done using intraoperative MRI to update the navigation; or other real-time exams, where ioMRI is not available, can be per-

Ultrasound is a fast, cheap, real-time, and commonly available exam. Although its image quality is not comparable to MRI, it plays an important role in brain tumor surgery. After craniotomy, for example, brain shift can occur even if brain deformation is still not present. Placing the probe directly on the dura and superimposing identifiable structures on both techniques can confirm if neuronavigation is still adequate. The main concept of using intraoperative US is that the focus is not on diagnosis but on localization. Undoubtedly, MRI is the gold standard exam to analyze brain lesions and define diagnosis. But to locate lesions and some structures, US is sometimes enough with the advantage of being easily and real-time performed. ioUS can affect the decision of further resection in 59% of cases [9]. Association of these two techniques offers the possibility to overcome the limitations of each one separately improving the safety of the procedure (**Figure 2**). Another important intraoperative use of navigation is in the association with other tools such as awake surgery and transcranial magnetic stimulation (TMS). Navigated TMS-based DTI-fiber tracking in awake surgery has been demonstrated

*MRI of a hemorrhagic tumor with ioUS view. Easy identification of both limits and differences of cystic and* 

formed to compare and adjust it such as ultrasound (US) [2, 8].

**64**

**Figure 2.**

*solid components.*

Biopsy of intracranial lesion is an important diagnostic tool in neurosurgery. With the progression of genetic and molecular characterization of tumors, biopsy becomes even more important in deep-seated lesions with difficult access such as in the thalamus, brainstem, and pineal gland.

Frame-based intracranial biopsy has been the gold standard technique for intracranial biopsy for a long time. The stereotactic system provides excellent precision of target achievement. After development of neuronavigation, the frameless intracranial biopsy, guided by neurosurgery, has evolved a lot. Both methods have similar effectiveness to histological diagnosis. But a frameless system has become increasingly the first choice among neurosurgeons due to reduced equipment size; reduced work of calculations to define targets, entry point, and trajectory; patient's comfort; reduced surgical time with navigation; and the absence of the need to redo image examination after placement of the frame (**Figure 3**) [12].

The use of real-time ioMRI-guided biopsy has also been compared to framebased and frameless neuronavigation-guided biopsy with comparable diagnostic yield in patients with no prior treatment. ioMRI-guided biopsy was associated with short hospital stay [12]. But ioMRI is not available in many places, and navigationguided frameless biopsy continues as the first option in most departments.

In pineal tumors, as some patients have hydrocephalus, endoscopic biopsy associated with third ventriculostomy is a feasible option, as cited before.

The most common complications of deep biopsies are brain shift, hemorrhage, and failure in representativeness of samples. Brain shift was discussed before in Section 2.2. Hemorrhage can be directly related to biopsy (intratumoral) or to the trajectory

#### **Figure 3.**

*MRI of a frameless-based biopsy (neuronavigation guided) of a deep-seated lesion. Trajectory planning without any passage through ventricular system to avoid CSF drainage and brain shift.*

(needle track). Hemorrhage is avoided with preoperative evaluation of coagulation marking a trajectory that avoids any arterial or venous structure that is achieved by using multiplanar reconstruction of image [13]. Representativeness of sample has been traditionally analyzed with adequate target definition in image and intraoperative pathology/frozen section. More recently fluorescence has been associated with biopsy procedures with good correlation compared to frozen section to check acquisition of relevant samples. Both 5-ALA and fluorescein were evaluated [14, 15].

#### **3. Magnet resonance imaging (MRI)**

In neuro-oncological surgery, complete resection with preservation of functions and quality of life is normally the goal of the procedure. Defining complete resection intraoperatively is easier in extra-axial tumors than in intra-axial tumors such as low-grade gliomas. A surgeon's perception of gross total resection (GTR) usually relies on the visual and tactile aspects of tumor boundaries. Studies compared the surgeon's perception with imaging findings and determined inaccuracy and overestimation of intraoperative EOR by up to a factor of 3 [16–18]. Young adult patients with low-grade glioma who undergo a neurosurgeon-determined GTR have a higher than 50% risk of tumor progression in 5 years postoperatively [18].

The surgeon's experience also was not significant to define additional resection. The positive predictive value (PPV) of the surgeon's expectation was shown to be high (93.1%). On the other hand, and most importantly, the ability to exclude additional resection from the intraoperative impression was very low (43.6%) [19].

This is a major concern specially in tumors that EOR is proven to be related with recurrence and survival.

Intraoperative or transoperative MRI emerges exactly in this context to clearly determine if GTR was achieved or not. Literature suggests rates of further operative resection secondary to ioMRI evaluation range from 13.3 to 59.37%, confirming the impact of this tool on the extent of tumor resection [20, 21].

Analysis comparing EOR, GTR, and progression-free survival (PFS) and overall survival (OS) in patients with gliomas that underwent ioMRI also confirmed the benefit with improvement of these aspects. The author showed an increase in GTR rate of 24.1%. In 59.37% of cases that underwent ioMRI, further resection was needed [21]. Certainty of ioMRI can make surgeon more tolerant and relaxed, ending resection early relying on ioMRI evaluation. But even if this is considered, the improve in resection is substantial.

In complex located tumors, for example, insular gliomas, ioMRI check during awake craniotomy increased EOR in 15.1%. Considering that median EOR on ioMRI was 51.2% and after further resection was 84.5%, it is clear that ioMRI really impacts outcome [22].

Identification of margins is not always simple. It depends on the tumor type, MRI sequence analyzed, and surgical trauma with blood-brain barrier break. In cases of high-grade glioma surgery, PWI helps in identification of tumoral x nontumoral tissue. Another option is the use of a single layer of oxidized regenerated cellulose covering the cavity to enhance margin visualization in ioMRI. Being a hemostatic agent, it accelerates oxidation of oxyhemoglobin to metahemoglobin, which is paramagnetic, and, so, it has a hyperintense signal in T1 sequences. This layer of hyperintense line observed may be a useful marker of tumor resection borders in cerebral glioma surgery [23].

