WO2024039764A2 - Epitranscriptomic analysis of glioma - Google Patents

Epitranscriptomic analysis of glioma Download PDF

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WO2024039764A2
WO2024039764A2 PCT/US2023/030443 US2023030443W WO2024039764A2 WO 2024039764 A2 WO2024039764 A2 WO 2024039764A2 US 2023030443 W US2023030443 W US 2023030443W WO 2024039764 A2 WO2024039764 A2 WO 2024039764A2
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pathway
regulation
glioma
protein
altered
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WO2024039764A3 (en
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Jose OTERO
Wesley WANG
Pierre GIGLIO
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Ohio State Innovation Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present disclosure relates to the field of epitranscriptomic signatures to distinguish between glioma progression versus glioma pseudoprogression.
  • IDH-wildtype glioblastoma accumulates mutations affecting aspects of immune response, cancer cell proliferation and stem cell-like activity. Notably, such aspects are not unique to lesions between patients, but also during the subsequent evolution of primary GB lesions to recurrent lesions.
  • modulation of CD -8 + T-cell activity, tumor associated macrophages, and microglia has been noted in a subset of patients. Following gross total resection, patients are treated with chemoradiation and undergo a surveillance by serial radiological imaging and clinical neurological examination.
  • GB patients may show new areas of contrast enhancement in brain MR imaging that could indicate tumor recurrence (termed progressive disease (PD), and which is treated aggressively with new treatment plans and palliation if indicated) or treatment reaction (termed pseudo-progressive disease (psPD) and which is treated conservatively with steroids).
  • PD progressive disease
  • psPD pseudo-progressive disease
  • the present disclosure provides methods of distinguishing between the disease states such as progressive gliomas and pseudoprogressive glioma for the purpose of determining the appropriate treatment methods based on the identified disease state.
  • a method of identifying whether a subject has a progressive glioma or a pseudoprogressive glioma comprising isolating a tissue biopsy from the subject, detecting an altered N 6 -Methyladenosine (m 6 A) epigenetic modification on an mRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from at least one pathway selected from mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, regulation of cAMP-dependent protein kinase activity (PKA) pathway, epidermal growth factor receptor 3 (ERBB3) signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway,
  • PDA cAMP-dependent protein
  • a method of treating either a progressive glioma or a pseudoprogressive glioma in a subject comprising isolating a tissue biopsy from the subject, detecting an altered N°-Methyladenosine (m 6 A) epigenetic modification on an mRNA from the tissue biopsy, wherein the mRNA is encoded by a gene from at least one pathway selected from mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, regulation of cAMP- dependent protein kinase activity (PKA) pathway, epidermal growth factor receptor 3 (ERBB3) signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway, cardiac vent
  • the method detects the altered m 6 A epigenetic modification in 5 or more genes from the at least one pathway. In some embodiments, the method detects the altered m 6 A epigenetic modification in 10 or more genes from the at least one pathway. In some embodiments, the method detects the altered m 6 A epigenetic modification in 50 or more genes from the at least one pathway. In some embodiments, the method detects the altered m 6 A epigenetic modification in 100 or more genes from the at least one pathway. In some embodiments, the method detects the altered m 6 A epigenetic modification in 250 or more genes from the at least one pathway. In some embodiments, the method detects the altered m 6 A epigenetic modification in 500 or more genes from the at least one pathway.
  • the mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, or regulation of cAMP-dependent PKA pathway comprises at least one gene selected from ABCD4, AHSA1, APBA2, ATP6AP1, ATXN7L3B, AUNIP, BACH1, BICD1, CABLES!, CALR.
  • the ERBB3 signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway, cardiac ventricle morphogenesis pathway, regulation of receptor recycling pathway, protein mannosylation pathway, or protein O-linked mannosylation pathway comprises at least one gene selected from ACTN4, AP2A2, ARHGEF25, ATP10A, BTN3A1, BUB3, C14ORF93, C2ORF42, CAPZA1, CASP8, CDCA2, COL6A1, CPSF7, DDR1, DOK1, DPM3, DSP, DUSP8, ECE1, EIF3G, ETV3, FAM83G, FBL, FUK, FTSJ3, GATA2, HIP1, INAFM2, JMJD4, JTB, KHNYN, KIF3B, KLHDC10, KNOP1, LARP4, LRP12, MAP3K10, MEGF6, MPHOSPHIO, MRTFB, NAD
  • the presynaptic active zone membrane pathway, presynaptic active zone pathway, regulation of cell population proliferation pathway, regulation of mitochondrial translation pathway, regulation of BMP signaling pathway, histone kinase activity pathway, oogenesis pathway, nuclear receptor activity pathway, ligand-activated transcription factor activity pathway, or regulation of T cell cytokine production pathway comprises at least one gene selected from ABHD5, ACBD3, ACSL3, ACVR1B, ADI1, ADIPOR2, AFG3L2, AHSA2P, AK2, AKIRIN1, AKT1P, ALDH1B1, AMIGO2, ANKRD50, ANKRD54, APOL6, APPL2, AR, ARHGAP26, ATP5F1 A, ATP5IF1, AVL9, AZI2, B4GALT2, BCL2, BICRAL, BIRC3, BRWD1, BTBD2, BTG1, BTN2A1, C16ORF72, C3ORF18, C3ORF38, C5ORF15
  • the translational initiation pathway, structural constituent of ribosome pathway, protein folding pathway, ribosome pathway, SRP-dependent cotranslational protein targeting to membrane pathway, cytoplasmic translation pathway, large ribosomal subunit pathway, protein folding chaperone pathway, or protein targeting pathway comprises at least one gene selected from ALMS1, ANTXR2, AUTS2, B3GNT10, KIAA1 I09, BOD1LI, Cl ORF 174, C5ORF24, C8ORF58, CRYBG1, CUL1, DIAPHl, EIF3H, EIF4E2, ELM01, ETS1, FAM156B, FNDC3A, GGA2, HERC1, HMGN4, HSDI7B12, HSP90AA1, HSPA1L, HSPA8, KIDINS220, LEMD2, LURAP1L, MAML2, MAPK4, MARCH7, MARK3, MC AT, MFSD6, MPH0SPH8, MRPL12, MRPL41, M TMR
  • the gene comprises the altered m 6 A epigenetic modification.
  • the progressive glioma comprises an increased alteration of m 6 A epigenetic modifications relative to the pseudoprogressive glioma. In some embodiments, the progressive glioma comprises a decreased alteration of m 6 A epigenetic modifications relative to the pseudoprogressive glioma. In some embodiments, the progressive glioma comprises a highgrade glioma.
  • the high-grade glioma compri ses a glioblastoma.
  • the chemotherapy is selected from an alkylating agent, an antimetabolite, an anthracycline, a topoisomerase inhibitor, or a corticosteroid.
  • the immunotherapy comprises a PD1 antibody therapy, a PD-L1 antibody therapy, or combinations thereof.
  • the pseudoprogressive glioma comprises an inflammatory' response to a cancer treatment given after the tissue biopsy.
  • the inflammatory' response is treated with a steroid.
  • the steroid is selected from prednisolone, methylprednisolone, dexamethasone, hydrocortisone, or combinations thereof.
  • the tissue biopsy comprises a brain tissue. In some embodiments, the subject comprises a human.
  • FIG. 1 shows the general workflow for the biochemical extractions.
  • FIGS. 2A and 2B show hierarchical clustering analyses.
  • FIG. 2A shows the heatmap of m6A modifications using hierarchical clustering.
  • FIG. 2B shows the heat map of gene expression with differentially methylated probes.
  • FIGS. 3A and 3B show hierarchical clustering analyses.
  • FIG. 3 A shows the heatmap of m6A modifications using hierarchical clustering.
  • FIG. 3B shows the heat map of gene expression with differentially methylated probes.
  • FIG. 4 shows the dendrogram of the cases clustered in the weighted gene correlation network analyses (WCGNA), demonstrating that the cases cluster along the diagnoses of progressive disease and pseudoprogressive disease.
  • WGNA weighted gene correlation network analyses
  • FIGS. 5A and 5B shows the WGCNA dendrogram of the gene expression modules.
  • FIG. 5B shows the correlation of these modules to the clinical phenotypes of progressive disease and pseudoprogressive disease.
  • the high correlation (red) shows positive correlation with progressive disease, whereas the blue show's negative correlation with progressive disease.
  • an agent includes a plurality of agents, including mixtures thereof.
  • the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur.
  • the statement that a formulation "may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
  • administering refers to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir.
  • parenteral includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.
