Classification of gliomas and key research targets of their molecular pathology

Gliomas are solid tumors

Figure 1.

Figure 3.

Figure 4a.

Figure 4c.

Figure 5.
Gliomas are classified
according to cell-derived type, degree of differentiation, and degree of malignancy.
The type, grade, and molecular characteristics of gliomas determine the diagnosis and treatment options
.
Based on comprehensive molecular analysis, adult diffuse invasive gliomas were divided into three overall groups
based on origin, isocitric acid dehydrogenase (IDH) mutations, ATRX, and 1p/19q expression.
In the pediatric population, diffuse gliomas are divided into low-grade gliomas and high-grade gliomas
.

Many reports highlight significant differences in adult and pediatric gliomas, based primarily on histological observation, frequency, location, and pathological spectrum
.
For example, pediatric high-grade gliomas usually appear in brain regions
that are rarely targeted by adult disease.
Pediatric diffuse gliomas are more complex and molecularly different
from adults.
Pediatric gliomas are usually classified as low and high grades, characterized by localized growth and frequent fusion or mutation
of the H3 and BRAF genes.

On the other hand, PTEN mutations and EGFR amplification, which are common in primary glioblastoma in adults, are less common in children (Pollack 2006
).
In addition, in adults, secondary glioblastoma rarely contains EGFR amplification
.
Mutations in TP53, CDKN2A, and PIK3CA are common
in adult and pediatric high-grade gliomas.
In addition, platelet-derived growth factor receptor-α (PDGFR-α) is the main target for the amplification of glioma lesions in high-grade children and may be an important target in pediatric populations
.

Changes in the number of gene copies can significantly distinguish glioblastoma
in children and adults.
No IDH1 hotspot mutations were found in pediatric tumors, highlighting molecular differences with secondary glioblastoma in adults
.
The frequent increase in chromosome 1q and the lower frequency of increase in chromosome 7 and deletion of 10q also clearly distinguish high-grade gliomas
in childhood and adults.

Glioma's Proteome
Glioma Proteome is available on the Human Protein Atlas (HPA) website
.
It builds on GBM data provided by the Cancer Genome Atlas Project (TCGA) and combines
transcriptome data with antibody-based protein data.

Transcriptome analysis showed that 72% (n=14370) of all human genes (n=20090) were expressed
in gliomas.
Figure 6 shows the number of elevated and prognostic (unfavorable and favorable) genes in gliomas
.

Figure 6.
Elevated and prognostic genes in gliomas
.

The distribution of the most common mutant genes in high-grade (GBM) and low-grade gliomas is expressed as a percentage
of total cases (n).
Source TCGA
.
The glioma proteome is constructed using TCGA transcriptomics data and antibody-based protein data
.
It showed elevated expression of 895 genes in gliomas, of which 267 were thought to be prognostic.

Of the prognostic genes, 200 were associated with a poor prognosis and 67 were associated with a good prognosis
.

For unfavorable genes, a higher relative expression level at the time of diagnosis significantly reduces the overall survival rate
of the patient.
For favorable genes, a higher relative expression level at the time of diagnosis significantly improves the overall survival of patients
.
Figure 7 shows the different compositions of mutated genes in high-grade (GBM) and low-grade gliomas
.

Figure 7.
The distribution of the most common mutation genes in high-grade and low-grade gliomas into different high-grade (GBM) and low-grade gliomas is expressed as a percentage
of total cases (n).

The first 20 genes most significantly associated with a poor PROGNOSIS for GBM:

The first 20 genes most significantly associated with a good GBM prognosis:

The dynamic interaction between
tumor cells and the surrounding tumor microenvironment (TME) plays a vital role
in the continuous growth, proliferation and invasion, survival, evasion of cell death, metabolism, migration and metastasis of gliomas.

Glioma TME is highly heterogeneous and consists of a variety of cancer cells and non-cancer cells, including endothelial cells (EC), immune cells, glioma stem cells (GSCs), and astrocytes, as well as non-cellular components such as the extracellular matrix (ECM) (Boyd 2021; Yekula 2020)
。 Several cell-to-cell communication patterns between glioma tumor cells and TME have been documented
.
In 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
).

Figure 8.
Understanding the dynamic glioma tumor microenvironment is necessary
to develop new immunotherapy strategies.