Pituitary tumors also benefit from ioMRI. A systematic review observed that complete radiological resection in patients whose procedure involved intraoperative ultrasound was 67.1% (range 63.5–77.8%) and endocrine remission was 88.4% (range

**67**

*Neurosurgical Tools to Improve Safety and Survival in Patients with Intracranial Tumors…*

76–100%) [24]. Studies with ioMRI also evidences the benefits with intraoperative unexpected residuals in up to 42% (range 15–83%) of cases, of which re-exploration was attempted in 36% (range 9–83%) and further tumor resection occurred in 33% (range 9–83%) of the cases [25]. But this paper considered low- and high-field ioMRI. In a study with 3 T ioMR, a complete resection was observed in 69% of the

Intraoperative image interpretation is even more difficult in transsphenoidal pituitary surgery than in glioma surgery, for example. This evaluation should only be done by an experienced neuroradiologist, because the literature shows relevant cases of false-positive leading to resection of normal tissue, in both ioMRI and ioUS [24, 25]. The Congress of Neurological Surgeons (CNS) suggested in 2016 that intraoperative images in nonfunctioning adenomas may help to improve overall gross total resection but at the cost of removing normal tissue [26]. So, we suggest weighting cost-benefit relation differently in nonfunctioning x functioning adeno-

In the beginning, ioMRI started with low-field strengths of 0.2–0.5 T. These units, although cheaper and requiring less spaces, take longer to perform scanning and produce low-quality images when compared to high-field (1.5 T and higher) equipment. Besides this, the possibility of advanced images such as DTI favors the

Cost is one of the most limiting factors to the spread of ioMRI. Additionally,

In order to overcome this limitation, the concept of "outside MRI" was proposed by Ramina et al. in 2010. In this strategy after completing the resection, oxidized regenerated cellulose is put to cover surgical cavity, and a partial closure of the dura is performed. The exposed dura is covered with cottonoid plates, and the skin is closed with running suture. A sterile plastic sheet covers the entire head to assure sterility and complete the preparation for MRI. The patient is conducted in the MRI-compatible bed through an internal special lift, designed for this purpose, to the MRI facilities. Time required to whole exam, since patient left OR and came back, was 25 min. No infection was observed [29]. Ahmadi et al. recently confirmed that inside ioMRI did not increase complications (hemorrhage, wound healing, and infection) in glioma surgery. In their publication the ioMRI procedure time was higher with a mean of 57 min [30]. "Outside MRI" has all advantages of "inside models" and the additional advantage of integrating neurosurgery/neuroradiology

5-ALA is a prodrug and leads to accumulation of protoporphyrin IX (PPIX) in gliomas and other tumor cells by an interaction with heme biosynthesis process. With special filters and blue/violet light, it is possible to see fluorescence of PPIX as light red or an intense pinkish color in a dark blue background. These filters and lights are usually part or an upgrade of surgical microscope. Normal brain tissue does not induce PPIX expression after ALA administration, and a high selectivity of malignant glioma cells is observed. When density of tumor cells in the tissue is

the price of the whole equipment and software and surgical and anesthesia equipment should be developed to be compatible with ioMRI environment. These adapted equipment are also expensive, which increases even more the investment on a magnet dedicated exclusively to intraoperative images. Besides this, in few years MRI equipment becomes obsolete with the need to change to maintain it

mas. But in an experienced team, good results can be achieved.

*DOI: http://dx.doi.org/10.5772/intechopen.81211*

use of high-field equipment [27, 28].

teams, which may lead to better results [29].

above 10%, fluorescence is expected to be present [31].

**4. 5-Aminolevulinic acid**

cases.

updated.

#### *Neurosurgical Tools to Improve Safety and Survival in Patients with Intracranial Tumors… DOI: http://dx.doi.org/10.5772/intechopen.81211*

76–100%) [24]. Studies with ioMRI also evidences the benefits with intraoperative unexpected residuals in up to 42% (range 15–83%) of cases, of which re-exploration was attempted in 36% (range 9–83%) and further tumor resection occurred in 33% (range 9–83%) of the cases [25]. But this paper considered low- and high-field ioMRI. In a study with 3 T ioMR, a complete resection was observed in 69% of the cases.

Intraoperative image interpretation is even more difficult in transsphenoidal pituitary surgery than in glioma surgery, for example. This evaluation should only be done by an experienced neuroradiologist, because the literature shows relevant cases of false-positive leading to resection of normal tissue, in both ioMRI and ioUS [24, 25]. The Congress of Neurological Surgeons (CNS) suggested in 2016 that intraoperative images in nonfunctioning adenomas may help to improve overall gross total resection but at the cost of removing normal tissue [26]. So, we suggest weighting cost-benefit relation differently in nonfunctioning x functioning adenomas. But in an experienced team, good results can be achieved.

In the beginning, ioMRI started with low-field strengths of 0.2–0.5 T. These units, although cheaper and requiring less spaces, take longer to perform scanning and produce low-quality images when compared to high-field (1.5 T and higher) equipment. Besides this, the possibility of advanced images such as DTI favors the use of high-field equipment [27, 28].

Cost is one of the most limiting factors to the spread of ioMRI. Additionally, the price of the whole equipment and software and surgical and anesthesia equipment should be developed to be compatible with ioMRI environment. These adapted equipment are also expensive, which increases even more the investment on a magnet dedicated exclusively to intraoperative images. Besides this, in few years MRI equipment becomes obsolete with the need to change to maintain it updated.

In order to overcome this limitation, the concept of "outside MRI" was proposed by Ramina et al. in 2010. In this strategy after completing the resection, oxidized regenerated cellulose is put to cover surgical cavity, and a partial closure of the dura is performed. The exposed dura is covered with cottonoid plates, and the skin is closed with running suture. A sterile plastic sheet covers the entire head to assure sterility and complete the preparation for MRI. The patient is conducted in the MRI-compatible bed through an internal special lift, designed for this purpose, to the MRI facilities. Time required to whole exam, since patient left OR and came back, was 25 min. No infection was observed [29]. Ahmadi et al. recently confirmed that inside ioMRI did not increase complications (hemorrhage, wound healing, and infection) in glioma surgery. In their publication the ioMRI procedure time was higher with a mean of 57 min [30]. "Outside MRI" has all advantages of "inside models" and the additional advantage of integrating neurosurgery/neuroradiology teams, which may lead to better results [29].

#### **4. 5-Aminolevulinic acid**

5-ALA is a prodrug and leads to accumulation of protoporphyrin IX (PPIX) in gliomas and other tumor cells by an interaction with heme biosynthesis process. With special filters and blue/violet light, it is possible to see fluorescence of PPIX as light red or an intense pinkish color in a dark blue background. These filters and lights are usually part or an upgrade of surgical microscope. Normal brain tissue does not induce PPIX expression after ALA administration, and a high selectivity of malignant glioma cells is observed. When density of tumor cells in the tissue is above 10%, fluorescence is expected to be present [31].