  • detect or “detecting” refers to an output signal released for the purpose of sensing of physical phenomenon. An event or change in environment is sensed and signal output released in the form of light.
  • a “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease, or any amount above, below 7 , or imbetw ? een those values, so long as the decrease is statistically significant.
  • An “increase” can refer to any change that results in larger amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to increase the genetic output of a gene when the genetic output of the gene product with the substance is more relative to the output of the gene product without the substance.
  • an increase can be a change in the symptoms of a disorder such that the symptoms are more than previously observed.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount.
  • the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase, or any amount above, below, or i n -between those values, so long as the increase is statistically significant.
  • prevent refers to a method of partially or completely delaying or precluding the onset or recurrence of a disorder or conditions and/or one or more of its attendant symptoms or barring a subject from acquiring or reacquiring a disorder or condition or reducing a subject’s risk of acquiring or reacquiring a disorder or condition or one or more of its attendant symptoms.
  • reduce or other forms of the word, such as “reducing” or “reduction,” means lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary' for the standard or relative value to be referred to.
  • reduced bacterial growth means reducing the rate of growth of a bacterium relative to a standard or a control.
  • cancer is used to address any neoplastic disease and is not limited to epithelial neoplasms (surface and glandular cancers; such a squamous cancers or adenomas)). It is used here to describe both solid tumors and hematologic malignancies, including epithelial (surface and glandular) cancers, glial tumors, soft tissue, and bone sarcomas, angiomas, mesothelioma, melanoma, lymphomas, leukemias and myeloma.
  • chemical compound and “compound,” refers to a chemical substance consisting of two or more different types of atoms or chemical elements in a fixed stoichiometric proportion. These compounds have a unique and defined chemical structure held together in a defined spatial arrangement by chemical bonds. Chemical compounds can be held together by covalent bonds, ionic bonds, metallic ions, or coordinate covalent bonds.
  • composition refers to any agent that has a beneficial biological effect.
  • beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition.
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • composition includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
  • a “control” is an alternative subject or sample used in an experiment for comparison purpose.
  • a control can be "positive” or “negative.”
  • the purpose of the experiment is to determine a correlation of an altered expression level of a proto-oncogene with a particular type of cancer, it is generally preferable to use a positive control (a subject or a sample from a subject, carrying such alteration and exhibiting syndromes characteristic of that disease), and a negative control (a subject or a sample from a subject lacking the altered expression and clinical syndrome of that disease).
  • a “subject” means an individual.
  • the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, chickens, ducks, geese, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds.
  • “Subject” can also include a mammal, such as a primate or a human.
  • the subject can be a human or veterinary patient.
  • database denotes a set of stored data that represent a collection of sequences, which in turn represent a collection of biological reference materials.
  • a “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotides sequences described herein may be used to identify larger fragments or full- length coding sequences of the gene with which they are associated.
  • a "probe” when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target.
  • a probe wall comprise a label or a means by which a label can be attached, either before or subsequent to the hybridization reaction.
  • Suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.
  • treat include partially or completely delaying, alleviating, mitigating, or reducing the intensity of one or more attendant symptoms of a disorder or condition and/or alleviating, mitigating, or impeding one or more causes of a disorder or condition.
  • Treatments according to the invention may be applied preventively, prophylactic-ally, palliatively, or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g., upon initial signs and symptoms of cancer), or after an established development of cancer. Prophylactic administration cart occur for several days to years prior to the manifestation of symptoms of an infection.
  • monitoring refers to the actions of observing and checking the progress or quality of a treatment or procedure over a period of time. “Monitoring” also refers to observing the course of a disease or condition, such as a cancer, over a period of time.
  • a “fluorophore” is a fluorescent chemical compound that can re-emit light upon light excitation.
  • the chemicals are sometimes used alone as a tracer in fluids, as a due for staining certain structures, as an enzyme substrate, or as a probe/indicator. More commonly they are covalently bonded to a macromolecule to serve as a marker for bioactive reagents (i.e.: antibodies, peptides, nucleic acids, etc.)
  • Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods such as fluorescent imaging and spectroscopy.
  • epigenetic modification refers to the heritable genetic changes the affect gene expression activity without altering the DNA or RNA sequence. These genetic changes include but are not limited to DNA or RNA methylation and histone modifications (i.e.: methylation and/or acetylation) that alter DNA or RNA accessibility and structure, thereby regulating gene expression patterns.
  • An m 6 A methylation modification is a methylation modification occurring on the sixth nitrogen on an adenosine nucleotide, also known as a chemical derivative of adenosine. This modification can occur on mRNA and DNA molecules in most eukaryotes regulating gene expression, alternative DNA splicing events, alternative polyadenylation, nuclear export, protein translation, mRNA degradation, and mRNA stabilization.
  • a “nucleotide” is a compound consisting of a nucleoside, which consists of a nitrogenous base and a 5-carbon sugar, linked to a phosphate group forming the basic structural unit of nucleic acids, such as DNA or RNA.
  • the four types of nucleotides are adenine (A), cytosine (C), guanine (G), and thymine (T), each of which are bound together by a phosphodiester bond to form a nucleic acid molecule.
  • a nucleotide can comprise an epigenetic modification, such as methylation, acetylation, sumolaytion, or any variant thereof.
  • nucleic acid is a chemical compound that serves as the primary information-carrying molecules in cells and make up the cellular genetic material. Nucleic acids are comprised nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). Examples of RNA include but are not limited to messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and heterogenous nuclear RNA (hnRNA). “genome” refers to a complete set of genes or genetic material present within a cell, tissue, or organism, A genome can be nuclear (found within the cell nucleus) or mitochondrial (found with the cell mitochondria).
  • mRNA refers to messenger ribonucleic acid, or single stranded molecule of RNA that corresponds to the genetic sequence of a gene and is translated by a ribosome in the process of synthesizing a protein.
  • mRNA is created during the process of transcription, where a gene is converted into a primary transcript mRNA (or pre-mRNA). The primary' transcript is further processed through RNA splicing to only contain regions that will encode protein. mRNA can also be targeted for epigenetic modifications, such as methylation, to impact mRNA translation, nuclear retention, nuclear export, processing, and splicing.
  • a “site” refers to a specific nucleotide or group of nucleotides along the 5’ to 3’ or 3’ to 5’ sequence of a nucleic acid molecule. These “sites” can be targeted for chemical modifications, such as methylation, acetylation, sumolaytion, or any mutation and variation thereof.
  • a “endoribonuclease” refers to an endonuclease enzyme that specifically degrades or cleaves ribonucleic acid molecules into smaller segments.
  • the cleavage point on the RNA molecule usually occurs within the sequence rather than at the end of the sequence.
  • methylation refers to the chemical modification to a molecule by adding a methyl group on a DNA, RNA, or protein molecule. This modification is usually performed by enzymes to regulate gene expression, protein function, and RNA processing.
  • An example of methylation includes but is not limited to CpG methylation occurring on DNA and m 6 A methylation occurring on mRNA.
  • the terms “cell,” “cell line” and “cell culture” include progeny. It is also understood that all progenies may not. be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that has the same function or biological property, as screened for in the originally transformed cell, are included.
  • the "host cells” used in the present invention generally are prokaryotic or eukaryotic hosts.
  • a “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotides sequences described herein may be used to identify larger fragments or full- length coding sequences of the gene with which they are associated.
  • an “oncogene” refers to a polynucleotide containing at least one open reading frame that is capable of transforming a normal cell to a cancerous (or neoplastic or tumor) cell when introduced into a host cell. Oncogenes are often altered forms of the cellular counterpart, namely the "proto-oncogenes” that are incapable of cell transformation when expressed at the level present in a non-cancer cell.
  • module refers to a subset of genes identified to comprise m6A epigenetic modifications. Each module of genes identified represent specific group or groups of mechanistic pathways and processes performed within a cell, tissue, or organ.
  • a “progressive disease” refers to a disease or physical ailment whose course in most cases is the worsening, growth, or spread of disease. This worsening will continue until serious debility, organ failure, or death occurs.
  • Some progressive diseases can be halted and/or reversed by treatment (surgical, dietary, or lifestyle interventions).
  • Some examples of progressive diseases include, but are not limited to various cancers, Huntington’s disease, Alzheimer’s disease, multiple sclerosis, and Parkinson’s disease.
  • progressive disease commonly refers to a phenomenon in which an initial increase in tumor size is observed or new lesions appear, followed by a decrease in tumor burden. This phenomenon can benefit patients receiving immunotherapy but often leads to premature discontinuation of treatment owing to the false judgement of progression.
