New Insights into Glioblastoma: Diagnosis, Therapeutics and Theranostics

New Insights into Glioblastoma: Diagnosis, Therapeutics and Theranostics provides a compendium of recent diagnostic and therapeutic advances in GBM, encompassing a pipeline of compounds and (bio) nanotechnology strategies that have stood out with potential increased antitumoral activity and capability to cross the blood-brain barrier. Issues and challenges related to their translation into the clinical practice and their contribution to the increase in survival rates and well-being of patients are addressed. This is a valuable resource for graduate students, oncologists, cancer researchers and members of the biomedical field who need to learn more on recent developments on the management of glioblastoma.

The book is split in three parts: Diagnosis, focusing on biomarkers and techniques such PET/MRI, infrared thermography, and deep neural networks; Therapeutics, discussing new chemical entities, as natural products and repurposed drugs, and new formulation approaches, as nanotechnology-based and microRNA approaches; and Theranostics, explaining the role of omics, system-based approaches, and glioblastoma microenvironment.

• Provides guidance towards recent advances of new chemical entities and delivery strategies targeted to glioblastoma
• Includes overviews to help readers apply information in their research
• Encompasses summarizing diagrams and real-world examples to facilitate comprehension and enhance the applicability of the content

• Language: English
• Publisher: Academic Press
• Release date: May 17, 2023
• ISBN: 9780323999427

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Part I

Introduction

Chapter 1: Introduction: A brief outlook into glioblastoma diagnosis and therapeutics

Carla Vitorinoa,b; Carmen Balañac; Célia Cabrald    a Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal

b Department of Chemistry, Faculty of Sciences and Technology, Coimbra Chemistry Centre, Institute of Molecular Sciences, University of Coimbra, Coimbra, Portugal

c Catalan Institute of Oncology, Badalona, Barcelona, Spain

d Coimbra Institute for Clinical and Biomedical Research (iCBR), Clinic Academic Center of Coimbra (CACC), Center for Innovative Biomedicine and Biotechnology (CIBB), Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Keywords

Glioblastoma; Diagnosis; Therapeutics; New chemical entities; Biomarkers; Nanotechnology; Theranostics; Radiogenomics

1.1: Introduction

Glioblastoma (GB) is the most frequent and malignant tumor of the central nervous system (CNS), being considered an unmet medical need.

The average incidence of primary malignant CNS tumors is 7.08 per 100,000 inhabitants in the United States. GB accounts for 48.6% of all malignant tumors, with an incidence of 3.23 per 100,000 inhabitants with a median observed survival of only 8 months for all diagnosed patients [1].

Although it can be diagnosed in children and adolescents (2.9% of all brain and other CNS tumors among those aged 0–19 years), the median age of incidence is 65 years old, and the incidence increases with age being maximum for patients over 75 years old (15.30 cases per 100,000 inhabitants) [1].

Median observed survival is only 8 months with a 5-year survival rate of 7.2 months (95% CI: 7–7.4), representing 61.3% of all deaths for a CNS tumor. Less than 50% of patients are fit enough to undergo postsurgical oncological treatment that is based on radiation therapy combined with concurrent and adjuvant temozolomide, with the possibility of adding tumor-treating fields (TTF) in a few countries [2–5]. In that case, median overall survival can increase to 14–16 months.

Significant advances have been made in diagnosis, identification of prognostic factors, symptomatic management, molecular biology, image interpretation, and radiogenomics but no new effective therapies have succeeded to increase the overall survival of these tumors in more than a decade.

Once in relapse, which happens in most patients, we have treatment with second surgery, re-irradiation, and re-treatment with temozolomide, nitrosoureas, and bevacizumab [6]. All of them have no impact on survival. Therefore, the best option should always be the clinical trial. However, the number of patients eligible to participate in clinical trials is decreasing as time progresses after diagnosis and the neurological damage caused by the disease and treatments worsens [7]. All this, in addition to other factors, makes it difficult to make progress in improving the treatment of the disease. It is therefore important to be aware of all the lines of research that are open so that we can work together to search for novel therapeutic alternatives. This may range from the discovery of new chemical entities, and the repurposing of drugs until the new formulation approaches.

Since ancient times, natural products have been used in traditional medicine to treat several illnesses and are important sources of biologically active molecules as drug leads. Currently, they are responsible for a large number of the available drugs, with over 60% of the anticancer drugs used in the clinic derived from natural sources [8].

Advanced therapies are at the forefront, with gene therapy, cell therapy, and immunotherapy taking a leading position [9–11]. New drug delivery systems, including nanotechnology, irrefutably offer a variety of options to address the multiple challenges posed by brain tumors, wherein GB is no exception [12]. A wide variety of nanocarriers have been described, ranging from organic to inorganic materials, as well as systems gathering materials from both natures, termed hybrid nanosystems, some of which have already entered clinical trial programs. Nanoparticulate delivery systems, such as liposomes, solid lipid matrix nanoparticles, micelles, polymeric nanoparticles, iron oxide, and gold particles, among others, can come in a diversity of designs, exhibiting distinct compositions, colloidal and loading properties, upheld by flexible and robust often upscalable production methods. With regard to the specificity of nanosystems for tumor cells and their microenvironment, advanced strategies for surface chemistry (e.g., aptamers, antibodies, peptides, proteins, carbohydrates, and small molecules, such as folate and vitamins) have been developed. Such approaches take into consideration the overexpressed receptors in tumor cells to mediate the internalization of their associated therapeutic cargoes and/or endo−/exogenous stimuli-triggered responses [13].

The combination of nanotechnology and advanced therapies in a biomimetic approach is also a way of boosting the performance of both therapeutic approaches [14].

Physiological surrogates, such as intranasal delivery, have also been highlighted as a reliable and direct route to cross the blood-brain barrier [15]. Because of the unique direct connection between the brain and the nasal cavity interceded by the olfactory epithelium, intranasal delivery is the only route by which the brain interlinks to the external environment, hypothetically expanding the application of therapies for GB.

More recently, theranostics has also opened a new dimension in GB therapy by enabling both diagnosis and treatment in a single system, offering a new reality of real-time disease monitoring [16].