*Primary Intracranial Tumors*

recurrence and survival.

improve in resection is substantial.

borders in cerebral glioma surgery [23].

impacts outcome [22].

**3. Magnet resonance imaging (MRI)**

(needle track). Hemorrhage is avoided with preoperative evaluation of coagulation marking a trajectory that avoids any arterial or venous structure that is achieved by using multiplanar reconstruction of image [13]. Representativeness of sample has been traditionally analyzed with adequate target definition in image and intraoperative pathology/frozen section. More recently fluorescence has been associated with biopsy procedures with good correlation compared to frozen section to check acquisi-

tion of relevant samples. Both 5-ALA and fluorescein were evaluated [14, 15].

than 50% risk of tumor progression in 5 years postoperatively [18].

impact of this tool on the extent of tumor resection [20, 21].

In neuro-oncological surgery, complete resection with preservation of functions and quality of life is normally the goal of the procedure. Defining complete resection intraoperatively is easier in extra-axial tumors than in intra-axial tumors such as low-grade gliomas. A surgeon's perception of gross total resection (GTR) usually relies on the visual and tactile aspects of tumor boundaries. Studies compared the surgeon's perception with imaging findings and determined inaccuracy and overestimation of intraoperative EOR by up to a factor of 3 [16–18]. Young adult patients with low-grade glioma who undergo a neurosurgeon-determined GTR have a higher

The surgeon's experience also was not significant to define additional resection.

Intraoperative or transoperative MRI emerges exactly in this context to clearly determine if GTR was achieved or not. Literature suggests rates of further operative resection secondary to ioMRI evaluation range from 13.3 to 59.37%, confirming the

Analysis comparing EOR, GTR, and progression-free survival (PFS) and overall survival (OS) in patients with gliomas that underwent ioMRI also confirmed the benefit with improvement of these aspects. The author showed an increase in GTR rate of 24.1%. In 59.37% of cases that underwent ioMRI, further resection was needed [21]. Certainty of ioMRI can make surgeon more tolerant and relaxed, ending resection early relying on ioMRI evaluation. But even if this is considered, the

In complex located tumors, for example, insular gliomas, ioMRI check during awake craniotomy increased EOR in 15.1%. Considering that median EOR on ioMRI was 51.2% and after further resection was 84.5%, it is clear that ioMRI really

Identification of margins is not always simple. It depends on the tumor type, MRI sequence analyzed, and surgical trauma with blood-brain barrier break. In cases of high-grade glioma surgery, PWI helps in identification of tumoral x nontumoral tissue. Another option is the use of a single layer of oxidized regenerated cellulose covering the cavity to enhance margin visualization in ioMRI. Being a hemostatic agent, it accelerates oxidation of oxyhemoglobin to metahemoglobin, which is paramagnetic, and, so, it has a hyperintense signal in T1 sequences. This layer of hyperintense line observed may be a useful marker of tumor resection

Pituitary tumors also benefit from ioMRI. A systematic review observed that complete radiological resection in patients whose procedure involved intraoperative ultrasound was 67.1% (range 63.5–77.8%) and endocrine remission was 88.4% (range

The positive predictive value (PPV) of the surgeon's expectation was shown to be high (93.1%). On the other hand, and most importantly, the ability to exclude additional resection from the intraoperative impression was very low (43.6%) [19]. This is a major concern specially in tumors that EOR is proven to be related with

**66**

This is another tool to go further with the concept that tumor tissues are many times much more than what we see with normal light surgical microscopy or even contrast-enhanced MRI. A high association between contrast enhancement and PPIX fluorescence is observed. But it was shown that PPIX fluorescence in noncontrast-enhanced areas can be present with good correlation with the presence of tumor tissue. So, PPIX accumulations seem to be more sensitive to glioma detection than contrast-agent accumulation (**Figure 4**) [31, 32].

Fluoroethyl tyrosine PET has been demonstrated to have a good correlation with PPIX fluorescence in gliomas without typical glioblastoma imaging features [33]. Also, areas with high atypia in low grade or non-contrast enhancing in high grade suggested by PET could be confirmed with 5-ALA fluorescence. The explanation to these findings may be in the mechanism of each method. Contrast enhancement and sodium-fluorescein fluorescence have intraoperative correlation, and both occur due to disruption of blood-brain barrier, which is not specific from tumors. 5-ALA fluorescence and PET tracer uptake, in turn, occur due to specific metabolism of tumor tissue. 5-ALA may be even more special than PET because it does not consider only the general quantitative aspect of metabolism and goes beyond. Its mechanism relates to a metabolic phenomenon of a pathway typical from a tumor tissue and not from a normal tissue [34].

Other tumors than WHO IV gliomas have also been tested regarding fluorescence after 5-ALA administration. Literature shows results with approximately 15–20% of fluorescence with 5-ALA in low-grade gliomas, 85–100% in high-grade gliomas, and 55–80% in metastasis [32, 35, 36]. In our most recent data analysis from INC, we could observe 5-ALA-positive fluorescence in 97.7% cases of WHO IV gliomas, 90% cases of WHO III gliomas, 22.2% cases of WHO II gliomas, and 85.7% in cases of metastasis. The quality of fluorescence differs among tumor types. In low-grade gliomas, for example, with positive fluorescence we observed usually weak to mild with stronger foci in some cases (higher atypia); metastasis, on the other hand, usually shows mild to strong fluorescence (**Figure 5**).

During the procedure, the surgeon alternates between white light resection and blue/violet light resection. This is important because white light shows anatomy, structures, and blood better. Only the resection, specially boundaries, is guided by fluorescence. Blood, inclusive, may be a confounding factor, because it prevents the

#### **Figure 4.**

*Glioma patient operated on using 5-ALA. A and B show white light and blue-filter images with identifiable tumoral tissue on the cortex, clearly visible with blue filter and difficult to identify with white light. C and D show areas of tumor with intense fluorescence in blue filter, corresponding to contrast-enhanced area shown by neuronavigation in E.*

**69**

tumors [9, 31, 40].