Glioma cells coexist
with normal non-tumor cells.
Glioma cells proliferate in hypoxic immunosuppressed TME, the vascular system is abnormal, the extracellular matrix is rich and unique, and the blood-brain tumor barrier is damaged
.
The four mechanisms in TME are critical for us to understand gliomas and for developing new therapeutic strategies such as immunomodulation, angiogenesis, cancer stem cells, and drug resistance
.
Many of these mechanisms are interdependent and carry specific molecular characteristics
.

Immunomodulation
In recent years, immunotherapy has become one
of the main options for anti-cancer treatment.
However, the complex mechanisms of glioma immunogenicity and microenvironmental interactions are only partially elucidated (Desland 2020
).
The ability of tumor cells to suppress local and systemic immune responses and hijack communication with TME severely limits the therapeutic effect
.

Immune cells are an important component of gliomaTM because they can reach tumor mass levels
of up to 50%.
Immune cells in gliomas turn on inflammatory processes in TME, thereby promoting tumor development (Gieryng 2017; Strepkos 2020
).
At the same time, high-grade gliomas have more immunosuppressive features than low-grade gliomas
.

In the immune-associated gene 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 reduced
.

Cytokines and chemokines secreted by glioma cells induce infiltration of immunosuppressive cells (MDSCs, Tregs, and TAMs) and bone marrow cells acquire pro-tumor phenotypes (M2 phenotype differentiation and PDL-1 and B7 expression).

Part of the reason for a poor prognosis for angiogenesis
glioblastoma is the inability of the drug to successfully cross the blood-brain tumor barrier
.
Glioblastoma is a highly vascularized tumor, and the growth of a glioma depends on the formation
of new blood vessels.
Angiogenesis is a complex process involving the proliferation, migration, and differentiation
of vascular endothelial cells stimulated by specific signals.

For example, the high metabolic demand for high-grade gliomas creates hypoxic regions that trigger the expression of PLVAP (a vascular marker of blood-brain barrier destruction) and angiogenesis, leading to the formation of abnormal blood vessels and dysfunction of the blood-brain tumor barrier
.
Endoglin (CD105), a marker of immature blood vessels, is significantly higher in glioblastoma than in normal tissues
around the tumor.

Glioma Cancer Stem Cells and Drug Resistance
Chemotherapy resistance and recurrence are major problems in the management of glioma patients and a major cause of
death.
The recurrence of gliomas is largely related
to the ability of tumor cells to regenerate from and around the primary tumor after initial treatment.
Glioma cancer stem cells (GSCs) are the primary driver of uncontrolled cell growth in advanced gliomas and are resistant to treatment
.

GSCs function through a core set of neurodevelopmental transcription factors and oncogenes
.
High-grade gliomas show elevated stem cell transcription, such as ALDH1A1, EZH2, GFAP, SALL4, NANOG, and POSTN
.
Molecular surface markers such as CD133, CD44, A2B5, CD15, and CD171 are also associated
with recurrence of gliomas and increased aggressiveness of glioblastoma.

Finally, I would like to introduce a few studies on related TME markers in different levels of gliomas:

1.
Chi3l1
The phenotypic plasticity of human tumors can be driven by activating the epithelial-interstitial transformation (EMT) process through which cells acquire plasticity and acquire the properties of
stem cells.

CHI3L1 is a secreted protein that acts as a regulator of stem cell state and is highly expressed
in gliomas.
Glioblastoma is a highly vascularized tumor, and the growth of a glioma depends on the formation
of new blood vessels.
CHI3L1 is used as a marker of glioblastoma invasion, migration, and angiogenesis
.

Figure 9.
Human normal cortex (left) and astrocytoma (right) stained with monoclonal anti-anti-CHI3L1 (AMAb91777).

Fluorescent immunohistochemical images of the sample show strong protein expression
in astrocytoma (green).
The nucleus is counterstained by DAPI (blue
).

2.
METTL21B
TME, including infiltration of bone marrow cells such as microglia and macrophages, plays an important role
in the progression of glioma.
Microglia/macrophages accumulated by gliomas are mixed cell populations with pro-tumor and anti-tumor properties in which microglia that present antigens to CD4-positive T cells express the HLADRA protein belonging to HLA class II proteins
.
HLA expression is increased in patients with glioblastoma, and there is evidence that the HLA family can be used as a specific molecular target for the treatment of glioblastoma
.