This book brings together contributions from experts in various complementary fields who share an interest in developing new techniques and strategies for the diagnosis and treatment of GB. Aspects related to the translation from research to clinical setting are also holistically addressed, ultimately instilling fresh hope for patients with GB.

References

[1] Ostrom Q.T. CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2013–2017. Neuro Oncol. 2020;22(Suppl. 1):iv1–iv96.

[2] Fabbro-Peray P. Association of patterns of care, prognostic factors, and use of radiotherapy-temozolomide therapy with survival in patients with newly diagnosed glioblastoma: a French national population-based study. J Neurooncol. 2019;142(1):91–101.

[3] Hansen S. Treatment and survival of glioblastoma patients in Denmark: the Danish Neuro-Oncology Registry 2009-2014. J Neurooncol. 2018;139(2):479–489.

[4] Stupp R. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352(10):987–996.

[5] Stupp R. Effect of tumor-treating fields plus maintenance temozolomide vs maintenance temozolomide alone on survival in patients with glioblastoma: a randomized clinical trial. JAMA. 2017;318(23):2306–2316.

[6] Weller M. EANO guidelines on the diagnosis and treatment of diffuse gliomas of adulthood. Nat Rev Clin Oncol. 2021;18(3):170–186.

[7] Skaga E. Real-world validity of randomized controlled phase III trials in newly diagnosed glioblastoma: to whom do the results of the trials apply?. Neurooncol Adv. 2021;3(1):vdab008.

[8] Magalhães M. Chemoprevention and therapeutic role of essential oils and phenolic compounds: modeling tumor microenvironment in glioblastoma. Pharmacol Res. 2021;169:105638.

[9] Ma Y. Advanced immunotherapy approaches for glioblastoma. Adv Ther. 2021;4(8):2100046.

[10] Luginbuehl V. Better by design: what to expect from novel CAR-engineered cell therapies?. Biotechnol Adv. 2022;58:107917.

[11] Nowak B. Mesenchymal stem cells in glioblastoma therapy and progression: how one cell does it all. Biochim Biophys Acta Rev Cancer. 2021;1876(1):188582.

[12] Shabani L. The brilliance of nanoscience over cancer therapy: novel promising nanotechnology-based methods for eradicating glioblastoma. J Neurol Sci. 2022;440:120316.

[13] Khan I. Nanomedicine for glioblastoma: progress and future prospects. Semin Cancer Biol. 2022.

[14] Han S., Lee Y., Lee M. Biomimetic cell membrane-coated DNA nanoparticles for gene delivery to glioblastoma. J Control Release. 2021;338:22–32.

[15] Morales D.E., Mousa S. Intranasal delivery in glioblastoma treatment: prospective molecular treatment modalities. Heliyon. 2022;8(5):e09517.

[16] Mendes M. Targeted theranostic nanoparticles for brain tumor treatment. Pharmaceutics. 2018;10(4).

Part II

Diagnosis

Part II.1

Biomarkers

Chapter 2: Practice guidelines for the diagnosis of glioblastoma

Iban Aldecoaa,b,c,d; Ivan Archillaa,b; Teresa Ribaltaa,b,d    a Department of Pathology, Biomedical Diagnosis Center, Hospital Clinic of Barcelona, Barcelona, Spain

b Institut d’Investigació Biomèdica August Pi i Sunyer (IDIBAPS), Hospital Clinic of Barcelona, Barcelona, Spain

c Neurological Tissue Bank of the Biobank, Barcelona, Spain

d Faculty of Medicine and Health Sciences, Universitat de Barcelona, Barcelona, Spain

Abstract

Glioblastoma is both the most common and the most malignant primary brain neoplasm in adults and remains almost universally incurable in both adults and children. Recent progress in genetic and epigenetic research has significantly expanded our understanding of its underlying molecular characteristics. Glioblastomas are being divided into meaningful biological subgroups and the new molecular biomarkers allow a clinically useful reclassification of these highly aggressive tumors across all ages based on a combination of classical histopathology and defining or characteristic molecular alterations. This new approach represents a paradigm shift, not only for the pathological classification of infiltrating gliomas but also for radiological characterization and therapeutic strategies. Current pathology reports document both the histopathological criteria as well as the molecular profile of the tumor and aim to provide an integrated diagnosis.

Keywords

Tumor; Central nervous system neoplasm; Glioma; Glioblastoma; High grade glioma; Astrocytoma; Oligodendroglioma

2.1: Introduction and historical perspective of high-grade gliomas

Called in the past glioblastoma multiforme, glioblastoma (GB) is the most common and the most aggressive primary brain neoplasm of the central nervous system (CNS) in adults and constitutes a significant group of tumors in pediatric age [1]. For many years, gliomas have been classified according to the characteristics such as their location, growth pattern, morphological characteristics, immunohistochemical expression of antigens (IHC), and biological behavior. In 2016, the fourth revised edition of the World Health Organization (WHO) classification of the central nervous system tumors already introduced a major restructuring of the diagnostic approach to brain tumors by complementing histological features with molecular parameters for the first time in an integrative format [2]. More recently, molecular biomarkers have gained importance in providing information with diagnostic and therapeutic utility, and therefore, already in 2016, in view of the speed of acquisition of new knowledge, a consortium of expert neuropathologists and oncologists called cIMPACT-NOW[3] was created to continuously update the criteria and clarify diagnostic doubts until the latest official publication of the WHO classification of the central nervous system tumours in 2021 (WHO2021) [4].

WHO2021 collects all the c-IMPACT-NOW recommendations and changes that advance the role of molecular parameters in tumor classification, emphasizing the importance of their integration with histology and IHC. In pathology reports, histological and molecular information is currently presented in a layered format [4]. According to the WHO2021, gliomas are classified into four grades (WHO grade 1–4) based on the histological and molecular criteria [4]. As a consequence of this new classification, the classical morphological glioblastoma multiforme diagnosis has been split into several high-grade glioma (HGG) histomolecular types and subtypes. In the WHO2021 classification, the GB term has been narrowed in adults to grade 4, IDHwildtype (IDH-wt) and Histone H3 wildtype (H3-wt) infiltrating HGG. Other gliomas in WHO grades 3 and 4 with characteristic driver mutations which vary with patient age and location of the tumor also belong to the HGG group [5].