*Neurosurgical Tools to Improve Safety and Survival in Patients with Intracranial Tumors…*

visualization of fluorescence. So, an adequate size of craniotomy (allowing light to enter the deep surgical field) and hemostasis (to avoid a blood layer over tumor area) in 5-ALA-guided surgery are more than ever must-do concepts. More common collateral effects are transient increase in liver enzymes and light sensitivity of the

*High-grade glioma patient operated on using 5-ALA. A and B show white light and blue-filter images with clearly identifiable tumoral tissue. In blue-filter image, a reddish color is observed, confirming the presence of tumoral tissue. The pinkish image demonstrates areas with tumoral infiltration. C and D show complete resection without any identifiable tumor in both white light and blue-filter images. Normal tissue appears* 

Other non-fluorescence techniques can also help in combination with

5-ALA. Intraoperative cortical stimulation added new advantages to resection about the function of tissues and provided additional safety for resection of primary malignant tumor in eloquent areas [39]. Intraoperative 3D US, as well as ioMRI, also was demonstrated to bring different information that when combined with 5-ALA fluorescence can improve the extent of resection, especially in non-enhancing

A combination of techniques may be the future of fluorescence-guided surgery. Dual-labeling surgery using 5-ALA and fluorescein has been tested with interesting results. Fluorescein created a useful background for 5-ALA fluorescence. It appeared as orange to red surrounded by greenly fluorescent normal brain and edematous tissue. Unspecific extravasation of fluorescein at resection margins was also observed, which did not interfere with 5-ALA fluorescence detection [37]. EOR and 6-month PFS have been proven to increase with the use of 5-ALA in cases of malignant gliomas. PFS at 6 months was 41% in 5-ALA group x 21.1% in the group operated only with white light-based resection. EOR improved from 36% in white light-based resection to 65% in 5-ALA [38]. EOR has also been analyzed in a systematic review with 22 series from the literature, including 1163 patients, with a

*DOI: http://dx.doi.org/10.5772/intechopen.81211*

skin until 24 h after administration.

**Figure 5.**

*blueish.*

GTR rate of 66.2% in gliomas using 5-ALA [35].

*Neurosurgical Tools to Improve Safety and Survival in Patients with Intracranial Tumors… DOI: http://dx.doi.org/10.5772/intechopen.81211*

#### **Figure 5.**

*Primary Intracranial Tumors*

This is another tool to go further with the concept that tumor tissues are many times much more than what we see with normal light surgical microscopy or even contrast-enhanced MRI. A high association between contrast enhancement and PPIX fluorescence is observed. But it was shown that PPIX fluorescence in noncontrast-enhanced areas can be present with good correlation with the presence of tumor tissue. So, PPIX accumulations seem to be more sensitive to glioma detection

Fluoroethyl tyrosine PET has been demonstrated to have a good correlation with PPIX fluorescence in gliomas without typical glioblastoma imaging features [33]. Also, areas with high atypia in low grade or non-contrast enhancing in high grade suggested by PET could be confirmed with 5-ALA fluorescence. The explanation to these findings may be in the mechanism of each method. Contrast enhancement and sodium-fluorescein fluorescence have intraoperative correlation, and both occur due to disruption of blood-brain barrier, which is not specific from tumors. 5-ALA fluorescence and PET tracer uptake, in turn, occur due to specific metabolism of tumor tissue. 5-ALA may be even more special than PET because it does not consider only the general quantitative aspect of metabolism and goes beyond. Its mechanism relates to a metabolic phenomenon of a pathway typical from a tumor

Other tumors than WHO IV gliomas have also been tested regarding fluorescence after 5-ALA administration. Literature shows results with approximately 15–20% of fluorescence with 5-ALA in low-grade gliomas, 85–100% in high-grade gliomas, and 55–80% in metastasis [32, 35, 36]. In our most recent data analysis from INC, we could observe 5-ALA-positive fluorescence in 97.7% cases of WHO IV gliomas, 90% cases of WHO III gliomas, 22.2% cases of WHO II gliomas, and 85.7% in cases of metastasis. The quality of fluorescence differs among tumor types. In low-grade gliomas, for example, with positive fluorescence we observed usually weak to mild with stronger foci in some cases (higher atypia); metastasis, on the

During the procedure, the surgeon alternates between white light resection and blue/violet light resection. This is important because white light shows anatomy, structures, and blood better. Only the resection, specially boundaries, is guided by fluorescence. Blood, inclusive, may be a confounding factor, because it prevents the

*Glioma patient operated on using 5-ALA. A and B show white light and blue-filter images with identifiable tumoral tissue on the cortex, clearly visible with blue filter and difficult to identify with white light. C and D show areas of tumor with intense fluorescence in blue filter, corresponding to contrast-enhanced area shown by* 

other hand, usually shows mild to strong fluorescence (**Figure 5**).

than contrast-agent accumulation (**Figure 4**) [31, 32].

tissue and not from a normal tissue [34].

**68**

**Figure 4.**

*neuronavigation in E.*

*High-grade glioma patient operated on using 5-ALA. A and B show white light and blue-filter images with clearly identifiable tumoral tissue. In blue-filter image, a reddish color is observed, confirming the presence of tumoral tissue. The pinkish image demonstrates areas with tumoral infiltration. C and D show complete resection without any identifiable tumor in both white light and blue-filter images. Normal tissue appears blueish.*

visualization of fluorescence. So, an adequate size of craniotomy (allowing light to enter the deep surgical field) and hemostasis (to avoid a blood layer over tumor area) in 5-ALA-guided surgery are more than ever must-do concepts. More common collateral effects are transient increase in liver enzymes and light sensitivity of the skin until 24 h after administration.

A combination of techniques may be the future of fluorescence-guided surgery. Dual-labeling surgery using 5-ALA and fluorescein has been tested with interesting results. Fluorescein created a useful background for 5-ALA fluorescence. It appeared as orange to red surrounded by greenly fluorescent normal brain and edematous tissue. Unspecific extravasation of fluorescein at resection margins was also observed, which did not interfere with 5-ALA fluorescence detection [37].

EOR and 6-month PFS have been proven to increase with the use of 5-ALA in cases of malignant gliomas. PFS at 6 months was 41% in 5-ALA group x 21.1% in the group operated only with white light-based resection. EOR improved from 36% in white light-based resection to 65% in 5-ALA [38]. EOR has also been analyzed in a systematic review with 22 series from the literature, including 1163 patients, with a GTR rate of 66.2% in gliomas using 5-ALA [35].

Other non-fluorescence techniques can also help in combination with 5-ALA. Intraoperative cortical stimulation added new advantages to resection about the function of tissues and provided additional safety for resection of primary malignant tumor in eloquent areas [39]. Intraoperative 3D US, as well as ioMRI, also was demonstrated to bring different information that when combined with 5-ALA fluorescence can improve the extent of resection, especially in non-enhancing tumors [9, 31, 40].