  • a “glioma” refers to a primary brain or spinal cord tumors affecting the glial cells that surround nerve endings in the brain and spinal cord. Glial cells function as a support to neurons, however dysregulated cellular growth can lead to glioma formation. Gliomas are categorized by grade wherein slower growing gliomas are referred to as “low grade” or Grade 1 or 2. Fast growing or aggressive gliomas are referred to as “high grade” or Grade 3 or 4. An example of a high-grade glioma is a glioblastoma, typically categorized as a Grade 4.
  • epigenetic refers to a field of study where the genes of an organism is changed or altered due to complex interactions between the genome and the environment that are involved in development, differentiation, ageing, and some diseases. These gene changes occur when specific nucleotides sites are chemically modified by adding or removing one or more methyl, acety l, or sumo groups. The processes of chemically modifying nucleotides are also referred to as methylation, acetylation, and sumolaytion.
  • Messenger RNA mRNA
  • m6A N6-methyladenine epigenetic modifications are the most abundant form of RNA modification in cellular biological processes. It has been found that the effect of m6A modification of RNA involves processing, nuclear export, translation, and even cell death. m6A modifications can also have a key role in the development of gliomas, or cancer of the glial cells.
  • the methods disclosed herein provides valuable insight into determining whether a patient’s future contrast enhancing lesions represents progressive disease or pseudoprogressive disease.
  • a method of determining, identifying, and/or diagnosing whether a subject has a progressive glioma or a pseudoprogressive glioma comprising collecting a tissue biopsy form the subject, detecting an altered N 6 -Methladenosine (m 6 A) epigenetic modification on an mRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from Table 1, Table 2, Table 3, and/or Table 4, and diagnosing the subject with either the progressive glioma or the pseudoprogressive glioma.
  • m 6 A N 6 -Methladenosine
  • Gliomas are the most common form of malignant tumor in the central nervous system.
  • glioblastomas an aggressive high-grade glioma, comprise the highest malignancy rate and account for almost half of all brain tumors. Given the aggressiveness of these tumors, surgical removal does not always promise complete remission.
  • treatments, such as chemotherapy is commonly followed by showing new areas of contrast enhancement in brain MR imaging that could indicate tumor recurrence (termed progressive disease (PD)) requiring further treatment, or a treatment reaction (termed pseudo-progressive disease (psPD)).
  • PD tumor recurrence
  • psPD pseudo-progressive disease
  • a method of identifying whether a subject has a progressive glioma or a pseudoprogressive glioma comprising isolating a tissue biopsy from the subject, detecting an altered N 6 -Mothyladenosine (m 6 A) epigenetic modification on an mRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from at least one pathway selected from mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, regulation of cAMP-dependent protein kinase activity (PKA) pathway, epidermal growth factor receptor 3 (ERBB3) signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway
  • PDA cAMP-dependent protein
  • the method comprises a method of identifying whether a subject has a progressive glioma or a pseudoprogressive glioma, the method comprising isolating a tissue biopsy from the subject, detecting an altered N 6 -Methyladenosine (m 6 A) epigenetic modification on an mRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from at least one pathway selected from Table 1 , Table 2, Table 3, and/or Table 4 and diagnosing the subject with either the progressive glioma or the pseudoprogressive glioma.
  • m 6 A N 6 -Methyladenosine
  • a method of treating either a progressive glioma or a pseudoprogressive glioma in a subject comprising isolating a tissue biopsy from the subject, detecting an altered N°-Methyladenosine (m 6 A) epigenetic modification on an mRNA from the tissue biopsy, wherein the mRNA is encoded by a gene from at least one pathway selected from mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, regulation of cAMP- dependent protein kinase activity (PKA) pathway, epidermal growth factor receptor 3 (ERBB3) signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway, cardiac vent
  • a method of identifying, diagnosing, treating, preventing, ameliorating, and/or decreasing either a progressive glioma or a pseudoprogressive glioma in a subject comprising isolating a tissue biopsy from the subject, detecting an altered N b -Methyladenosine (m 6 A) epigenetic modification on an mRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, or more pathways.
  • m 6 A N b -Methyladenosine
  • a method of treating either a progressive glioma or a pseudoprogressive glioma in a subject comprising isolating a tissue biopsy from the subject, detecting an altered N 6 -Methyl adenosine (m 6 A) epigenetic modification on an rnRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from at least one pathway selected from Table 1, Table 2, Table 3, and/or Table 4, diagnosing the subject with either the progressive glioma or the pseudoprogressive glioma, and treating the subject with a chemotherapy, an immunotherapy, or a palliative therapy if diagnosed with the progressive glioma or treating with a steroid if diagnosed with the pseudoprogressive glioma.
  • m 6 A N 6 -Methyl adenosine
  • any of the genes of Table 1 comprise the altered m 6 A epigenetic modification.
  • any of the genes of Table 2 comprise the altered m 6 A epigenetic modification.
  • any of the genes of Table 3 comprise the altered m 6 A epigenetic modification.
  • any of the genes of Table 4 comprise the altered m 6 A epigenetic modification.
  • the method detects the altered m 6 A epigenetic modification in 5 or more genes from the at least one pathway. In some embodiments, the method detects the altered m 6 A epigenetic modification in 10 or more genes from the at least one pathway. In some embodiments, the method detects the altered m 6 A epigenetic modification in 50 or more genes from the at least one pathway. In some embodiments, the method detects the altered m 6 A epigenetic modification in 100 or more genes from the at least one pathway. In some embodiments, the method detects the altered m 6 A epigenetic modification in 250 or more genes from the at least one pathway. In some embodiments, the method detects the altered m 6 A epigenetic modification in 500 or more genes from the at least one pathway.
  • the method detects the altered m 6 A epigenetic modification in 5 or more genes from Table 1. In some embodiments, the method detects the altered m 6 A epigenetic modification in 10 or more genes from Table 1. In some embodiments, the method detects the altered m 6 A epigenetic modification in 50 or more genes from Table 1. In some embodiments, the method detects the altered m 6 A epigenetic modification in 100 or more genes from Table 1. In some embodiments, the method detects the altered m 6 A epigenetic modification in 250 or more genes from Table 1. In some embodiments, the method detects the altered m 6 A epigenetic modification in 500 or more genes from Table 1.
  • the method detects the altered m 6 A epigenetic modification in 5 or more genes from Table 2. In some embodiments, the method detects the altered m 6 A epigenetic modification in 10 or more genes from Table 2. In some embodiments, the method detects the altered m 6 A epigenetic modification in 50 or more genes from Table 2. In some embodiments, the method detects the altered m 6 A epigenetic modification in 100 or more genes from Table 2. In some embodiments, the method detects the altered m 6 A epigenetic modification in 250 or more genes from Table 2. In some embodiments, the method detects the altered m 6 A epigenetic modification in 500 or more genes from Table 2.
  • the method detects the altered m 6 A epigenetic modification in 5 or more genes from Table 3. In some embodiments, the method detects the altered m 6 A epigenetic modification in 10 or more genes from Table 3. In some embodiments, the method detects the altered m 6 A epigenetic modification in 50 or more genes from Table 3. In some embodiments, the method detects the altered m 6 A epigenetic modification in 100 or more genes from Table 3. In some embodiments, the method detects the altered m 6 A epigenetic modification in 250 or more genes from Table 3. In some embodiments, the method detects the altered m 6 A epigenetic modification in 500 or more genes from Table 3.
  • the method detects the altered m 6 A epigenetic modification in 5 or more genes from Table 4. In some embodiments, the method detects the altered m 6 A epigenetic modification in 10 or more genes from Table 4. In some embodiments, the method detects the altered m 6 A epigenetic modification in 50 or more genes from Table 4. In some embodiments, the method detects the altered m 6 A epigenetic modification in 100 or more genes from Table 4. In some embodiments, the method detects the altered m 6 A epigenetic modification in 250 or more genes from Table 4. In some embodiments, the method detects the altered m 6 A epigenetic modification in 500 or more genes from Table 4. In some embodiments, the method detects the altered m 6 A epigenetic modification in a gene sequence.
  • the method detects the altered m 6 A epigenetic modification in one or more of 1997 gene sequences. In some embodiments, the method detects the altered m 6 A epigenetic modification in two or more of 1997 gene sequences. In some embodiments, the method detects the altered m 6 A epigenetic modification in five or more of 1997 gene sequences. In some embodiments, the method detects the altered m 6 A epigenetic modification in ten or more of 1997 gene sequences. In some embodiments, the method detects the altered m 6 A epigenetic modification in 50 or more of 1997 gene sequences. In some embodiments, the method detects the altered m 6 A epigenetic modification in 100 or more of 1997 gene sequences.