2.2: Glioblastoma and other high-grade gliomas. Current classification and genetic driver mutations

Many molecular markers have been described in GB, other HGGs, and low-grade diffuse gliomas in recent years. Their knowledge is necessary not only to propose adequate differential diagnoses but also to understand the globality of the current WHO classification of HGGs. The WHO2021 draws a complex classification of gliomas that are based on clinical, radiological, morphological, and molecular data [4]. Moreover, each one of these items has a different weight depending on the glioma type and subtype. Briefly, IDH mutations have been cemented as a basic classifier of infiltrating gliomas in the adult; items have been specified for GB with more stringent diagnostic criteria; a redefinition of Histone 3 K27-altered gliomas has been introduced, and a histomolecular classification has been set for IDH-wt and H3-wt pediatric HGG (see Box 2.1).

BOX 2.1

New and revised high-grade gliomas in the 2021 WHO classification of CNS tumors [4].

Classical entities without significant changes

Oligodendroglioma, IDH-mutant, and 1p/19q-codeleted

High-grade gliomas with revised terminology

Glioblastoma, IDHwt

Astrocytoma, IDH-mutant, grades 3 or 4 (instead of Glioblastoma IDH-mutant in WHO2016)

Diffuse midline glioma, H3 K27-altered (instead of DMG, H3 K27M)

Four new high-grade glioma types (formerly called entities)

Diffuse hemispheric glioma, H3.3 G34-mutant

Diffuse pediatric-type high-grade glioma, H3-wild type and IDH-wildtype

Infant-type hemispheric glioma

High-grade astrocytoma with piloid features (circumscribed)

WHO2021 has incorporated the definition of new types of glioma and a revision of terminology. In line with the previous 2016 classification, the need for molecular studies has been deepened for a greater number of tumor types, with the need for methylation profiling in selected entities. One of the most notable changes is that the term astrocytoma grade 4, IDH mutant replaces the term glioblastoma, IDH mutant.

2.3: Glioblastoma, IDH-wildtype (IDHwt GB)

GB accounts for about 45% of primary malignant CNS tumors and 54% of all gliomas [6]. The median survival of GB patients is approximately 15 months, even after receiving multimodal therapies that include maximal surgical resection with the preservation of neurological functions, followed by adjuvant radiotherapy and chemotherapy [7]. GB may involve any neuroanatomical level or structure but is most common in the cerebral hemispheres. Neuroimaging findings are usually an aid to pathological diagnosis. GBs can be visualized on magnetic resonance imaging as contrast-enhancing tumors, but neoplastic cells invading brain parenchyma extend well beyond the area of enhancement, frequently crossing the midline through the corpus callosum which gives rise to the pattern in butterfly wings or involves other lobes or far away anatomical regions. Newly diagnosed GB usually presents on imaging as a single peripherally enhancing lesion, but multiple enhancing lesions can occur, termed multifocal or multicentric depending on whether a connection is observed between the lesions or not, respectively [8,9]. Rarely, does GB present with a growth pattern of gliomatosis. Historically, gliomatosis cerebri has been considered a unique glial tumor entity, but evidence from molecular studies supports that gliomatosis cerebri is just an exceptionally diffuse pattern of involvement of the nervous tissue with minimal or no central focal tumor mass [10]. Even in usual GB, due to this biological behavior, complete microscopic resection can never be achieved, and remnant neoplastic cells are frequently the source of disease recurrence. Moreover, extensive brainstem infiltration has been described as a common feature in end-stage cerebral GBs, even more than the mass effect [11].

GB has historically been divided into two clinical subtypes based on the presence or absence of a precursor lesion. Primary GB is the most common type (90%); it arises de novo, without evidence of a precursor lesion, and is common in older adults (over 50-years old). Secondary GB represents a progression from a pre-existent, lower-grade astrocytoma and generally has a better outcome than primary GB [2,12]. Currently, according to WHO2021, many of these secondary GBs belong to the category of astrocytoma IDH-mutant, grade 4, while most of the primary GBs are defined as glioblastoma, IDH-wildtype [4,13].

Primary GBs in adults are characterized by EGFR amplification, PTEN mutation, TERT promoter mutation, and absence of IDH and Histone 3 mutations [14]. In fact, according to WHO2021 criteria, IDH-wt infiltrating astrocytomas that harbor either EGFR amplification, TERT promoter mutation, or the combination of gain of chromosome 7 and loss of chromosome 10 (+7/−10), can be diagnosed in adults as IDH-wt GB even if they lack histological hallmarks such as necrosis or microvascular proliferation [4].

IDH-wt GBs lack a single defining mutation, although studies based on analysis of mutations, mRNA expression, and alterations in DNA copy number in adult GB have revealed that very often these IDH-wt GBs show alterations in at least three key genetic pathways [15]: (1) inactivation mutations in the p53 pathway or inactivation mutations in the retinoblastoma (RB) pathway; (2) activation of the PI3K pathway and (3) amplification and mutational activation of the receptor tyrosine kinase (RTK) genes. Below we review some of the mutations commonly studied in the diagnostic field:

TP53/MDM2 Pathway: TP53 mutations are found in approximately two-thirds of IDH-mutant astrocytomas and in one-third of IDH-wt GBs. Astrocytomas with IDH mutations typically have TP53 hotspot mutations, however, in IDH-wt GBs, TP53 mutations occur at more widespread loci [16]. Strong p53 reactivity by IHC in >10% of cells provides an accurate prediction of mutation [17]. TP53 mutations are mutually exclusive with MDM2 amplification, an inhibitor of p53 present in 10–14% of GBs [18].