A comparison of combined ioMRI + 5-ALA versus ioMRI isolated in patients with high grade (WHO IV) gliomas showed that in combined group EOR above 95% was reached in all cases. In the ioMRI group, 18% of EOR were below 95% with a minimum EOR of 87% in this group versus a minimum EOR of 97% in the combined group [40]. Considering that EOR of 78% is the cutoff to improve survival in high-grade gliomas, both methods were efficient. But the association of 5-ALA and ioMRI leads to a higher rate, possibly having a greater impact on survival. But this is still to be proven, demanding further studies.

Despite drawbacks of being only a 2D information, hidden 5-ALA fluorescence by blood or hemostatic agents, and regulatory issues in many countries, 5-ALAguided resection is a very useful tool offering real-time information from the tissue (not indirectly not from images), without the influence of brain shift avoiding second-look procedures or even new complementary resections, which are usually much more expensive than the costs of 5-ALA (**Figure 5**).

### **5. Conclusion**

Every tool that can add data to surgical planning or intraoperative evaluation is valid. Neuronavigation is very useful in surgical strategy (planning and intraoperative steps) improving efficacy and safety of the procedure. 5-ALA-guided resection and intraoperative image (such as ioUS and ioMRI) are proven to be cost-effective with increased GTR rates and an impact on survival. The future probably will prove that combination of these tools, selected case by case, is the best way to achieve the best results regarding safety and effectiveness.

### **Conflict of interest**

Authors have no conflict of interest.

### **Author details**

Luis Fernando Moura da Silva1,2\*, Guilherme Augusto de Souza Machado1 and Ricardo Ramina1

1 Neurological Institute of Curitiba—INC, Curitiba, Brazil

2 NOZ Neurocentro, São Luis, Brazil

\*Address all correspondence to: luisfernando@inc-neuro.com.br

© 2019 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.

**71**

*Neurosurgical Tools to Improve Safety and Survival in Patients with Intracranial Tumors…*

die Echtzeit-Neuronavigation in der Neurochirurgie von Hirntumoren.

[9] Moiyadi AV, Shetty PM. Usefulness of three-dimensional navigable intraoperative ultrasound in resection

Ultraschall in der Medizin.

of brain tumors with a special emphasis on malignant gliomas. Acta Neurochirurgica. 2013;**155**:2217-2225

[10] Sollmann N, Kelm A, Ille S, Schröder A, Zimmer C, Ringel F, et al. Setup presentation and clinical outcome analysis of treating highly language-eloquent gliomas via preoperative navigated transcranial magnetic stimulation and tractography. Neurosurgical Focus. 2018;**44**(6):E2

[11] Rozumenko A, Kliuchka V,

[12] Lu Y, Yeung C, Radmanesh A, Wiemann R, Black PM, Golby AJ, et al. Comparative effectiveness of framebased, frameless and intraoperative MRI guided brain biopsy techniques. World Neurosurgery. 2015;**83**(3):261-268

[13] Abdel A, Shakal S, Abdel E, Mokbel H. Hemorrhage after stereotactic biopsy from intra-axial brain lesions: Incidence and avoidance. Journal of Neurological Surgery Part A: Central European Neurosurgery. 2014;**75**:177-182

[14] Kiesel B, Millesi M, Woehrer A, Furtner J, Bavand A, Roetzer T, et al. 5-ALA-induced fluorescence as a marker for diagnostic tissue in stereotactic biopsies of intracranial lymphomas: Experience in 41 patients. Neurosurgical

Focus. 2018;**44**(6):E7

2018;**41**(4):1045-1052

Rozumenko V, Semenova V, Kolesnyk S, Fedorenko Z. Image-guided resection of glioblastoma in eloquent brain areas facilitated by laser surface thermal therapy: Clinical outcomes and longterm results. Neurosurgical Review.

2015;**36**:174-186

*DOI: http://dx.doi.org/10.5772/intechopen.81211*

[1] Lee JJ. Surgical treatment of multiple brain metastases. Journal of Neurosurgery. 1993;**79**:210-216

[2] Hu S, Kang H, Baek Y, El FG, Kuang A, Choi HS. Real-time imaging of brain tumor for image-guided

Aug 2018;**7**(16):e1800066

surgery. Advanced Healthcare Materials.

[3] Bir SC, Konar SK, Maiti TK, Thakur JD, Guthikonda B, Nanda A. Utility of neuronavigation in intracranial meningioma resection: A singlecenter retrospective study. World Neurosurgery. 2016;**90**:546-555

[4] da Silva EB, Leal AG, Milano JB, da Silva LFM, Clemente RS, Ramina R. Image-guided surgical planning using anatomical landmarks in the retrosigmoid approach. Acta Neurochirurgica. 2010;**152**(5):905-910

[5] Ricciardi L, Maria G, Pepa D, Izzo A, Simboli GA, Polli FM, et al. Use of neuronavigation system for superficial vein identification: Safe and quick method to avoid intraoperative bleeding and vein closure: Technical note. World

Neurosurgery. 2018;**117**:92-96

[6] Gerard IJ, Kersten-oertel M, Petrecca K, Sirhan D, Hall JA, Collins DL. Brain shift in neuronavigation of brain tumors: A review. Medical Image

[7] Elias WJ, Fu K-M, Frysinger

[8] Prada F, Del Bene M, Mattei L, Lodigiani L, DeBeni S, Kolev V, et al. Preoperative magnetic resonance and intraoperative ultrasound fusion imaging for real-time neuronavigation in brain tumor surgery—Präoperative MRI-und intraoperative Ultraschallfusion für

RC. Cortical and subcortical brain shift during stereotactic procedures. Journal of Neurosurgery. 2007;**107**:983-988

Analysis. 2017;**35**:403-420

**References**

*Neurosurgical Tools to Improve Safety and Survival in Patients with Intracranial Tumors… DOI: http://dx.doi.org/10.5772/intechopen.81211*

#### **References**

*Primary Intracranial Tumors*

still to be proven, demanding further studies.

best results regarding safety and effectiveness.

Authors have no conflict of interest.

much more expensive than the costs of 5-ALA (**Figure 5**).

**70**

**Author details**

**Conflict of interest**

**5. Conclusion**

Ricardo Ramina1

provided the original work is properly cited.