METTL21B is involved in cell adhesion, angiogenesis, and cell proliferation
.
Its expression is positively correlated
with protein-level glioma grading.
METTL21B promotes immune evasion of tumors and affects prognosis by mediating the polarization of macrophages from M1 to M2 and modulating the expression of immune checkpoints
.
Nonetheless, patients with high METTL21B levels may respond
better to immune checkpoint blockade therapy.
Due to its substrate specificity, METTL21B is a promising target for the treatment of gliomas
.

The figure below shows the high expression of METTL21B in glioblastoma tumor cells and the increase in
the number of activated microglia (HLA-DRA) compared to controls.

Figure 10.
Multiplex IHC-IF staining
of human normal cortical (left) and glioblastoma (right) samples using anti-HLA-DRA monoclonal AMAb91674 (cytoplasmic, green) and anti-METTL21B polyclonal HAP043020 (nucleus, red).
Arrows indicate activated microglia (HLA-DRA-positive staining
).

3.
SALL4
targeting GSCs is an extremely important aspect of clinical treatment of gliomas
.
A better understanding of glioma GSCs provides functional insights into the dynamic communication of cells during glioma development, creating new opportunities
for diagnosis and treatment.

Transcription factor SALL4 is involved in cell proliferation, apoptosis, cycles, invasion, evolution, and drug resistance
.
SALL4 is overexpressed in patients with gliomas and is associated with adverse outcomes
.
SALL4 works by strengthening the PI3K/AKT signaling pathway, a well-known pathway that regulates tumorigenesis and is significantly activated in gliomas, thereby reducing the expression
of the tumor suppressor PTEN.

Figure 11.
Characterization of SALL4 in human and mouse tissues
.
Immunohistochemical staining using anti-SALL4 (AMAb91769) monoclonal antibody showed nuclear positivity in germ cells in human tissues in testicles (A), oocytes (B), and embryonic testicular cancer (C), as well as nuclear-positive mouse embryos E11(D)
in cells in a subset of the developing brain.

Figure 12.
SALL4 is more expressed in glioma samples
.
Multiplex IHC-IF staining
of human normal cortical and glioblastoma samples using anti-SALL4 (AMAb91769) monoclonal antibody (nuclear, red).
and anti-PTEN monoclonal antibody (AMAb91736) (cytoplasm, green
).
Nuclei are counterstained with DAPI (blue).

4.
EZH2/ALDH1A3/Shugoshin 2

The transcription factor EZH2 is highly expressed in gliomas and is a potential target for
immunotherapy.
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 has been linked to several immune checkpoints, cell cycles, DNA replication, mismatch repair, p53 signaling, and tumor-infiltrating lymphocytes
.

The aldehyde dehydrogenase ALDH1A3 is associated with cell adhesion and tumor invasion and is a marker
of the glioma Mes subtype.
ALDH is a marker of cancer stem cells associated with malignant phenotypes of gliomas
.
In the ALDH isoform, ALDH1A3 is overexpressed in high-grade gliomas than in low-grade gliomas, while ALDH1A1 is overexpressed
in low-grade gliomas than in high-grade gliomas.
Most patients with mes subtypes have high ALDH1A3 mRNA expression, suggesting that ALDH1A3 is a useful marker for glioma mes subtypes
.

Shugoshin 2 is essential
in cell division and cell cycle processes.
Shugoshin 2 (SGO2) plays a key role
in glioma cell proliferation.
The data showed that SGO2 expression was positively correlated
with who's grading of human gliomas.

Figure 13.
Multiple IHC-IF stained monoclonal antibodies
were performed on normal cortical samples of human glioblast granuloma cells, astroma-shaped, oligodendrocyte glioma using anti-EZH2 (AMAb91752) (green, nuclear), anti-PARP1 (AMAb90959) (red, nuclear), and anti-ALDH1A3 (AMAb91754) (blue, cytoplasm).

Figure 14 Multiplex IHC-IF stained (magenta, nuclear) polyclonal antibodies were performed on human normal cortical and glioblastoma samples using anti-EZH2 (AMAb91750) (green, nuclear) monoclonal and
anti-SGO2 (HPA03516).

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