  • the method detects the altered m 6 A epigenetic modification in 250 or more of 1997 gene sequences. In some embodiments, the method detects the altered m 6 A epigenetic modification in 500 or more of 1997 gene sequences. In some embodiments, the method detects the altered m 6 A epigenetic modification in 750 or more of 1997 gene sequences. In some embodiments, the method detects the altered m 6 A epigenetic modification in 1000 or more of 1997 gene sequences. In some embodiments, the method detects the altered m 6 A epigenetic modification in 1500 or more of 1997 gene sequences.
  • the method detects the altered m 6 A epigenetic modification in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
  • the altered m 6 A epigenetic modification is either increased or decreased in the progressive glioma compared to the pseudoprogressive glioma. Definitions of “increase” and “decrease” are found above.
  • the mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, or regulation of cAMP-dependent PKA pathway comprises at least one gene selected from ABCD4, AHSA1, APBA2, ATP6AP1, ATXN7L3B, AUNIP, BACH L BICD1, CABLES!, CALR.
  • the ERBB3 signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway, cardiac ventricle morphogenesis pathway, regulation of receptor recycling pathway, protein mannosylation pathway, or protein O-linked mannosylation pathway comprises at least one gene selected from ACTN4, AP2A2, ARHGEF25, ATP10A, BTN3A1, BUB3, C14ORF93, C2ORF42, CAPZA1, CASP8, CDCA2, COL6A1, CPSF7, DDR1, DOK1, DPM3, DSP, DUSP8, ECE1, EIF3G, ETV3, FAM83G, FBL, FUK, FTSJ3, GATA2, HIP1, INAFM2, JMJD4, JTB, KHNYN, KIF3B, KLHDC10, KNOP1, LARP4, LRP12, MAP3K10, MEGF6, MPHOSPHIO, MRTFB, NAD
  • the presynaptic active zone membrane pathway, presynaptic active zone pathway, regulation of cell population proliferation pathway, regulation of mitochondrial translation pathway, regulation of BMP signaling pathway, histone kinase activity pathway, oogenesis pathway, nuclear receptor activity pathway, ligand-activated transcription factor activity pathway, or regulation of T cell cytokine production pathway comprises at least one gene selected from ABHD5, ACBD3, ACSL3, ACVR1B, ADI1, ADIPOR2, AFG3L2, AHSA2P, AK2, AKIRIN1, AKTIP, ALDH1B1, AMIG02, ANKRD50, ANKRD54, APOL6, APPL2, AR, ARHGAP26, ATP5F1A, ATP5IF1, AVIN, AZI2, B4GALT2, BCL2, BICRAL, BIRC3, BRWD1, BTBD2, BTG1, BTN2A1, C16ORF72, C3ORF18, C3ORF38, C5ORF15, C
  • the translational initiation pathway, structural constituent of ribosome pathway, protein folding pathway, ribosome pathway, SRP-dependent cotranslational protein targeting to membrane pathway, cytoplasmic translation pathway, large ribosomal subunit pathway, protein folding chaperone pathway, or protein targeting pathway comprises at least one gene selected from ALMS1, ANTXR2, AUTS2, B3GNT10, KIAA1109, BOD1L1, C1ORF174, C5ORF24, C8ORF58, CRYBG1, Cl.
  • genes of any preceding aspects or any preceding embodiments can be grouped into a group of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
  • the m 6 A epigenetic modification occurs at any group of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 genes. In some embodiments, the m 6 A epigenetic modification occurs at any group of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or 1900 genes. In some embodiments, the specific m 6 A epigenetic modification occurs on any one of 2590 nucleotide sites. In some embodiments, the specific m 6 A epigenetic modification occurs at any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
  • the m 6 A epigenetic modification occurs at any group of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 sites.
  • the gene comprises the altered m 6 A epigenetic modification.
  • the progressive glioma comprises an increased alteration of m 6 A epigenetic modifications relative to the pseudoprogressive glioma. In some embodiments, the progressive glioma comprises a decreased alteration of m 6 A epigenetic modifications relative to the pseudoprogressive glioma.
  • the pseudoprogressive glioma comprises an inflammatory response to a cancer treatment given after the tissue biopsy.
  • the inflammatory response is treated with a steroid.
  • the steroid is selected from prednisolone, methylprednisolone, dexamethasone, hydrocortisone, or combinations thereof.
  • a glioma is a type of tumor that occurs in the brain and spinal cord that begin in the glial cells that surround nerve cells.
  • a progressive glioma is a tumor that occurs in the brain wherein the tumor growth worsens over time, thus resulting in decline of general overall health.
  • Gliomas are classified into four grades (I, II, III, or IV), wherein the treatment and prognosis depend upon the glioma grade.
  • grade IV gliomas are also referred to as high-grade gliomas.
  • the progressive glioma comprises a high-grade glioma.
  • the high-grade glioma is a glioblastoma.
  • a glioblastoma refers to an aggressive, fast growing type of glioma that occurs in the brain and spinal cord.
  • chemotherapy and/or immunotherapy is given as a treatment for progressive glioma.
  • Such chemotherapy can be selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, topoisomerase inhibitors, or corticosteroids.
  • the immunotherapy is either a PD1 antibody therapy or a PD-L1 antibody therapy.
  • standard first-line chemotherapy can consist of temozolomide (75 mg/m 2 daily) during radiotherapy followed by a further six cycles of temozolomide (150-200 mg/m 2 on days 1- 5 every 28 days).
  • Lomustine, carmustine, and rechallenge with temozolomide are all potential options for treatment of progressive glioma as well. Further options include further surgical resection, reirradiation, systemic therapies such as lomustine or bevacizumab, combined approaches, or supportive care alone. Other options include radiotherapy, surgery, or palliative care.
  • single-agent bevacizumab can be used for the treatment of progressive glioma.
  • Bevacizumab in combination with lomustine improves PFS compared with lomustine alone.
  • the combination of bevacizumab with chemotherapy is recommended after failure on bevacizumab alone, which is different than the treatment for pseudoprogressive glioma.
  • those with progressive glioma should not be treated the same as those with pseudoprogressive glioma, particularly concerning bevacizumab. This is due to its effect on inflammatory and radio chemotherapy-induced changes in GBM vasculature.
  • Brandsma discloses that in patients managed with temozolomide chemoradiotherapy who have clinically asymptomatic progressive lesions at the end of treatment, adjuvant temozolomide should be continued; in clinically symptomatic patients, surgery' should be considered. If mainly necrosis is noted during surgery, continuation of adjuvant temozolomide is logical. Trials on the treatment of recurrent malignant glioma should exclude patients with progression within the first 3 months after temozolomide chemoradiotherapy unless histological confirmation of tumor recurrence is available.
  • a pseudoprogressive disease refers to an occurrence in which an initial increase in tumor size is observed or new lesions appear, followed by a decrease in tumor burden following the subject receiving a cancer therapy.
  • a pseudoprogressive glioma refers to a transient imaging pattern that mimics tumor progression but not presenting with the accompanied decline of health. The pseudo-progression of brain tumors is attributed to damage from a cancer treatment leading to local inflammation and edema.
  • the pseudoprogressive glioma is an inflammatory response to a cancer treatment given after the tissue biopsy. In other embodiments, the inflammatory response is treated with a steroid.
  • the steroid is selected from the group consisting of prednisolone, methylprednisolone, dexamethasone, hydrocortisone, or any combination thereof.
  • Further treatments for pseudoprogression include, but are not limited to, Apatinib and bevacizumab (at different doses than those given for progressive glioma). (Wang et al., Apatinib treatment for symptomatic pseudoprogression after standard treatment for glioblastoma multiforme: a case report, APM Vol. 8, No, 5, Nov. 2019, herein incorporated by reference for its teaching concerning treating pseudo progression).
  • pseudoprogression The etiology of pseudoprogression is thought to be due to vascular and oligodendroglia cell injury, leading to inflammation and increased BBB permeability. Because the enhancement seen in pseudoprogression can be mistaken for actual tumor progression, patients are often routed to bevacizumab as second-line therapy for recurrence. Bevacizumab has been observed to decrease the permeability of not only tumor-related leaky vasculature but also of radiation-induced leaky vasculature, thereby “curing” biopsy-proven pseudoprogression (Weinstein JS, Varallyay CG, Dosa E, et al.