RB/CDK4/CDKN2A-p16INK4a Pathway: The cell growth inhibition mechanism is found to be defective in GB most commonly through alterations in RB1, CDK4, and CDKN2A, being the homozygous deletion of CDKN2A the most frequent (present in more than half of the cases). These alterations lead to dysregulation of the cell cycle and uncontrolled proliferation. These alterations can affect both IDH-mutant high-grade astrocytomas and IDH-wt GB [19]. CDKN2A homozygous deletion can be studied by fluorescent in situ hybridization (FISH) or other molecular techniques and, in IDH-mutant astrocytomas, is a marker of the highest malignancy grade [4].

TERT: Approximately 70% of all adult primary GBs harbor a mutation in the TERT promoter (pTERTmut) [20–22]. TERT encodes the catalytic subunit of telomerase, a protein responsible for the normal gradual shortening of telomeres in successive cell divisions until they reach a certain length, at which point the cell stops dividing and falls into senescence. The most common oncogenic mutations in the TERT promoter occur at C228 and C250, or less frequently at C229. Activation or increased TERT expression is associated with shorter survival in gliomas [23]. TERT promoter mutation is one of the three genetic parameters that WHO2021 uses to upgrade astrocytoma, IDH-wt to glioblastoma, IDH-wt [4]. Care must be taken when only TERT mutations are analyzed in an HGG. Other IDH-mutant gliomas, especially IDH-mutated and 1p/19q-codeleted oligodendrogliomas, and other non-glial tumors harbor frequent TERT mutations. TERT has also been described in some lower-grade IDH-mutated astrocytomas [24]. Hence, TERT mutations must be interpreted in context, always in conjunction with the IDH status and age of the patient.

MGMT: O6-methylguanine-DNA methyltransferase (MGMT) encodes a repair enzyme for DNA damage induced by alkylating agents. Hypermethylation of the MGMT promoter causes what is known as gene silencing and consequently MGMT down-regulation, an effect of paramount clinical importance as it predicts a better response to temozolomide chemotherapy [25]. MGMT methylation is more common in IDH-mutant astrocytomas than in IDH-wt GB [26] and can be determined by quantitative pyrosequencing or other molecular techniques such as bisulfite sequencing.

Other chromosomal and epigenetic alterations: Other chromosomal changes accumulate in GB, the most relevant of which, due to their role in assisting in the diagnosis, is the gain of chromosome 7 and loss of chromosome 10. These changes seem to occur very early in the gliomagenesis of IDH-wt GBs. IDH-wt GB molecular profiling analyses have also identified co-gain 19/20 as a chromosomal alteration significantly associated with better long-term survival in these patients [27].

2.3.1: Selected subtypes of glioblastoma

Epithelioid GB (E-GB), giant cell GB (GC-GB), and gliosarcoma are rare variants of IDH-wt GB [28].

E-GB is histologically characterized by malignant glial cells with a polygonal or rounded morphology similar to carcinoma cells, but its genetic characteristics are variable. Based on methylation patterns, cytogenetic analyses, and mutation analysis data in combination with clinical findings, Korshunov et al. [29] disclosed three different, well-established tumor molecular subtypes: (1) Anaplastic PXA-like tumors with more favorable prognosis, predominantly in children and young adults, and frequent BRAFV600E mutation and homozygous CDKN2A deletions; (2) IDH-wt GB-like forms with a poor prognosis, which primarily occurs in older adults (median age of 50 years), albeit with more frequent BRAF mutations; and (3) RTK1 pediatric GB-like neoplasms of intermediate prognosis in children and young adults, which are associated with chromothripsis and frequent PDGFRA amplification, sometimes combined with MYCN amplification. The authors concluded that histopathologically defined E-GB does not represent a single diagnostic entity, but rather at least three molecularly and biologically distinct categories.

GC-GB is a variant of IDH-wt GB showing cells with exaggeratedly large and aberrant-appearing nuclei. Although they are aggressive tumors, they appear to have a slightly better prognosis than conventional GB. The spectrum of genetic alterations differs somewhat from those seen in IDH-wt GB. Mutations of the TERT promoter are present in <25% of GC-GB [30] and instead are frequently associated with loss of expression of ATRX by IHC, which is considered a surrogate for gene mutation [31]. Likewise, TP53 mutations are present in an elevated number of cases [32]. In contrast, EGFR amplification and homozygous CDKN2A deletion are rare [32]. Frequently seen chromosome abnormalities include the gain of chromosome 7 and loss of chromosome 10 as in conventional IDH-wt GB, but also the gain of chromosome 20 and loss of the long arm of chromosome 22 [33].

Gliosarcoma shows areas with histological and IHC characteristics of sarcoma intertwined with others with a malignant astrocytic appearance, which gives it a biphasic appearance [28]. In this variant, TERT promoter mutations, PTEN mutation or deletion, and homozygous CDKN2A deletion are common, but they rarely show EGFR amplification [34].

2.4: IDH-mutated gliomas

Isocitrate dehydrogenase (IDH) enzymes, of which there are three isoforms (IDH 1/2/3), are essential enzymes that participate in several major metabolic processes, playing key roles in the Krebs cycle and cellular homeostasis. IDH mutations play a crucial role in glioma classification, especially in adult-type diffuse gliomas where the mutational status of the IDH gene must be defined since the WHO2016 classification of gliomas [2,12]. The discovery of IDH mutations in a subgroup of GBs [14] was a key fact for the first molecular stratification with a clinical interest in two main subtypes: the formerly known as GB with IDH mutations (IDH-mutant), now called astrocytoma IDH-mutant grade 4, frequently arises from a previous low-grade astrocytoma, occur typically in patients aged 30–55 years and generally exhibits a better disease outcome with an average survival of 2–3 years, while IDH-wt GBs tend to occur in patients often older than 50 years, with a median of 65 years and their median survival is usually around 1 year [35].

