Gliomas are one of the most aggressive and lethal solid tumors of the central nervous system. Despite the alarming numbers, the molecular pathogenetic mechanisms of gliomas are not yet fully elucidated. Nowadays, several approaches aim to understand the biology of the disease and identify promising markers, which may provide effective novel therapies.
This white paper summarizes the recent developments in glioma classification and the key molecular markers for glioma stratification. Next, it highlights the glioma proteome with a list of relevant genes with favorable and unfavorable prognostic values in glioma. It then focuses on the glioma tumor microenvironment that may provide helpful insights when developing novel therapeutic strategies.
Gliomas are solid tumors of the central nervous system (CNS). They arise from the neoplastic transformation of glial cells, namely astrocytes, oligodendrocytes, and ependymal cells.
Brain tumors comprise approximately 2% of all adult cancers but form a larger fraction within the group of childhood tumors. Gliomas account for about 80% of all malignant brain tumors and are classified according to the cell type of origin, differentiation, and malignancy grade. Gliomas show considerable variability in age of onset, grade of severity, histological features, and ability to progress, as well as to metastasize.
Survival time after diagnosis with glioma varies significantly depending on grade. According to the World Health Organization (WHO) classification, gliomas are divided into four grades. Grade 1, 2, and 3 are classified as low-grade gliomas, while grade 4 are classified as high-grade gliomas. Unfortunately, the prognosis for high-grade gliomas is poor due to limited possibilities of curative treatment.
The most common form of glioma - astrocytoma
The most common form of glioma is an astrocytoma, representing approximately 50% of all gliomas. Grade 4 astrocytoma is also known as glioblastoma or glioblastoma multiforme (GBM).
GBM is the most frequent and malignant histological type, accounting for 65% of gliomas with a very poor 5-year survival (less than 5%). GBM is typically classified into three subtypes, namely proneural, classical and mesenchymal, according to the gene expression of various biomarkers, including platelet-derived growth factor receptor (PDGFR), neurofilament light chain (NF-L), epidermal growth factor receptor (EGFR), and CD44, respectively. Pro-neural to mesenchymal transition of glioma is associated with aggressive phenotypes, unfavorable prognosis, and treatment resistance.
Gliomas are well-known tumors at the molecular level. However, these advances have not been translated into therapeutic benefits for the patients.
While our knowledge about the molecular biology of gliomas is rapidly expanding and is, to some extent, already assisting us in the design of tumor-tailored therapeutics, such as immunotherapy treatments, we are still struggling to develop efficient treatments. As a result, the overall survival in patients with gliomas has not significantly increased in the last decades.
Typical treatments for gliomas involve surgical resection of the tumor mass, radiotherapy, and chemotherapy treatments. However, relapse or even recurrence of gliomas is common and mainly due to their infiltrative growth and their high proliferative abilities.
Due to the absence of definitive surgical and medical treatments currently available, an early diagnosis coupled with an accurate tumor classification is crucial to select a personalized treatment.
Moreover, the functional heterogeneity in gliomas is defined not only by the genetic makeup of glioma cells but also through microenvironment-driven epigenetic influences that regulate glioma cell stemness.
Hence, elucidating the state transition programs and mechanisms driving cellular plasticity is essential to overcome current therapeutic limitations in GBM.
We must expand our knowledge as a step toward the design of practical and safe treatments. Therefore, the identification of molecular biomarkers is unquestionably essential and urgent for an accurate prognosis and development of critical therapeutic targets in gliomas.
Glioma classification and key molecular pathology
Since 2008 the classification of brain tumors by the World Health Organization (WHO) has continued to evolve up to today with the latest fifth edition (WHO CNS5, 2021)(Louis 2021).
The updated classification emphasizes the need for a classification of gliomas based on histological and molecular genetic features for clinical diagnosis and outcome prediction.
With the WHO CNS5 2021 edition, several IHC diagnostic and prognostic markers have been added to the diagnostic criteria of gliomas. The new seven-layers approach for glioma classification is shown in (figure 1).