  • Bevacizumab may decrease the correlation between MGMT status and pseudoprogression (Thompson EM, Frenkel EP, Neuwelt EA. The paradoxical effect of bevacizumab in the therapy of malignant gliomas. Neurology. 2011 Jan 4;76(1):87-93.)
  • a range of chemotherapy partners including lomustine, carmustine, and temozolomide, can also be used in tandem with chemotherapy.
  • Immunotherapy is also an option to be used alone or in combination with a chemotherapy and/or a palliative therapy.
  • Immune checkpoint inhibitors predominantly targeting PD-1/PD-L1 and/or CTLA-4, can be used.
  • Chimeric antigen receptor (CAR) T-cell therapy with genetically modified T cells can also be used. Examples of treatment types can be found in Tan et al. (Tan, AC, Ashley, DM, Lopez, GY, Malinzak, M, Friedman, HS, Khasraw, M. Management of glioblastoma: State of the art and future directions. CA Cancer J Clin. 2020: 70: 299- 312), herein incorporated by reference in its entirety for its teaching concerning treatment of glioma.
  • the tissue biopsy is a brain tissue. In other embodiments, the tissue biopsy is a spinal cord tissue. In one embodiment, the subject is a human.
  • Example 1 Rationale for epitranscriptomic analysis in recurrent glioma
  • RNA modification on mRNA by methylation of adenosine on position 6 shows high prevalence, with classical studies indicating that on average each mRNA transcript containing three m6A modifications with some transcripts containing over twenty m6A sites which, when located in clusters of less than 100 nucleotides, are deemed “clustered m6A” sites.
  • m6A modification of RNA Single and clustered m6A sites promote noncanonical cap-independent translation, modulate translation-elongation dynamics, promote IncRNA decay in dendritic cell migration, and promote tumor cell proliferation.
  • m6A modification and the machinery that promotes the “writing” and “reading” of this RNA modification have been implicated in the initiation, progression, maintenance, and drug resistance of various types of disease.
  • Many cancers immune-evasion processes have been shown to be modified by ni6A RNA modification.
  • m6A RNA modifications are mapped out in primary glioblastoma, so as to predict from the first, biopsy which patients would ultimately undergo pseudoprogression and which would undergo progression.
  • Gene expression module 1 listing genes involved in the following pathways: mesangial cell development, glomerular mesangial cell development, positive regulation of phagocytosis, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation, cell cycle, regulation of platelet activation, regulation of cohesion loading, pericyte cell differentiation, and regulation of c AMP-dependent protein kinase activity.
  • Gene expression module 2 listing genes involved in the following pathways: ER.BB3 signaling pathway, receptor metabolic process, positive regulation of ERBB signaling pathway, regulation of secretion, regulation of exocytosis, cardiac chamber development, cardiac ventricle morphogenesis, positive regulation of receptor recycling, protein mannosylation, and protein O- linked mannosylation.
  • Gene expression module 3 listing genes involved in the following pathways: presynaptic active zone membrane, presynaptic active zone, negative regulation of cell population proliferation, positive regulation of mitochondrial translation, regulation of BMP signaling pathway, histone kinase activity, oogenesis, nuclear receptor activity, ligand-activated transcription factor activity, and regulation of T cell cytokine production.
  • Gene expression module 1 listing genes involved in the following pathways: translational initiation, structural constituent of ribosome, protein folding, ribosome, SRP-dependent cotranslational protein targeting to membrane, cytoplasmic translation, large ribosomal subunit, protein folding chaperone, protein targeting, cotranslational protein targeting to membrane.

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Abstract

The present disclosure relates to the field of epitranscriptomic signatures to determine glioma progression versus glioma pseudoprogression.

Description

EPITRANSCRIPTOMIC ANALYSIS OF GLIOMA
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63/398,582, filed August 17, 2022, entitled “EPITRANSCRIPTOMIC ANALYSIS OF GLIOMA,” which is incorporated by reference herein in its entirety.
FIELD
The present disclosure relates to the field of epitranscriptomic signatures to distinguish between glioma progression versus glioma pseudoprogression.
BACKGROUND
IDH-wildtype glioblastoma (GB) accumulates mutations affecting aspects of immune response, cancer cell proliferation and stem cell-like activity. Notably, such aspects are not unique to lesions between patients, but also during the subsequent evolution of primary GB lesions to recurrent lesions. In particular modulation of CD -8+ T-cell activity, tumor associated macrophages, and microglia has been noted in a subset of patients. Following gross total resection, patients are treated with chemoradiation and undergo a surveillance by serial radiological imaging and clinical neurological examination. GB patients may show new areas of contrast enhancement in brain MR imaging that could indicate tumor recurrence (termed progressive disease (PD), and which is treated aggressively with new treatment plans and palliation if indicated) or treatment reaction (termed pseudo-progressive disease (psPD) and which is treated conservatively with steroids). Some patients undergo surgical biopsy to confirm PD histopathologically, where the neuropathological spectrum ranges from purely reactive/inflammatory architecture to aggressively dividing neoplastic cells. Complicating the picture is that most cases contain a mixture of both reactive and recurrent processes noted on histology. In many situations, unequivocally attributing the radiological changes to tumor recurrence versus inflammatory processes is impossible, and no clear clinical practice guidelines are available to standardize diagnostic workflow's.
Given the difficulty of differentiating between tumor recurrence or an inflammatory/ response, there is need to address the aforementioned problems mentioned above by developing methods to clearly distinguish between the two outcomes.
The methods disclosed herein address these and other needs. SUMMARY
The present disclosure provides methods of distinguishing between the disease states such as progressive gliomas and pseudoprogressive glioma for the purpose of determining the appropriate treatment methods based on the identified disease state.
In one aspect, disclosed herein is a method of identifying whether a subject has a progressive glioma or a pseudoprogressive glioma, the method comprising isolating a tissue biopsy from the subject, detecting an altered N6-Methyladenosine (m6A) epigenetic modification on an mRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from at least one pathway selected from mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, regulation of cAMP-dependent protein kinase activity (PKA) pathway, epidermal growth factor receptor 3 (ERBB3) signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway, cardiac ventricle morphogenesis pathway, regulation of receptor recycling pathway, protein mannosylation pathway, protein O-linked mannosylation pathway, presynaptic active zone membrane pathway, presynaptic active zone pathway, regulation of cell population proliferation pathway, regulation of mitochondrial translation pathway, regulation of bone morphogenetic protein (BMP) signaling pathway, histone kinase activity pathway, oogenesis pathway, nuclear receptor activity pathway, ligand-activated transcription factor activity pathway, regulation of T cell cytokine production pathway, translational initiation pathway, structural constituent of ribosome pathway, protein folding pathway, ribosome pathway, signal-recognition particle (SRP)-dependent cotranslational protein targeting to membrane pathway, cytoplasmic translation pathway, large ribosomal subunit pathway, protein folding chaperone pathway, or protein targeting pathway and diagnosing the subject with either the progressive glioma or the pseudoprogressive glioma.
In some aspects, disclosed herein is a method of treating either a progressive glioma or a pseudoprogressive glioma in a subject, the method comprising isolating a tissue biopsy from the subject, detecting an altered N°-Methyladenosine (m6A) epigenetic modification on an mRNA from the tissue biopsy, wherein the mRNA is encoded by a gene from at least one pathway selected from mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, regulation of cAMP- dependent protein kinase activity (PKA) pathway, epidermal growth factor receptor 3 (ERBB3) signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway, cardiac ventricle morphogenesis pathway, regulation of receptor recycling pathway, protein mannosylation pathway, protein O-linked mannosylation pathway, presynaptic active zone membrane pathway, presynaptic active zone pathway, regulation of cell population proliferation pathway, regulation of mitochondrial translation pathway, regulation of bone morphogenetic protein (BMP) signaling pathway, histone kinase activity pathway, oogenesis pathway, nuclear receptor activity pathway, ligand-activated transcription factor activity pathway, regulation of T cell cytokine production pathway, translational initiation pathway, structural constituent of ribosome pathway, protein folding pathway, ribosome pathway, signal-recognition particle (SRP)- dependent cotranslational protein targeting to membrane pathway, cytoplasmic translation pathway, large ribosomal subunit pathway, protein folding chaperone pathway, or protein targeting pathway, diagnosing the subject with either the progressive glioma or the pseudoprogressive glioma, and treating the subject with a chemotherapy, an immunotherapy, or a palliative therapy if diagnosed with the progressive glioma or treating with a steroid if diagnosed with the pseudoprogressive glioma.