2 NOZ Neurocentro, São Luis, Brazil

© 2019 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,

Luis Fernando Moura da Silva1,2\*, Guilherme Augusto de Souza Machado1

A comparison of combined ioMRI + 5-ALA versus ioMRI isolated in patients with high grade (WHO IV) gliomas showed that in combined group EOR above 95% was reached in all cases. In the ioMRI group, 18% of EOR were below 95% with a minimum EOR of 87% in this group versus a minimum EOR of 97% in the combined group [40]. Considering that EOR of 78% is the cutoff to improve survival in high-grade gliomas, both methods were efficient. But the association of 5-ALA and ioMRI leads to a higher rate, possibly having a greater impact on survival. But this is

Despite drawbacks of being only a 2D information, hidden 5-ALA fluorescence by blood or hemostatic agents, and regulatory issues in many countries, 5-ALAguided resection is a very useful tool offering real-time information from the tissue (not indirectly not from images), without the influence of brain shift avoiding second-look procedures or even new complementary resections, which are usually

Every tool that can add data to surgical planning or intraoperative evaluation is valid. Neuronavigation is very useful in surgical strategy (planning and intraoperative steps) improving efficacy and safety of the procedure. 5-ALA-guided resection and intraoperative image (such as ioUS and ioMRI) are proven to be cost-effective with increased GTR rates and an impact on survival. The future probably will prove that combination of these tools, selected case by case, is the best way to achieve the

1 Neurological Institute of Curitiba—INC, Curitiba, Brazil

\*Address all correspondence to: luisfernando@inc-neuro.com.br

and

[1] Lee JJ. Surgical treatment of multiple brain metastases. Journal of Neurosurgery. 1993;**79**:210-216

[2] Hu S, Kang H, Baek Y, El FG, Kuang A, Choi HS. Real-time imaging of brain tumor for image-guided surgery. Advanced Healthcare Materials. Aug 2018;**7**(16):e1800066

[3] Bir SC, Konar SK, Maiti TK, Thakur JD, Guthikonda B, Nanda A. Utility of neuronavigation in intracranial meningioma resection: A singlecenter retrospective study. World Neurosurgery. 2016;**90**:546-555

[4] da Silva EB, Leal AG, Milano JB, da Silva LFM, Clemente RS, Ramina R. Image-guided surgical planning using anatomical landmarks in the retrosigmoid approach. Acta Neurochirurgica. 2010;**152**(5):905-910

[5] Ricciardi L, Maria G, Pepa D, Izzo A, Simboli GA, Polli FM, et al. Use of neuronavigation system for superficial vein identification: Safe and quick method to avoid intraoperative bleeding and vein closure: Technical note. World Neurosurgery. 2018;**117**:92-96

[6] Gerard IJ, Kersten-oertel M, Petrecca K, Sirhan D, Hall JA, Collins DL. Brain shift in neuronavigation of brain tumors: A review. Medical Image Analysis. 2017;**35**:403-420

[7] Elias WJ, Fu K-M, Frysinger RC. Cortical and subcortical brain shift during stereotactic procedures. Journal of Neurosurgery. 2007;**107**:983-988

[8] Prada F, Del Bene M, Mattei L, Lodigiani L, DeBeni S, Kolev V, et al. Preoperative magnetic resonance and intraoperative ultrasound fusion imaging for real-time neuronavigation in brain tumor surgery—Präoperative MRI-und intraoperative Ultraschallfusion für

die Echtzeit-Neuronavigation in der Neurochirurgie von Hirntumoren. Ultraschall in der Medizin. 2015;**36**:174-186

[9] Moiyadi AV, Shetty PM. Usefulness of three-dimensional navigable intraoperative ultrasound in resection of brain tumors with a special emphasis on malignant gliomas. Acta Neurochirurgica. 2013;**155**:2217-2225

[10] Sollmann N, Kelm A, Ille S, Schröder A, Zimmer C, Ringel F, et al. Setup presentation and clinical outcome analysis of treating highly language-eloquent gliomas via preoperative navigated transcranial magnetic stimulation and tractography. Neurosurgical Focus. 2018;**44**(6):E2

[11] Rozumenko A, Kliuchka V, Rozumenko V, Semenova V, Kolesnyk S, Fedorenko Z. Image-guided resection of glioblastoma in eloquent brain areas facilitated by laser surface thermal therapy: Clinical outcomes and longterm results. Neurosurgical Review. 2018;**41**(4):1045-1052

[12] Lu Y, Yeung C, Radmanesh A, Wiemann R, Black PM, Golby AJ, et al. Comparative effectiveness of framebased, frameless and intraoperative MRI guided brain biopsy techniques. World Neurosurgery. 2015;**83**(3):261-268

[13] Abdel A, Shakal S, Abdel E, Mokbel H. Hemorrhage after stereotactic biopsy from intra-axial brain lesions: Incidence and avoidance. Journal of Neurological Surgery Part A: Central European Neurosurgery. 2014;**75**:177-182

[14] Kiesel B, Millesi M, Woehrer A, Furtner J, Bavand A, Roetzer T, et al. 5-ALA-induced fluorescence as a marker for diagnostic tissue in stereotactic biopsies of intracranial lymphomas: Experience in 41 patients. Neurosurgical Focus. 2018;**44**(6):E7

[15] Thien A, Han JX, Kumar K, Ng YP, Rao JP, Ng WH. Investigation of the usefulness of fluorescein sodium fluorescence in stereotactic brain biopsy. Acta Neurochirurgica. 2018;**160**(2):317-324

[16] Renfrow JJ, Strowd RE, Laxton AW, Tatter SB, Geer CP, Lesser GJ. Surgical considerations in the optimal management of patients with malignant brain tumors. Current Treatment Options in Oncology. 2017;**18**(8):46

[17] Orringer D, Lau D, Khatri S, Zamora-Berridi GJ, Zhang K, Wu C, et al. Extent of resection in patients with glioblastoma: Limiting factors, perception of resectability, and effect on survival. Journal of Neurosurgery. 2012;**117**:851-859

[18] Shaw EG, Berkey B, Coons SW, Bullerd D, Brachman D, Buckner JC, et al. Recurrence following neurosurgeon-determined gross-total resection of adult supratentorial lowgrade glioma: Results of a prospective clinical trial. Journal of Neurosurgery. 2008;**109**:835-841