Antibodies against ATRX, the mutated forms of IDH1 (IDH R132H), and histone H3 (H3K27M) are used to detect the respective mutations. Analysis of deletion of 1p and 19q chromosome arms is done by multiplex ligation-dependent probe amplification (MLPA) or, less commonly, by fluorescent in situ hybridization.
The distinction between the different forms of glioma is mainly based on morphological features, mutations and chromosomal aberrations (figure 2).
In gliomas neuropathological diagnostics, antibodies directed towards proteins such as IDH (isocitrate dehydrogenase), ATRX (alpha thalassemia/mental retardation syndrome X-linked), GFAP (glial fibrillary acidic protein), SYN (synaptophysin), EGFR (epidermal growth factor receptor), p53 (tumor suppressor protein 53), and the proliferation marker Ki-67 (MKI67) are the gold standard and routinely used.
Immunohistochemistry (IHC) plays a vital role in distinguishing between different tumor types. Figures 3-6 show some examples of IDH1, ATRX, and GFAP multiplexed IHC-IF staining in different glioma grades.
Adult and pediatric gliomas: distinctive features
An essential update in the WHO CNS5 2021 classification of brain tumors is the distinction of diffuse gliomas into adult-type and pediatric-type, all with distinct responses to treatment and outcomes (figure 7).
The distinction between different forms of gliomas is mainly based on morphological features; however, IHC analysis plays a vital role in distinguishing between different tumor types, particularly when the tumor is poorly differentiated.
Numerous reports have highlighted substantial differences in adult and pediatric gliomas mainly based on histological observations, frequency, location, and pathologic spectrum (Broniscer 2007). Pediatric high-grade gliomas, for example, often arise in brain regions that are rarely targeted in adult disease.
Pediatric diffuse gliomas are more complex and molecularly distinct from those in adults. Pediatric gliomas are generally classified as low-grade and high-grade and are characterized by circumscribed growth and frequent H3 and BRAF gene fusions or mutations (Louis 2021).
On the other hand, PTEN mutations and EGFR amplification, frequent in adult primary glioblastoma, are less common in children (Pollack 2006). Furthermore, in adults, secondary glioblastomas rarely contain EGFR amplification (Furnari 2007).
Mutations in TP53, CDKN2A, and PIK3CA are common in adult and pediatric high-grade gliomas (Gallia 2006). Furthermore, the platelet-derived growth factor receptor-α (PDGFR-α) is the predominant target of focal amplification in high-grade childhood glioma and may be a valuable target for the pediatric population (Paugh 2010).
Significant differences in copy number alterations distinguish childhood and adult glioblastoma. No IDH1 hotspot mutations were found in pediatric tumors, highlighting molecular differences with adult secondary glioblastoma. Frequent gain of chromosome 1q and lower frequency of chromosome 7 gain and 10q loss also clearly distinguished childhood from adult high-grade gliomas (Paugh 2010).
The glioma proteome
The glioma proteome is revealed and accessible on the Human Protein Atlas (HPA) website (proteinatlas. org). It is built on the GBM data available from The Cancer Genome Atlas program (TCGA) and combines transcriptomic data with antibody-based protein data.
The transcriptome analysis shows that 72% (n=14370) of all human genes (n=20090) are expressed in glioma. Figure 8 shows the number of elevated and prognostic (unfavorable and favorable) genes in glioma.
The top 20 significant genes related to unfavorable and favorable prognosis in GBM are listed in Table 1 and Table 2, respectively. For unfavorable genes, higher relative expression levels at diagnosis give significantly lower overall survival for the patients. For favorable genes, higher relative expression levels at diagnosis give significantly higher overall survival for the patients.
Figure 9 shows the different composition of mutated genes across high-grade (GBM) and low-grade gliomas.
The glioma proteome is built using TCGA transcriptomics data and antibody-based protein data. It shows that 895 genes have elevated expression in glioma, 267 of which are suggested as prognostic. Among the prognostic genes, 200 are associated with unfavorable prognosis and 67 are associated with favorable prognosis.