In some embodiments, the method detects the altered m6A epigenetic modification in 5 or more genes from the at least one pathway. In some embodiments, the method detects the altered m6A epigenetic modification in 10 or more genes from the at least one pathway. In some embodiments, the method detects the altered m6A epigenetic modification in 50 or more genes from the at least one pathway. In some embodiments, the method detects the altered m6A epigenetic modification in 100 or more genes from the at least one pathway. In some embodiments, the method detects the altered m6A epigenetic modification in 250 or more genes from the at least one pathway. In some embodiments, the method detects the altered m6A epigenetic modification in 500 or more genes from the at least one pathway.
In some embodiments, the mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, or regulation of cAMP-dependent PKA pathway comprises at least one gene selected from ABCD4, AHSA1, APBA2, ATP6AP1, ATXN7L3B, AUNIP, BACH1, BICD1, CABLES!, CALR. CCAR2, CDCA5, CDCA8, CDK4, CDR2, CLCN7, CNNM4, DNMBP, DOHH, DXO, EIF2S3, ELF1, EPAS1, EX0C7, FEM1A, FKBP1A, FOSB, FUNDCI, GCNT4, HMG20A, HR, HTT, C6ORF106, INTS11, JPT1 , KIAA0930, KXD1, LETM1, LOC100129034, LYAR, VIE.X3D, MIIP, VIKR X L MMADHC, MRPL54, MTRNR2L2, NLRXI, NOTCH!, NRBF2, PAK4, PAXIP1-AS1, PCDH17, PDGFB, PIGO, PPP2R1A, PRKARIA, PRKCQ, PTK7, PTPN18, PTPN7, RAB39B, RABL6, RAC1, RAPGEF1, RAVER 1, RBBP5, RND1, RNF14, RNF167, SEC23IP, SERTAD1, SIK2, SLC25A19, SLC43A2, SLC7A6, SNRNP25, SNTA1, SOCS3, SPIN4, STAGE SYNDIG1, SYNGR2, TBC1D9, TP53, TRIM65, TSC22D1, TTC28, TTYH3, TUBB6, TULP3, UBE2L3, UBE3B, WAPL, ZBED3, ZBTB48, ZSWIM1, LOCI 00507437, or combinations thereof.
In some embodiments, the ERBB3 signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway, cardiac ventricle morphogenesis pathway, regulation of receptor recycling pathway, protein mannosylation pathway, or protein O-linked mannosylation pathway comprises at least one gene selected from ACTN4, AP2A2, ARHGEF25, ATP10A, BTN3A1, BUB3, C14ORF93, C2ORF42, CAPZA1, CASP8, CDCA2, COL6A1, CPSF7, DDR1, DOK1, DPM3, DSP, DUSP8, ECE1, EIF3G, ETV3, FAM83G, FBL, FUK, FTSJ3, GATA2, HIP1, INAFM2, JMJD4, JTB, KHNYN, KIF3B, KLHDC10, KNOP1, LARP4, LRP12, MAP3K10, MEGF6, MPHOSPHIO, MRTFB, NADK, NCKAP5L, NCLN, NR2F2, NRG1, NSF, OGFOD3, OS9, PLEKHH3, PMVK, PNPO, POMGNT2, QSOX2, RAB15, RAB1A, RHOC, RIN3, RNF43, RTN4, SALL1 , SDF2, SOX4, SP140, SPTLC2, SRRM1, SYT17, TAB!, TBC1D22A, TBC1D8, TERF2, THRAP3, TIAL1, TOX, TPD52L2, TPRA1, TRABD, TRIP6, USP40, ZBTB26, ZBTB9, ZNF318, ZNF503, ZNF668, ZNF827, ZNF865, or combinations thereof.
In some embodiments, the presynaptic active zone membrane pathway, presynaptic active zone pathway, regulation of cell population proliferation pathway, regulation of mitochondrial translation pathway, regulation of BMP signaling pathway, histone kinase activity pathway, oogenesis pathway, nuclear receptor activity pathway, ligand-activated transcription factor activity pathway, or regulation of T cell cytokine production pathway comprises at least one gene selected from ABHD5, ACBD3, ACSL3, ACVR1B, ADI1, ADIPOR2, AFG3L2, AHSA2P, AK2, AKIRIN1, AKT1P, ALDH1B1, AMIGO2, ANKRD50, ANKRD54, APOL6, APPL2, AR, ARHGAP26, ATP5F1 A, ATP5IF1, AVL9, AZI2, B4GALT2, BCL2, BICRAL, BIRC3, BRWD1, BTBD2, BTG1, BTN2A1, C16ORF72, C3ORF18, C3ORF38, C5ORF15, C8ORF33, CACNB1, CCDC71L, CCNT2, CCT7, CDCA4, CDK2, CDKN1B, CDS2, CEACAM6, CEP78, CHST11, CHST12, CHTOP, CLN5, CLPB, CNOT6, CO A3, COLECI 2, COQ5, CRIM1, CTCF, CTR9, CWF19L1, DARS2, DCAF1, DCAF5, DCUN1D2, DDX19B, DDX20, DDX24, DDX42, DENND4A, DESI2, WDR92, DNAJA1, DNMTI, DTL, DYRK3, EBLN3P, EBP, ECPAS, EFL1, EIF1, EIF2A, E1F4G1, EL0F1, EMC1, ENG, EOMES, EPC 2, EPS 15, ERCC6, FAM107B, F AM193 A, FAM83D, FBXL5, FBXO30, FBXO34, FBXO5, FDFT1, FGL2, FNBP1L, FOXN2, FRMD8, FRS2, FSTL1, FZD8, GATA3, GFOD1, GJC1, GOLGA4, GOLPH3, GPATCH2, GPATCH2L, GVQW3, HASPIN, HAUS3, HAUS6, HDAC6, HIPK1, HKDC1, HLA-A, HNF4A, HNRNPUL2, HOTAIR, HOXB7, HOXC13, HTRA1, IDI1, IER5L, INIP, INO80C, INTS10, ITPRIP, IVD, KANSL3, KAT2A, KCNG1, KCTD3, KIAA0232, KLHL13, KLHL15, KLHL24, KLHL9, LARP1B, LLPH, LNPK, LOC107985939, LRFN3, LRRN2, LYRM2, I .YST. MAD2L1BP, MAGEE L MAML3, MAN1A1, MANBA, MAP3K7. MARF1, MARK1, MDM4, MDN1, MED13L, MIA3, MINPP1, MISI 8BP1, MLLT3, MOB3B, MRPL17, MRPS26, MT CHI, MTDH, MTFR1, MTSS1L, MYD88, NCALD, NCR3LG1, NDUFV3, NECTIN1, NETO2, NGRN, FAM129A, NKX3-1, NNT-AS1, NOL9, NOPI4-AS1, NOTCH2, NR1D2, NR2C2, NSL1, NTN3, NXTI, ORC2, OTUB2, PARP14, PBRM1, PCMTD1, PCOLCE, PDGFRL, PDXP. PGP, PGRMC2, PHB, PI4KB, PIK3R1, PLCL2, PLEKHM1, KDELC2, POLE, POLE3, PPARA, PPM1A, PPP1R9B, PPP2CB, PRR14L, PRSS23, PTGER4, PTGFRN, PTP4A2, PUS7, PVR, QSER1, RAB23, RABEP1, RAE1, RANBP3, RANBP6, RBBP8, RBM12B, RBM25, RCOR1, RLF RNASEH1, RNF2I2, RPIA, RPS6KA1, RPUSD4, RRP12, RSL1D1, SAAL1, SAR1A, SATB2, SEPHS1, SETD2, SF1, SFMBT1, SGMS2, SHQ1, SLC25A20, SLC35G1, SLC39A6, SLC41A1, SMAD6, SMARCA2, SMIM15, SMIM26, SNEDl, SOCS5, SOS1, SOX12, SRBD1, ST3GAL2, STARD10, STX2, SYDE1, SYMPK, SYNM, SYTL2, TAF1, TBC1D10A, TBCC, TCOF1, THBS 1 TIMM 13, TL.E3, TLR2, TMED8, TMEM131L, TMEM186, TMEM198B, TMEM44, TP73-AS1, TPCN2, TPM2, TRABD2A, TRAF2, TRANK 1, TRMT10C, TSPAN17, TTI1, TUT4, TUT7, TXNDC11, UBAP1, UBAP2L, UBQLN2, USF3, USP54, VANGL1, VELMA, WDR24, WDR6, WDR89, WSCD1, YWHAE, ZBTB14, LINGO 1137, ZC3H12C, ZC3H18, ZDHHC8, ZFP91, ZNF107, ZNF184, ZNF189, ZNF202, ZNF419, ZNF514, ZNF518A, ZNF527, ZNF567, ZNF627, ZNF629, ZNF641, ZNF669, ZNF701 , ZNF703, ZNF740, ZNF84I, ZNF862, ZSCAN29, ZSWIM4, ZYGI IB, or combinations thereof.