[19] Scherer M, Jungk C, Younsi A, Kickingereder P, Müller S, Unterberg A. Factors triggering an additional resection and determining residual tumor volume on intraoperative MRI: Analysis from a prospective singlecenter registry of supratentorial gliomas. Neurosurgical Focus. 2016;**40**(3):E4

[20] Swinney C, Li A, Bhatti I, Veeravagu A. Optimization of tumor resection with intra-operative magnetic resonance imaging. Journal of Clinical Neuroscience. 2016;**34**:11-14

[21] Familiari P, Frati A, Pesce A, Miscusi M, Cimatti M, Raco A. Real impact of intraoperative MRI in newly diagnosed glioblastoma multiforme resection: An observational analytic cohort study from a single surgeon

experience. World Neurosurgery. 2018;**116**:e9-e17

[22] Motomura K, Natsume A, Iijima K, Kuramitsu S, Fujii M, Yamamoto T, et al. Surgical benefits of combined awake craniotomy and intraoperative magnetic resonance imaging for gliomas associated with eloquent areas. Journal of Neurosurgery. 2017;**127**:790-797

[23] Ramina R, Coelho Neto M, Nascimento AB, Vosgerau R. Intraoperative MRI features of absorbable oxidized regenerated cellulose during cerebral glioma surgery. Brazilian Neurosurgery. 2013;**24**(1):16-10

[24] Marcus HJ, Vercauteren T, Ourselin S, Dorward NL. Literature review intraoperative ultrasound in patients undergoing transsphenoidal surgery for pituitary adenoma: Systematic review. World Neurosurgery. 2017;**106**:680-685

[25] Chittiboina P. iMRI during transsphenoidal surgery. Neurosurgery Clinics of North America. 2017;**28**(4):499-512

[26] Aghi MK, Chen CC, Fleseriu M, Lucas JW, Kuo JS, Barkhoudarian G, et al. Congress of neurological surgeons systematic review and evidence-based guidelines on the management of patients with nonfunctioning pituitary adenomas: Executive summary. Neurosurgery. 2016;**79**(4):521-523

[27] Bisdas S, Roder C, Ernemann U, Tatagiba MS. Intraoperative MR imaging in neurosurgery. Clinical Neuroradiology. 2015;**25**:237-244

[28] Rao G. Intraoperative MRI and maximizing extent of resection. Neurosurgery Clinics of North America. 2017;**28**(4):477-485

[29] Ramina R, Neto MC, Giacomelli A, Barros E Jr, Vosgerau R, Nascimento A, et al. Optimizing costs of intraoperative

**73**

*Neurosurgical Tools to Improve Safety and Survival in Patients with Intracranial Tumors…*

[36] Ramina R, da Silva EB Jr, Coelho Neto M, Ruschel LG, Navarrette FAC. 5-aminolevulinic acid—Protoporphyrin IX fluorescence-guided surgery for CNS tumors. First 41 cases in Latin America. Brazilian Neurosurgery.

[37] Molina ES, Wölfer J, Ewelt C, Ehrhardt A, Brokinkel B, Stummer W. Dual-labeling with 5-aminolevulinic acid and fluorescein for fluorescenceguided resection of high-grade gliomas: Technical note. Journal of Neurosurgery.

[38] Stummer W, Pichlmeier U, Meinel T, Wiestler OD, Zanella F, Reulen H. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: A randomised controlled multicentre phase III trial. The Lancet Oncology. 2006;**7**:392-401

[39] Feigl GC, Ritz R, Moraes M, Klein J, Ramina K, Gharabaghi A, et al. Resection of malignant brain tumors in eloquent cortical areas: A new multimodal approach combining 5-aminolevulinic acid and intraoperative monitoring. Journal of Neurosurgery. 2010;**113**:352-357

[40] Coburger J, Hagel V, Wirtz CR, König R. Surgery for glioblastoma: Impact of the combined use of 5-aminolevulinic acid and intraoperative MRI on extent of resection and survival. PLoS One.

2015;**10**(2):e0131872

2017;**27**(1):13-19

2018;**128**:399-405

*DOI: http://dx.doi.org/10.5772/intechopen.81211*

magnetic resonance imaging. A series of 29 glioma cases. Acta Neurochirurgica.

[30] Ahmadi R, Campos B, Haux D, Rieke J, Beigel B, Unterberg A. Assessing perioperative complications associated with use of intraoperative magnetic resonance imaging during glioma surgery—A single centre experience with 516 cases. British Journal of Neurosurgery. 2016;**30**(4):397-400

[31] Molina ES, Schipmann S, Stummer W. Maximizing safe resections: The roles of 5-aminolevulinic acid and intraoperative MR imaging in glioma surgery—Review of the literature. Neurosurgical Review. 2017 [Epub

[32] Stummer W, Suero Molina E. Fluorescence imaging/agents in tumor resection. Neurosurgery Clinics of North America. 2017;**28**(4):569-583

[33] Jaber M, Wölfer J, Ewelt C, Holling M, Hasselblatt M, Niederstadt T, et al. 5-Aminolevulinic acid in low-grade gliomas and high-grade gliomas lacking glioblastoma imaging features: An analysis based on fluorescence, magnetic resonance imaging, 18F-fluoroethyl tyrosine positron emission tomography, and tumor molecular factors. Neurosurgery.

[34] Yano H, Nakayama N, Ohe N, Miwa K, Shinoda J, Iwama T. Pathological analysis of the surgical margins of resected glioblastomas excised using photodynamic visualization with both 5-aminolevulinic acid and fluorescein sodium. Journal of Neuro-Oncology.

[35] Ferraro N, Barbarite E, Albert TR, Berchmans E, Shah AH, Bregy A, et al. The role of 5-aminolevulinic acid in brain tumor surgery: A systematic review. Neurosurgical Review.