The glioma tumor microenvironment
The dynamic communication between tumor cells and the surrounding tumor microenvironment (TME) plays a crucial role in the sustained growth, proliferation, and invasion of gliomas, survival, evasion of cell death, metabolism, migration, and metastasis.
The glioma TME is highly heterogeneous, consisting of a multiplex of both cancerous and non-cancerous cells, including endothelial cells (ECs), immune cells, glioma stem-like cells (GSCs), and astrocytes, as well as non-cellular components such as the extracellular matrix (ECM) (Boyd 2021; Yekula 2020).
Several means of intercellular communication between the glioma tumoral cells and the TME have been documented. In the glioma TME, cells communicate through endothelial cells, growth factors, cytokines, chemokines, monocytes, macrophages, mast cells, microglia, T-cells, astrocytes, oligodendrocytes, and cancer stem cells (Cole 2020; Radin 2020).
All the different means of communication show a multifaceted role in supporting several hallmarks of cancer (figure 10).
Figures 11-16 show examples of immunostainings using PrecisA Monoclonals antibodies against TME markers in different grades of gliomas. Table 3 lists the newly released PrecisA Monoclonals markers from Atlas Antibodies.
Figure 10. Understanding the dynamic glioma tumor microenvironment is necessary for the development of new immunotherapeutic strategies. Glioma cells coexist with normal non-neoplastic cells.
Glioma cells proliferate in an hypoxic immunosuppressive TME, with aberrant vasculature, abundant and distinct extracellular matrix and impaired blood-brain tumor barrier.
Four mechanisms in the TME have emerged as paramount for our understanding of gliomas and crucial for the development of new treatment strategies such as immunomodulation, angiogenesis, cancer stem cells, and drug resistance. Many of these mechansims are interdependent and carry specific molecular signatures.
In recent years, immunotherapy has become one of the major options for anti-cancer therapy. However, the complex mechanisms of glioma immunogenicity and microenvironment interactions have only partially been elucidated (Desland 2020). The ability of tumor cells to suppress both local and systemic immune responses and to hijack the communication with the TME, severely restrict treatment efficacy.
Immune cells constitute an important component of the glioma TME as they can reach up to 50% of the tumor mass content. Immune cells in glioma switch on the inflammatory process in the TME thus enhancing tumor development (Gieryng 2017; Strepkos 2020). Consistently, high-grade gliomas have a more immunosuppressive profile than low-grade gliomas.
Among the immune-related genes ADORA2A, CD160, CD276, NRP1 and VTCN1 are significantly overexpressed in low-grade gliomas. In high-grade gliomas, VTCN1, BTNL2 and METTL21B are overexpressed, while the expression of CD86, HAVCR2, LAIR1 and VSIR is significantly decreased.
Cytokines and chemokines secreted by glioma cells induce infiltration of immunosuppressive cells (MDSCs, Tregs and TAMs) and the acquisition of pro-tumoral phenotypes by myeloid cells (M2 phenotype differentiation and PDL-1 and B7 expression).
The poor prognosis for glioblastoma is also partly due to the lack of successful drug delivery across the blood-brain tumor barrier.
Glioblastomas are highly vascularized tumors, and glioma growth depends on the formation of new blood vessels. Angiogenesis is a complex process involving proliferation, migration, and differentiation of vascular endothelial cells under the stimulation of specific signals.
The high metabolic demands of high-grade glioma, for instance, create hypoxic areas, that trigger the expression of PLVAP (a vascular marker of disrupted blood-brain barrier) and angiogenesis, leading to the formation of abnormal vessels and a dysfunctional blood-brain tumor barrier. Endoglin (CD105), a marker for immature blood vessels, is significantly higher in glioblastoma compared with peritumoral normal tissue (Burghardt 2021).
Glioma cancer stem cells and drug resistance
Chemoresistance and reoccurence are major issues in the management of patients with glioma and the main cause of mortality. Reoccurence of gliomas is largely associated with the ability of tumor cells to regenerate from treatment-resistant cancer stem cells within and surrounding the primary tumor after initial treatment. Glioma cancer stem cells (GSCs) are the main drivers of uncontrolled cells growth in high-grade gliomas, but also the resistance to therapy (Boyd 2021; Ma 2018).