IDH-mutant astrocytomas often harbor the triad of IDH, ATRX, and TP53 mutations. Historically, especially when IDH mutations were not yet known, tumor grade was based solely on glioma morphology. The presence of mitotic figures in an astrocytic tumor was considered sufficient criteria for an anaplastic/WHO grade III astrocytoma, and microvascular proliferation and/or necrosis were the prerequisites for the diagnosis of GB. In the current WHO2021 classification, these morphological criteria are maintained for the classification of IDH-mutant astrocytoma so that if mitoses are identified it corresponds to a grade 3, and if necrosis and/or microvascular proliferation is also observed, the astrocytoma is grade 4 [13]. However, recent evidence has shown that the homozygous deletion of CDKN2A in grade 2 or 3 IDH-mutant astrocytoma has a negative impact on tumor biology [13,36], therefore its demonstration in an IDH-mutant astrocytoma, even in the absence of the high-grade histological features above mentioned, determines its classification as a grade 4 IDH-mutant astrocytoma [4].

IDH: IDH converts isocitrate to α-ketoglutarate (αKG) in the cytosol (IDH1) and mitochondria (IDH2). Mutations in the IDH gene occur in gliomas most frequently in IDH1 and consist of a single amino acid substitution at codon 132. These mutations are associated with the accumulation of 2-hydroxyglutarate (2HG) within the tumor altering cancer metabolism and influencing the hypoxia-inducible factor subunit HIF-1α, a transcription factor that promotes tumor growth when oxygen levels are low. NADPH production is also impaired in gliomas with IDH1 mutations, which may sensitize the tumors to radiation and chemotherapy, explaining why patients with IDH-mutant neoplasms live longer [37]. While the metabolic consequences and downstream molecular effects of these mutations are yet to be elucidated, their potential value as diagnostic and prognostic markers in gliomas has been established from their clear association with improved overall survival when outcomes are compared between IDH-mutant and IDH-wildtype tumors [14,38]. The most frequent IDH1 R132H mutation can be detected easily by IHC [39]. A positive IHC result in an astrocytic tumor is sufficient for the diagnosis of astrocytoma, IDH-mutant, while a negative result in patients under 55 years of age requires sequencing the genes to exclude less frequent variants that may be present in this age group, such as other IDH1 mutations, or mutations in IDH2 codons R140 and R172 [4]. Gene expression profiling studies suggest that there are distinctive molecular subclasses in both the IDH-wt GB subtype and the IDH-mutant astrocytoma [40]. In TCGA molecular classification [15], most IDH-mutant astrocytomas are ascribed to the proneural subtype and are characterized by TP53 mutations, PDGFRA gain, absence of EGFR mutation, and G-CIMP phenotype, whereas wt-IDH GB are mostly of the mesenchymal subtype.

ATRX: The X-linked α-thalassemia/mental retardation syndrome (ATRX) is a key component of a multiprotein complex that regulates chromatin remodeling, nucleosome assembly, telomere maintenance, and histone H3.3 deposition in transcriptionally silent genomic regions [41]. ATRX is mutated in about 89% of astrocytomas, IDH-mutant [42], and is usually mutually exclusive with TERT promoter mutations. Diffuse gliomas harboring IDH mutations can be diagnosed as astrocytoma, IDH-mutant if there is a loss of ATRX nuclear expression by IHC without the need for 1p/19q testing [4]. Moreover, loss of nuclear ATRX expression is one of the criteria to diagnose a newly recognized entity, the high-grade astrocytoma with piloid features which is defined by a characteristic DNA-methylation profile [4,43].

Oligodendrogliomas currently defined by the combination of IDH mutation and 1p/19q codeletion in a diffuse glioma are the other subtype of IDH-mutant glioma. In oligodendrogliomas thus defined there are also mutations of the TERT promoter. They are usually present in young adults, in a predominantly cortical location and with a typical microscopical morphology made up of rounded cells with a central nucleus and a clear perinuclear halo [44]. As in astrocytomas, the presence of homozygous CDKN2A deletions is associated with an unfavorable prognosis, so their presence or that of morphological signs of anaplasia (frequent mitoses, microvascular proliferation, and/or necrosis) are sufficient for a grade 3 oligodendroglioma [4].

2.5: High-grade gliomas in pediatric, adolescent, and young adult populations

The delay in the investigation of pediatric gliomas accumulated for decades because of the legal protection of children who participate in medical research has fortunately recovered in recent years, a fact that has made it possible to know that the pediatric malignant gliomas (pHGGs) represent entities different from their counterparts in adults. Indeed, pHGGs although morphologically similar to those of adults are totally different, genetically, biologically, and clinically. Biomarkers other than those common in adult malignant gliomas are required for their identification. It is important to keep in mind that adults can have malignant pediatric gliomas, especially young adults, and conversely, adolescent patients can have malignant adult-type gliomas. The term GB, which was traditionally used in pHGGs, is no longer applied to these entities.

According to WHO2021 [4], the group of pHGGs encompasses four entities that require molecular characterization and integration of histological data for diagnostic purposes. This category includes:

2.5.1: Diffuse midline glioma, Histone 3 K27M altered

Diffuse midline gliomas are gliomas that were originally based on a specific location and have a highly negative prognosis. These are gliomas located in midline structures such as the pons, midbrain, spinal cord, basal ganglia, and thalamus, and predominate in children, but can be seen in adults as well. Histologically, they are similar to infiltrating astrocytomas, with the presence of mitosis, vascular proliferation, and necrosis, but none of them are mandatory. In fact, if Histone 3 (H3) K27M mutation is confirmed, either by immunohistochemical studies or Sanger sequencing, the WHO directly confers a grade 4, considering its very negative prognosis [4].