In some embodiments, the translational initiation pathway, structural constituent of ribosome pathway, protein folding pathway, ribosome pathway, SRP-dependent cotranslational protein targeting to membrane pathway, cytoplasmic translation pathway, large ribosomal subunit pathway, protein folding chaperone pathway, or protein targeting pathway comprises at least one gene selected from ALMS1, ANTXR2, AUTS2, B3GNT10, KIAA1 I09, BOD1LI, Cl ORF 174, C5ORF24, C8ORF58, CRYBG1, CUL1, DIAPHl, EIF3H, EIF4E2, ELM01, ETS1, FAM156B, FNDC3A, GGA2, HERC1, HMGN4, HSDI7B12, HSP90AA1, HSPA1L, HSPA8, KIDINS220, LEMD2, LURAP1L, MAML2, MAPK4, MARCH7, MARK3, MC AT, MFSD6, MPH0SPH8, MRPL12, MRPL41, M TMR 1, MY09A, XC 'APD3. NCK1, NT5DC3, PLK4, P0LG2, PRPF38B, QRICH1, RBBP6, RGP1, RPL35A, RPL37A, RPLPO, RPP38, S100A1O, SFPQ, SLFN12L, SPART, SPOP, TPX2, TRAIP, TRIM2, UBALD1, USP15, USP9X, UTP3, ZDHHC16, ZKSCAN1, ZNF232, ZNF398, ZNF747, ZZZ3, or combinations thereof.
In some embodiments, the gene comprises the altered m6A epigenetic modification.
In some embodiments, the progressive glioma comprises an increased alteration of m6A epigenetic modifications relative to the pseudoprogressive glioma. In some embodiments, the progressive glioma comprises a decreased alteration of m6A epigenetic modifications relative to the pseudoprogressive glioma. In some embodiments, the progressive glioma comprises a highgrade glioma.
In some embodiments, the high-grade glioma compri ses a glioblastoma.
In some embodiments, the chemotherapy is selected from an alkylating agent, an antimetabolite, an anthracycline, a topoisomerase inhibitor, or a corticosteroid. In some embodiments, the immunotherapy comprises a PD1 antibody therapy, a PD-L1 antibody therapy, or combinations thereof.
In some embodiments, the pseudoprogressive glioma comprises an inflammatory' response to a cancer treatment given after the tissue biopsy. In some embodiments, the inflammatory' response is treated with a steroid. In some embodiments, the steroid is selected from prednisolone, methylprednisolone, dexamethasone, hydrocortisone, or combinations thereof.
In some embodiments, the tissue biopsy comprises a brain tissue. In some embodiments, the subject comprises a human.
BRIEF DESCRIPTION OF FIGURES
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain examples of the present, disclosure and together with the description, serve to explain, without limitation, the principles of the disclosure. Like numbers represent the same elements throughout, the figures.
FIG. 1 shows the general workflow for the biochemical extractions.
FIGS. 2A and 2B show hierarchical clustering analyses. FIG. 2A shows the heatmap of m6A modifications using hierarchical clustering. FIG. 2B shows the heat map of gene expression with differentially methylated probes. FIGS. 3A and 3B show hierarchical clustering analyses. FIG. 3 A shows the heatmap of m6A modifications using hierarchical clustering. FIG. 3B shows the heat map of gene expression with differentially methylated probes. These data serve as confirmation of findings in FIG. 2.
FIG. 4 shows the dendrogram of the cases clustered in the weighted gene correlation network analyses (WCGNA), demonstrating that the cases cluster along the diagnoses of progressive disease and pseudoprogressive disease.
FIGS. 5A and 5B shows the WGCNA dendrogram of the gene expression modules. FIG. 5B shows the correlation of these modules to the clinical phenotypes of progressive disease and pseudoprogressive disease. The high correlation (red) shows positive correlation with progressive disease, whereas the blue show's negative correlation with progressive disease.
DETAILED DESCRIPTION
The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant ait will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It w'ill also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.
Reference will now' be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
Terminology
In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:
As used in the specification and claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.
As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.
The term “administering” refers to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.
The term “detect” or “detecting” refers to an output signal released for the purpose of sensing of physical phenomenon. An event or change in environment is sensed and signal output released in the form of light.
A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease, or any amount above, below7, or imbetw?een those values, so long as the decrease is statistically significant.
An "increase" can refer to any change that results in larger amount of a symptom, disease, composition, condition, or activity. A substance is also understood to increase the genetic output of a gene when the genetic output of the gene product with the substance is more relative to the output of the gene product without the substance. Also, for example, an increase can be a change in the symptoms of a disorder such that the symptoms are more than previously observed. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase, or any amount above, below, or i n -between those values, so long as the increase is statistically significant. The terms “prevent,” “preventing,” “prevention,” and grammatical variations thereof as used herein, refer to a method of partially or completely delaying or precluding the onset or recurrence of a disorder or conditions and/or one or more of its attendant symptoms or barring a subject from acquiring or reacquiring a disorder or condition or reducing a subject’s risk of acquiring or reacquiring a disorder or condition or one or more of its attendant symptoms.
By “reduce” or other forms of the word, such as “reducing” or “reduction,” means lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary' for the standard or relative value to be referred to. For example, “reduces bacterial growth” means reducing the rate of growth of a bacterium relative to a standard or a control.
The term “cancer” is used to address any neoplastic disease and is not limited to epithelial neoplasms (surface and glandular cancers; such a squamous cancers or adenomas)). It is used here to describe both solid tumors and hematologic malignancies, including epithelial (surface and glandular) cancers, glial tumors, soft tissue, and bone sarcomas, angiomas, mesothelioma, melanoma, lymphomas, leukemias and myeloma.
As used herein, the term “chemical compound” and “compound,” refers to a chemical substance consisting of two or more different types of atoms or chemical elements in a fixed stoichiometric proportion. These compounds have a unique and defined chemical structure held together in a defined spatial arrangement by chemical bonds. Chemical compounds can be held together by covalent bonds, ionic bonds, metallic ions, or coordinate covalent bonds.
“Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
The term “comprising,” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed.
A "control" is an alternative subject or sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of an altered expression level of a proto-oncogene with a particular type of cancer, it is generally preferable to use a positive control (a subject or a sample from a subject, carrying such alteration and exhibiting syndromes characteristic of that disease), and a negative control (a subject or a sample from a subject lacking the altered expression and clinical syndrome of that disease).
As used herein, by a “subject” means an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, chickens, ducks, geese, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient.
An expression "database" denotes a set of stored data that represent a collection of sequences, which in turn represent a collection of biological reference materials.
A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotides sequences described herein may be used to identify larger fragments or full- length coding sequences of the gene with which they are associated.
A "probe" when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target. Usually, a probe wall comprise a label or a means by which a label can be attached, either before or subsequent to the hybridization reaction. Suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.
The terms “treat,” “treating,” “treatment,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating, or reducing the intensity of one or more attendant symptoms of a disorder or condition and/or alleviating, mitigating, or impeding one or more causes of a disorder or condition. Treatments according to the invention may be applied preventively, prophylactic-ally, palliatively, or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g., upon initial signs and symptoms of cancer), or after an established development of cancer. Prophylactic administration cart occur for several days to years prior to the manifestation of symptoms of an infection.
As used herein, “monitoring” refers to the actions of observing and checking the progress or quality of a treatment or procedure over a period of time. “Monitoring” also refers to observing the course of a disease or condition, such as a cancer, over a period of time.
A “fluorophore” is a fluorescent chemical compound that can re-emit light upon light excitation. The chemicals are sometimes used alone as a tracer in fluids, as a due for staining certain structures, as an enzyme substrate, or as a probe/indicator. More commonly they are covalently bonded to a macromolecule to serve as a marker for bioactive reagents (i.e.: antibodies, peptides, nucleic acids, etc.) Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods such as fluorescent imaging and spectroscopy.
As used herein, “epigenetic modification” refers to the heritable genetic changes the affect gene expression activity without altering the DNA or RNA sequence. These genetic changes include but are not limited to DNA or RNA methylation and histone modifications (i.e.: methylation and/or acetylation) that alter DNA or RNA accessibility and structure, thereby regulating gene expression patterns.