GSCs, acts through a core set of neurodevelopmental transcription factors and oncogenes. High-grade gliomas show elevated transcription of stem cells such as ALDH1A1, EZH2, GFAP, SALL4, NANOG, and POSTN. Molecular surface markers such as CD133, CD44, A2B5, CD15, CD171 have also been implicated in the reoccurrence of gliomas and increased aggressiveness of glioblastoma.
Chi3l1 as modulator of stem cells cellular states
Phenotypic plasticity in human tumors can be driven by activation of epithelial-to-mesenchymal transition (EMT) process by which cells acquire plasticity and gain the properties of stem cells.
CHI3L1 is a secreted protein acting as modulator of stem cell states and highly expressed in gliomas. CHI3L1 is used as a marker for invasion, migration and angiogenesis in glioblastoma (Guetta-Terrier 2021).
TME, including infiltration of myeloid cells such as microglia and macrophages, plays a significant role in glioma progression (Brandenburg 2020).
Glioma accumulated microglia/macrophages are a mixed cell population with pro- and anti-tumoral properties, although tumor-supporting effects generally predominate.
Microglial cells which present the antigens to the CD4-positive T-cells, express HLA-DRA protein which belongs to the HLA class II proteins (Neefjes 2011).
HLA expression is increased in glioblastoma patients, and there is some evidence suggesting that HLA-family can be used a specific molecular target in treatment of glioblastoma (Wang 2015).
METTL21B is involved in cell adhesion, angiogenesis and cell proliferation. Its expression is positively associated with glioma grade at the protein level. METTL21B facilitates immune evasion of tumor and affect prognosis by mediating macrophage polarization from M1 to M2 and regulating expression of immune checkpoints.
Nevertheless, patients with high METTL21B level are likely to have better response to immune checkpoints blockage therapy. Because of its substrate specificity, METTL21B is a promising target for the treatment of glioma (Shu 2021).
Figure 12 shows the high METTL21B expression in glioblastoma tumor cells as well as increased number of activated microglial cells (HLA-DRA) compared to controls.
Targeting GSCs is an extremely important aspect of the clinical treatment of gliomas. A better comprehension of glioma GSCs provide functional insights into the dynamic of cellular communication during gliomagenesis, creating new opportunities for diagnostics and therapeutics.
The transcription factor SALL4 participates in cell proliferation, apoptosis, cycle, invasion, evolution and drug resistance. SALL4 is overexpressed in glioma patients and correlated with poor outcome (Zhang L. 2015).
SALL4 acts by strengthening the PI3K/AKT signaling pathway (which is a well-known pathway in the regulation of tumorigenesis, significantly activated in glioma) thus reducing the expression of the tumor suppressor PTEN (Liu 2017).
The transcription factor EZH2 is highly expressed in gliomas and a potential target for immune therapy.
EZH2 is a histone H3 lysine methyltransferase that promotes tumorigenesis in a variety of human malignancies including gliomas by altering the expression of tumor suppressor genes. EZH2 overexpression in gliomas is associated with several immune checkpoints, cell cycle, DNA replication, mismatch repair, p53 signaling and tumor-infiltrating lymphocytes (Chen 2021).
The aldehyde dehydrogenases ALDH1A3 is associated with cell adhesion and tumor invasion and a marker of Mes-subtype of gliomas.
ALDH is a marker of cancer stem cells associated with the malignant phenotype in gliomas.
Among the ALDH isoforms, ALDH1A3 is overexpressed in high-grade than in low-grade gliomas, while ALDH1A1 is overexpressed in low-grade compared to high-grade gliomas.
Most of the Mes-subtype patients have high ALDH1A3 mRNA expression, indicating ALDH1A3 as a useful marker for Mes-subtype of gliomas (Zhang W. 2015).
Shugoshin 2 is critical in cell division and cell cycle progression.
Shugoshin 2 (SGO2) plays a crucial role in glioma cells proliferation. Data show that SGO2 expression positively correlates with WHO grading of human gliomas (Kao 2021).
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