Heterozygous H3 K27M mutation in HGGs causes a repressive epigenetic modification of the histone with loss of normal lysine trimethylation at position 27 (H3 K27me3) [45]. p53 overexpression/TP53 mutation and ATRX mutation often accompany the H3 mutation. H3 K27M mutation is associated with the loss of expression of H3K27me3 by IHC which is a consequence of the loss of the normal trimethylation of histone H3 at position K27. If immunostaining for H3K27M is negative, there are two less frequent alternative molecular alterations with a similar clinical significance [46]: EGFR mutation (subtype 2) present in bi-thalamic tumors, with occasional overlap with the H3 K27M mutation, and EZHIP overexpression (subtype 3). Because the three subtypes share the loss of expression of H3K27me3 by IHC, the WHO2021 groups these three subtypes under the designation of Diffuse midline glioma, H3 K27-altered, WHO grade 4 [4]. Nevertheless, H3-K27me3 loss may not always be related to the H3 K27-altered spectrum of gliomas. IDH-mutant and 1p/19q codeleted oligodendrogliomas also show H3-K27me3 loss, especially in IDH1 R132H mutation [47]. Hence, H3-K27me3 loss can act as a surrogate marker for H3 K27M mutation (or the diagnosis of H3 K27-altered HGG) in concordant clinical settings (i.e., midline lesions in non-adult population or when IDH mutations have been effectively discarded). On the other hand, H3 K27 alterations can be seen in other CNS tumors like posterior fossa ependymomas, pilocytic astrocytomas, and glioneuronal tumors, and even in non-midline diffuse gliomas [4], but the prognostic significance of an H3 K27 alteration in a non-canonical location or tumor entity is still unclear, so that the Diffuse midline glioma, H3 K27-altered term and the accompanying WHO grade 4 designation should be restricted to diffuse midline gliomas and not be applied to other tumors only harboring the mutation [48].

2.5.2: Diffuse glioma, H3 G34-mutant

In non-midline HGG in a child over 1 year of age or in a young adult, the histone H3 G34R/V mutation should be studied. This tumor is generally located in the cerebral hemispheres, and harbors a missense mutation in the H3F3A gene, exchanging glycine for arginine or valine at position 34 (G34R/V) of the histone H3.3 protein. The histone H3 G34R/V mutation can be studied by IHC or sequencing. Histologically, the high-grade diffuse infiltrating glioma shows a lack of expression of OLIG2 in 90% of the cases, as well as loss of ATRX and p53 overexpression in all cases [4]. One caveat is that some H3 G34-mutant gliomas show an embryonic-like appearance of tumor cells [49], so a high index of suspicion of this tumor type must be maintained, especially in adult patients.

2.5.3: Diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype

This pHGG is a newly recognized type that requires histological and molecular data integration. It is a highly heterogeneous entity and genome-wide studies have rendered three molecular profiles based on the different patterns of copy number alterations: PDGFRA amplification, MYCN amplification, and EGFR amplification. On this type of pHGG, it may be helpful to go directly to the performance of methylation profile analysis [50].

2.5.4: Infant-type hemispheric glioma

Infant-type hemispheric glioma is a new entity incorporated in the WHO2021 classification [4]. It presents in newborns and has a defined molecular profile, characterized by fusion genes involving ALK, ROS1, NTRK1/2/3, MET, and RAS/MAPK, with differentiated prognosis and treatment [51].

2.6: Molecular profiles and epigenetic studies applied to the diagnosis of glioblastoma

Whole-genome and epigenetic studies in GB have provided a dearth of information regarding GB subtypes. Gene expression profiling of GB identified several transcriptome subgroups. Two of the main subgroups sit at opposite ends: proneural and mesenchymal [52]. Proneural GB is common in young adults, corresponds to the secondary GB subtype (astrocytoma, IDH-mutant grade 4), and has neuronal differentiation. Is characterized by IDH/TP53 mutations/positivity for the glioma-CpG island methylator phenotype (G-CIMP) and normal EGFR/PTEN/Notch signaling. The G-CIMP phenotype, like IDH mutation, provides a molecular definition of secondary GB. The mesenchymal GB is common in older adults, has mesenchymal differentiation, is associated with worse outcomes, and is characterized by abnormal EGFR amplification/PTEN loss/NF1 mutations/Akt signaling.

Recent molecular studies have deepened the molecular architecture of GB. Califano and Alvarez [53] have applied their knowledge of the cancer interactome in elucidating the molecular drivers of the GB mesenchymal variant. They have succeeded in constructing the GB oncotecture, that is, the regulatory architecture that underpins the phenotype and nature of GB, using the Algorithm for the Accurate Reconstruction of Cellular Networks (ARACNe). With the built oncotecture, they have confirmed that the Master Regulator (MR) proteins of the mesenchymal phenotype, which are the proteins that participate in the modular regulatory structure necessary for the mesenchymal phenotype, are C/EBP, STAT3, FOSL2, RUNX1, and BHLHB2 [54]. Using other bioinformatic tools, they have also succeeded in defining the upstream driver mutations of the activation of this MR hub, namely C/EBPB and C/EBPD amplification and KLHL9 deletion [55]. Indeed, it was inferred that CEBPD amplification and KLHL9 deletion combined would produce a mesenchymal phenotype with an OR of 20, compared, for example, to EGFR amplification with a non-significant OR of 1.35.

These molecular studies have relied primarily on RNA studies, a complex technique that is not widely used in clinical laboratories, especially outside of tertiary academic centers, and is therefore rarely used in routine diagnosis of GB. On the other hand, efforts led by German researchers have enabled a practical classification of CNS tumors based on methylation studies [56]. Using formalin-fixed paraffin-embedded samples and methylation array techniques, they initially defined 82 classes of CNS tumors characterized by distinct DNA methylation profiles, including eight subclasses in the GB group. Two of them are driven by mutations in Histone 3, at K27 and G34, and are now considered by the WHO to be distinct HGG. In the remaining six, there were three variants linked to alterations in the receptor tyrosine kinase (RTKI, II, and III), the mesenchymal subtype, the midline subtype, and the MYCN-altered subtype. It is a feasible method to use in clinical practice, especially in cases that may represent a diagnostic challenge, such as astrocytomas of the IDH-wt type that do not have the molecular characteristics required to be diagnosed as GB IDH-wt (amplification of EGFR, TERT promoter mutation, or +7/−10); evidence shows that such tumors can be easily grouped by their methylation profile with GB or other low-grade glial/neuroglial profiles, providing information that is critical for prognosis and treatment decision.