2010;**152**:27-33

ahead of print]

2016;**78**(3):401-411

2017;**133**:389-397

2016;**39**(4):545-555

*Neurosurgical Tools to Improve Safety and Survival in Patients with Intracranial Tumors… DOI: http://dx.doi.org/10.5772/intechopen.81211*

magnetic resonance imaging. A series of 29 glioma cases. Acta Neurochirurgica. 2010;**152**:27-33

[30] Ahmadi R, Campos B, Haux D, Rieke J, Beigel B, Unterberg A. Assessing perioperative complications associated with use of intraoperative magnetic resonance imaging during glioma surgery—A single centre experience with 516 cases. British Journal of Neurosurgery. 2016;**30**(4):397-400

[31] Molina ES, Schipmann S, Stummer W. Maximizing safe resections: The roles of 5-aminolevulinic acid and intraoperative MR imaging in glioma surgery—Review of the literature. Neurosurgical Review. 2017 [Epub ahead of print]

[32] Stummer W, Suero Molina E. Fluorescence imaging/agents in tumor resection. Neurosurgery Clinics of North America. 2017;**28**(4):569-583

[33] Jaber M, Wölfer J, Ewelt C, Holling M, Hasselblatt M, Niederstadt T, et al. 5-Aminolevulinic acid in low-grade gliomas and high-grade gliomas lacking glioblastoma imaging features: An analysis based on fluorescence, magnetic resonance imaging, 18F-fluoroethyl tyrosine positron emission tomography, and tumor molecular factors. Neurosurgery. 2016;**78**(3):401-411

[34] Yano H, Nakayama N, Ohe N, Miwa K, Shinoda J, Iwama T. Pathological analysis of the surgical margins of resected glioblastomas excised using photodynamic visualization with both 5-aminolevulinic acid and fluorescein sodium. Journal of Neuro-Oncology. 2017;**133**:389-397

[35] Ferraro N, Barbarite E, Albert TR, Berchmans E, Shah AH, Bregy A, et al. The role of 5-aminolevulinic acid in brain tumor surgery: A systematic review. Neurosurgical Review. 2016;**39**(4):545-555

[36] Ramina R, da Silva EB Jr, Coelho Neto M, Ruschel LG, Navarrette FAC. 5-aminolevulinic acid—Protoporphyrin IX fluorescence-guided surgery for CNS tumors. First 41 cases in Latin America. Brazilian Neurosurgery. 2017;**27**(1):13-19

[37] Molina ES, Wölfer J, Ewelt C, Ehrhardt A, Brokinkel B, Stummer W. Dual-labeling with 5-aminolevulinic acid and fluorescein for fluorescenceguided resection of high-grade gliomas: Technical note. Journal of Neurosurgery. 2018;**128**:399-405

[38] Stummer W, Pichlmeier U, Meinel T, Wiestler OD, Zanella F, Reulen H. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: A randomised controlled multicentre phase III trial. The Lancet Oncology. 2006;**7**:392-401

[39] Feigl GC, Ritz R, Moraes M, Klein J, Ramina K, Gharabaghi A, et al. Resection of malignant brain tumors in eloquent cortical areas: A new multimodal approach combining 5-aminolevulinic acid and intraoperative monitoring. Journal of Neurosurgery. 2010;**113**:352-357

[40] Coburger J, Hagel V, Wirtz CR, König R. Surgery for glioblastoma: Impact of the combined use of 5-aminolevulinic acid and intraoperative MRI on extent of resection and survival. PLoS One. 2015;**10**(2):e0131872

**72**

*Primary Intracranial Tumors*

2018;**160**(2):317-324

2012;**117**:851-859

2008;**109**:835-841

2016;**40**(3):E4

[15] Thien A, Han JX, Kumar K, Ng YP, Rao JP, Ng WH. Investigation of the usefulness of fluorescein sodium fluorescence in stereotactic brain biopsy. Acta Neurochirurgica. experience. World Neurosurgery.

[22] Motomura K, Natsume A, Iijima K, Kuramitsu S, Fujii M, Yamamoto T, et al. Surgical benefits of combined awake craniotomy and intraoperative magnetic resonance imaging for gliomas associated with eloquent areas. Journal of Neurosurgery. 2017;**127**:790-797

[23] Ramina R, Coelho Neto M, Nascimento AB, Vosgerau R. Intraoperative MRI features of absorbable oxidized regenerated cellulose during cerebral glioma surgery. Brazilian Neurosurgery.

[24] Marcus HJ, Vercauteren T, Ourselin S, Dorward NL. Literature review intraoperative ultrasound in patients undergoing transsphenoidal surgery for pituitary adenoma: Systematic review. World Neurosurgery. 2017;**106**:680-685

transsphenoidal surgery. Neurosurgery

[26] Aghi MK, Chen CC, Fleseriu M, Lucas JW, Kuo JS, Barkhoudarian G, et al. Congress of neurological surgeons systematic review and evidence-based guidelines on the management of patients with nonfunctioning pituitary

adenomas: Executive summary. Neurosurgery. 2016;**79**(4):521-523

[27] Bisdas S, Roder C, Ernemann U, Tatagiba MS. Intraoperative MR imaging in neurosurgery. Clinical Neuroradiology. 2015;**25**:237-244

[28] Rao G. Intraoperative MRI and maximizing extent of resection.

2017;**28**(4):477-485

Neurosurgery Clinics of North America.

[29] Ramina R, Neto MC, Giacomelli A, Barros E Jr, Vosgerau R, Nascimento A, et al. Optimizing costs of intraoperative

[25] Chittiboina P. iMRI during

Clinics of North America. 2017;**28**(4):499-512

2013;**24**(1):16-10

2018;**116**:e9-e17

[16] Renfrow JJ, Strowd RE, Laxton AW, Tatter SB, Geer CP, Lesser GJ. Surgical

management of patients with malignant brain tumors. Current Treatment Options in Oncology. 2017;**18**(8):46

considerations in the optimal

[17] Orringer D, Lau D, Khatri S, Zamora-Berridi GJ, Zhang K, Wu C, et al. Extent of resection in patients with glioblastoma: Limiting factors, perception of resectability, and effect on survival. Journal of Neurosurgery.

[18] Shaw EG, Berkey B, Coons SW, Bullerd D, Brachman D, Buckner JC, et al. Recurrence following neurosurgeon-determined gross-total resection of adult supratentorial lowgrade glioma: Results of a prospective clinical trial. Journal of Neurosurgery.

[19] Scherer M, Jungk C, Younsi A, Kickingereder P, Müller S, Unterberg A. Factors triggering an additional resection and determining residual tumor volume on intraoperative MRI: Analysis from a prospective singlecenter registry of supratentorial gliomas. Neurosurgical Focus.

[20] Swinney C, Li A, Bhatti I, Veeravagu A. Optimization of tumor resection with intra-operative magnetic resonance imaging. Journal of Clinical

Neuroscience. 2016;**34**:11-14

[21] Familiari P, Frati A, Pesce A, Miscusi M, Cimatti M, Raco A. Real impact of intraoperative MRI in newly diagnosed glioblastoma multiforme resection: An observational analytic cohort study from a single surgeon

Section 3

Management of Specific

Tumors

75

### Section 3