Next-generation sequencing (NGS) approaches have also been developed not only for GB but also for other CNS tumors. For example, the Glioseq panel, developed by UPMC [57], is an example of a tailored NGS panel that can cover adult and pediatric populations. Extraction of RNA in addition to DNA is also needed to study gene fusions that are diagnostic in certain brain tumors, including gliomas. Therefore, NGS panels of CNS tumors are currently still of limited use. Similarly, in the United Kingdom, NGS approaches tend to focus more on the detection of clinically actionable mutations in the therapeutic context, and are therefore sometimes performed outside of diagnostic workflows. Interestingly, methylation profiles may be closely related to clinically relevant gene mutations, expression profiles, or copy number variations, thus methylation profiling may provide useful therapeutic information in conjunction with molecular diagnosis [58].

2.7: Recommended nomenclature for reporting glioblastoma and high-grade gliomas

Due to the increasing clinical impact of the molecular findings in the diagnosis and classification of CNS tumors, and the need to integrate them with histopathological, radiological, and clinical characteristics, the International Society of Neuropathology updated the way in which gliomas and other CNS should be reported to provide the full range of information available and to standardize diagnosis reports [59]. It is recommended to perform a layered diagnosis, in which the first layer, on the top of the diagnostic report, corresponds to the integrated diagnosis, combining histological and molecular data. The second layer refers to the microscopical findings based on routine histological and IHC. The third layer includes the CNS WHO Grade, and the fourth layer describes all the molecular information available, and techniques employed. Some examples of pathological reports are shown in Table 2.1.

Table 2.1

Examples of layered diagnosis of CNS tumors. The first layer represents the final integrated diagnosis by combining histological and molecular available information. The second layer describes the microscopic findings, the third layer specifies the CNS WHO Grade, and the fourth layer reports all the molecular information and the applied molecular techniques.

The not otherwise specified (NOS) designation should be applied when diagnoses lack necessary diagnostic information (e.g., molecular) for a more specific classification. The not elsewhere specified (NEC) qualifier can be applied when there is a mismatch between histological features and molecular results. Alternatively, NEC can be used when diagnostic tests show non-canonical results, precluding assignment to a known WHO entity and therefore suggestive of a new/emerging tumor type [60].

2.8: Pathology of IDH-wt glioblastoma and diagnostic workup

Before histomolecular classification, GBs were defined microscopically as infiltrating gliomas with atypical astrocytic-like cells at different stages of differentiation, mitotic figures, microvascular proliferation, and/or necrosis with or without pseudopalisading (Fig. 2.1).

Fig. 2.1

Fig. 2.1 Histological hallmarks of GB. GBs are composed of atypical tumor cells (A) with frequent mitotic figures (B, black arrow ). The presence of necrosis (C), surrounded or not by a peripheral pseudopalisade of pyknotic tumor cells, as well as microvascular proliferation (D) are prominent features. Scale bars figures A, C, D 50 µm, B 20 µm. GB , Glioblastoma.

Intra- and intertumoral morphological variability is characteristic, ranging from large, sometimes giant, differentiated astrocytes to small, undifferentiated, highly packed glial cells. Microvascular proliferation and necrosis may be absent in some GBs. Characteristically, GB cells migrate individually from the main tumor mass through the brain parenchyma, surrounding and dissecting normal CNS tissue components, such as neurons or vessels (perineuronal and perivascular satellitosis), accumulating in the subpial region and migrating to along white matter tracts (intrafascicular spread). All of these phenomena are historically known as secondary Scherer structures and are diagnostically useful in small peritumoral biopsies [61].

Some authors have attempted to identify distinctive morphological features based on driver mutations [62]. For example, high-grade astrocytomas with IDH mutation tend to show a more microcystic background or gemistocytic tumor cells, HGG with histone 3 K27 mutation are associated with marked nuclear pleomorphism, HGG with histone 3 G34 mutation have poorly differentiated areas, and GB with IDH/Histone 3-wt often show foci of epithelioid-like cells. However, in most cases, morphology does not allow the prediction of the molecular substrate. Therefore, the existence of GB variants (i.e., small cell GB, epithelioid GB, gliosarcoma), as well as HGG with morphologies that can mimic GB or other neuroectodermal tumors (e.g., H3 G34 mutant HGG that can mimic an embryonal tumor), emphasize the need for an integrated diagnosis, including IHC and molecular studies in addition to clinical-radiological information (Fig. 2.2).

Fig. 2.2

Fig. 2.2 Variants and mimickers of GB IDHwt. GBs can present different morphological patterns, such as small cells (A), epithelioid (B), or gliosarcoma (C), the latter characterized by an increase in the reticulin network (D). In some cases, the morphology presents a diagnostic challenge, as in this high-grade spindle cell glioma (E), which harbored an IDH1 R132H mutation (F) and a 1p/19q codeletion (data not shown), turning out to be an oligodendroglioma, IDH-mutant and 1p/19q-codeleted. (G) HGG with small cell and gemistocytic components in a cerebral hemisphere of a young adult. Molecular studies (H) revealed a missense mutation in the H3F3A gene, exchanging glycine for arginine at position 34 (G34R), corresponding to a diffuse hemispheric H3G34 mutant glioma. Scale bars figures A, B, E, F 50 µm, C, D, G 100 µm. GBs , Glioblastomas; HGG , High-grade glioma.

In the evaluation of an infiltrating glioma, initial IHC studies can provide useful molecular profiling information. By IHC, GB cells are usually diffuse and strongly positive for glial fibrillary acidic protein (GFAP) (Fig. 2.3A), which is the differentiation biomarker most used by pathologists, although immunoexpression may be weaker, focal, or rarely absent. Another biomarker of glial differentiation commonly used in clinical practice is OLIG2, which is useful in differentiating GB from its histological mimics such as ependymoma with anaplastic features, extraventricular neurocytoma, as well as other metastatic or primary non-glial poorly differentiated neoplasms [63]. However, care must be taken, as rare subtypes of HGG, such as the Histone 3 G34 mutant HGG, can be OLIG2 negative [64]. The demonstration of neurofilaments in the tumor background can also be useful to show the infiltrative nature of the glioma (Fig. 2.3B). The IDH R132H mutation can be easily diagnosed with the specific antibody, with IDH1 mutant cases being diffusely positive (Fig. 2.3C) while cases with IDH1 mutations other than the R132H mutation or IDH2 mutations are negative (Fig. 2.3D). Diffuse high-grade IDH-mutant gliomas with loss of nuclear immunoexpression of ATRX or ATRX mutation are considered to be of astrocytic lineage and are classified as IDH-mutant astrocytomas, grade 4 (Fig. 2.3E), whereas GB IDH-wt tend to retain ATRX expression (Fig. 2.3F) as do oligodendrogliomas. Astrocytomas with IDH mutation usually present a strong nuclear expression of p53 by IHC in >10% of tumor cells or TP53 mutation (Fig. 2.3G), while this mutation is less frequent in IDH-wt GB (Fig. 2.3H).

Fig. 2.3

Fig. 2.3 Immunohistochemical studies and profiles in infiltrating gliomas and GBs. Infiltrating gliomas are characteristically positive for GFAP (A), while neurofilament staining shows axons trapped between neoplastic cells (B), highlighting the infiltrating nature of the tumor. The IDH1 R132H mutation is the most frequent IDH1/2 mutation in IDH-mutated gliomas, showing diffuse immunohistochemical positivity (C), while IDHwt GBs are negative (D). On the other hand, ATRX mutation results in the loss of nuclear staining in tumor cells (E), whereas non-mutated engulfed normal and/or reactive cells show nuclear reactivity (F). The ATRX mutation is frequently associated with IDH-mutated astrocytomas and high-grade Histone 3 G34 gliomas. p53 staining in mutated tumors shows strong positivity in >10% of tumor cells (G), while the presence of occasional scattered positive cells (H) indicates a TP53wt tumor. Scale bars 50 µm. GFAP , Glial fibrillary acidic protein.

As shown in Fig. 2.4, WHO2021 divides diffuse gliomas into two main types according to the presence or absence of an IDH mutation. This first distinction is quick and easy by IHC using an antibody against IDH1 R132H. In patients aged 55 years or older, IHC negativity for IDH1 is sufficient to classify the glioma as IDH-wt GB, while in younger patients it is highly recommended to exclude non-canonical variants by sequencing or other molecular techniques since their detection has an impact on the clinical management. In adults, diffuse IDH-wt gliomas that do not show necrosis or microvascular proliferation changes must have at least one of the following three molecular alterations: TERT promoter mutation, EGFR gene amplification, or chromosome copy number changes with gains on chromosome 7 and loss of chromosome 10 (+7/−10). If these conditions are met, the glioma is designated IDH-wt GB grade 4 even if it has a lower grade histology. Histone H3 should be studied in all midline and hemispheric IDH-wt GBs, as explained in the High grade gliomas in pediatric, adolescent and young adult populations section. In IDH-wt and H3-wt GB of young patients or in selected cases, methylation profiling may provide useful information.

Fig. 2.4

Fig. 2.4 Diagnostic diagram of diffuse gliomas. In a diffuse glioma, the age of presentation, the location of the tumor, the radiological characteristics, and the microscopic morphology are the basic data on which the diagnosis is made. The most common IDH mutation (IDH1 R132H) can be rapidly and easily evaluated by IHC and is the first molecular study in adult cases. Reaching an accurate final diagnosis may require multiple other molecular techniques, whose integration with the rest of the clinical and morphological findings leads to the final histomolecular diagnosis. In some newly defined entities, characterized by an often complex molecular background, direct NGS panel or methylation profiling may be more appropriate, especially in the pediatric population and in small tumor samples. GB , Glioblastoma; MVP , Microvascular proliferation. Color codes for diagnostic procedures: Blue , IHC studies; Red , FISH (Fluorescent in situ hybridization) studies; Green , sequencing/molecular studies; White , histology; Yellow , methylation studies.

In summary, in the initial IHC workup, an IDH-wt GB will typically show a strong GFAP positivity, IDH1 R132H negativity, ATRX nuclear expression, and absence of p53 mutation. Different results for these stains, especially IDH and ATRX, should prompt doubts on the IDH-wt GB diagnosis and should lead to the search for alternate HGGs. In a patient of 55-years old or older with the IDH/ATRXwt pattern, the IDH-wt GB diagnosis can be readily made. If doubts arise, a diagnosis of HGG, IDH1 negative, NOS can be made, and the definitive integrated diagnosis can be held until IDH1/2 sequencing has been performed. However, it is a common practice to confirm the IDH-wt GB type by demonstrating the presence of either TERT promoter mutation, and/or EGFR alterations, and/or +7/−10 chromosome changes by any of the molecular biology techniques available in the laboratory. In addition, MGMT promoter methylation status is also routinely studied. As previously mentioned, MGMT status provides useful information regarding chemotherapy sensitivity. In addition, the combination of TERT/MGMT status may provide additional prognostic information, as pTERTmut and MGMT methylated IDH-wt GB have better prognosis than other pTERT/MGMT combinations [65]. Moreover, up to 20% of IDH-wt pTERT-wt GB can have BRAF V600 mutations, a mutation more common in pediatric gliomas and rarely found in adult gliomas (0.7%–2%) [22]. Hence, the absence of TERT promoter mutations, or EGFR or +7/−10 alterations in an IDH-wt GB should lead to analyzing BRAF mutations, which is a clinically actionable mutation. With those studies, the vast majority of IDH-wt GB can be diagnosed easily, quickly, and reliably. Nevertheless, ancillary techniques may be necessary for specific contexts. Methylation profiling and NGS panels may be useful tools in selected cases providing additional information for diagnostic and therapeutic purposes.

2.9: Final remarks and future trends

As the old term glioblastoma multiforme implies, IDH-wt GB and other HGGs constitute a heterogeneous group of tumors with different histological appearances and molecular alterations. Many of them develop through the activation of certain aberrant signaling pathways that lead tumors to diverse phenotypes. In the era of personalized medicine, the development of treatment strategies must consider the unique characteristics of each tumor so that each patient can be treated based on the individual characteristics of their specific tumor. In this context, the search for therapeutic targets in molecular analyses at the time of diagnosis will be of the utmost importance in all IDH-wt GB/HGG, regardless of their potential impact on the WHO diagnostic categorization.

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