An m6A methylation modification is a methylation modification occurring on the sixth nitrogen on an adenosine nucleotide, also known as a chemical derivative of adenosine. This modification can occur on mRNA and DNA molecules in most eukaryotes regulating gene expression, alternative DNA splicing events, alternative polyadenylation, nuclear export, protein translation, mRNA degradation, and mRNA stabilization.
A “nucleotide” is a compound consisting of a nucleoside, which consists of a nitrogenous base and a 5-carbon sugar, linked to a phosphate group forming the basic structural unit of nucleic acids, such as DNA or RNA. The four types of nucleotides are adenine (A), cytosine (C), guanine (G), and thymine (T), each of which are bound together by a phosphodiester bond to form a nucleic acid molecule. As used herein, a nucleotide can comprise an epigenetic modification, such as methylation, acetylation, sumolaytion, or any variant thereof.
A “nucleic acid” is a chemical compound that serves as the primary information-carrying molecules in cells and make up the cellular genetic material. Nucleic acids are comprised nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). Examples of RNA include but are not limited to messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and heterogenous nuclear RNA (hnRNA). “genome” refers to a complete set of genes or genetic material present within a cell, tissue, or organism, A genome can be nuclear (found within the cell nucleus) or mitochondrial (found with the cell mitochondria).
The term “mRNA” refers to messenger ribonucleic acid, or single stranded molecule of RNA that corresponds to the genetic sequence of a gene and is translated by a ribosome in the process of synthesizing a protein. mRNA is created during the process of transcription, where a gene is converted into a primary transcript mRNA (or pre-mRNA). The primary' transcript is further processed through RNA splicing to only contain regions that will encode protein. mRNA can also be targeted for epigenetic modifications, such as methylation, to impact mRNA translation, nuclear retention, nuclear export, processing, and splicing.
As used herein, a “site” refers to a specific nucleotide or group of nucleotides along the 5’ to 3’ or 3’ to 5’ sequence of a nucleic acid molecule. These “sites” can be targeted for chemical modifications, such as methylation, acetylation, sumolaytion, or any mutation and variation thereof.
A “endoribonuclease” refers to an endonuclease enzyme that specifically degrades or cleaves ribonucleic acid molecules into smaller segments. The cleavage point on the RNA molecule usually occurs within the sequence rather than at the end of the sequence.
The term “methylation” refers to the chemical modification to a molecule by adding a methyl group on a DNA, RNA, or protein molecule. This modification is usually performed by enzymes to regulate gene expression, protein function, and RNA processing. An example of methylation includes but is not limited to CpG methylation occurring on DNA and m6A methylation occurring on mRNA.
The terms "cell," "cell line" and "cell culture" include progeny. It is also understood that all progenies may not. be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that has the same function or biological property, as screened for in the originally transformed cell, are included. The "host cells" used in the present invention generally are prokaryotic or eukaryotic hosts.
A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotides sequences described herein may be used to identify larger fragments or full- length coding sequences of the gene with which they are associated.
An "oncogene" refers to a polynucleotide containing at least one open reading frame that is capable of transforming a normal cell to a cancerous (or neoplastic or tumor) cell when introduced into a host cell. Oncogenes are often altered forms of the cellular counterpart, namely the "proto-oncogenes" that are incapable of cell transformation when expressed at the level present in a non-cancer cell.
As used herein, the term “module” refers to a subset of genes identified to comprise m6A epigenetic modifications. Each module of genes identified represent specific group or groups of mechanistic pathways and processes performed within a cell, tissue, or organ.
As used herein, a “progressive disease” refers to a disease or physical ailment whose course in most cases is the worsening, growth, or spread of disease. This worsening will continue until serious debility, organ failure, or death occurs. Some progressive diseases can be halted and/or reversed by treatment (surgical, dietary, or lifestyle interventions). Some examples of progressive diseases include, but are not limited to various cancers, Huntington’s disease, Alzheimer’s disease, multiple sclerosis, and Parkinson’s disease.
The term “pseudoprogressive disease” commonly refers to a phenomenon in which an initial increase in tumor size is observed or new lesions appear, followed by a decrease in tumor burden. This phenomenon can benefit patients receiving immunotherapy but often leads to premature discontinuation of treatment owing to the false judgement of progression.
A “glioma” refers to a primary brain or spinal cord tumors affecting the glial cells that surround nerve endings in the brain and spinal cord. Glial cells function as a support to neurons, however dysregulated cellular growth can lead to glioma formation. Gliomas are categorized by grade wherein slower growing gliomas are referred to as “low grade” or Grade 1 or 2. Fast growing or aggressive gliomas are referred to as “high grade” or Grade 3 or 4. An example of a high-grade glioma is a glioblastoma, typically categorized as a Grade 4.
Methods of detecting, identifying, diagnosing, treating, and/or preventing progressive diseases
The term “epigenetic” refers to a field of study where the genes of an organism is changed or altered due to complex interactions between the genome and the environment that are involved in development, differentiation, ageing, and some diseases. These gene changes occur when specific nucleotides sites are chemically modified by adding or removing one or more methyl, acety l, or sumo groups. The processes of chemically modifying nucleotides are also referred to as methylation, acetylation, and sumolaytion. Messenger RNA (mRNA) can also be epigenetically modified to regulate gene expression. N6-methyladenine (m6A) epigenetic modifications are the most abundant form of RNA modification in cellular biological processes. It has been found that the effect of m6A modification of RNA involves processing, nuclear export, translation, and even cell death. m6A modifications can also have a key role in the development of gliomas, or cancer of the glial cells.
The methods disclosed herein provides valuable insight into determining whether a patient’s future contrast enhancing lesions represents progressive disease or pseudoprogressive disease. Specifically, disclosed herein is a method of determining, identifying, and/or diagnosing whether a subject has a progressive glioma or a pseudoprogressive glioma, the method comprising collecting a tissue biopsy form the subject, detecting an altered N6-Methladenosine (m6A) epigenetic modification on an mRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from Table 1, Table 2, Table 3, and/or Table 4, and diagnosing the subject with either the progressive glioma or the pseudoprogressive glioma.
Gliomas are the most common form of malignant tumor in the central nervous system. In addition, glioblastomas, an aggressive high-grade glioma, comprise the highest malignancy rate and account for almost half of all brain tumors. Given the aggressiveness of these tumors, surgical removal does not always promise complete remission. In addition, treatments, such as chemotherapy, is commonly followed by showing new areas of contrast enhancement in brain MR imaging that could indicate tumor recurrence (termed progressive disease (PD)) requiring further treatment, or a treatment reaction (termed pseudo-progressive disease (psPD)). There is currently a need to distinguish between the two outcomes to ensure proper treatment is administered, and unnecessary surgeries are avoided.
In one aspect, disclosed herein is a method of identifying whether a subject has a progressive glioma or a pseudoprogressive glioma, the method comprising isolating a tissue biopsy from the subject, detecting an altered N6-Mothyladenosine (m6A) epigenetic modification on an mRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from at least one pathway selected from mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, regulation of cAMP-dependent protein kinase activity (PKA) pathway, epidermal growth factor receptor 3 (ERBB3) signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway, cardiac ventricle morphogenesis pathway, regulation of receptor recycling pathway, protein mannosylation pathway, protein O-linked mannosylation pathway, presynaptic active zone membrane pathway, presynaptic active zone pathway, regulation of cell population proliferation pathway, regulation of mitochondrial translation pathway, regulation of bone morphogenetic protein (BMP) signaling pathway, histone kinase activity pathway, oogenesis pathway, nuclear receptor activity pathway, ligand-activated transcription factor activity pathway, regulation of T cell cytokine production pathway, translational initiation pathway, structural constituent of ribosome pathway, protein folding pathway, ribosome pathway, signal-recognition particle (SRP)-dependent cotranslational protein targeting to membrane pathway, cytoplasmic translation pathway, large ribosomal subunit pathway, protein folding chaperone pathway, protein targeting pathway and diagnosing the subject with either the progressive glioma or the pseudoprogressive glioma, combinations of pathways.
In some embodiments, the method comprises a method of identifying whether a subject has a progressive glioma or a pseudoprogressive glioma, the method comprising isolating a tissue biopsy from the subject, detecting an altered N6-Methyladenosine (m6A) epigenetic modification on an mRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from at least one pathway selected from Table 1 , Table 2, Table 3, and/or Table 4 and diagnosing the subject with either the progressive glioma or the pseudoprogressive glioma.
In some aspects, disclosed herein is a method of treating either a progressive glioma or a pseudoprogressive glioma in a subject, the method comprising isolating a tissue biopsy from the subject, detecting an altered N°-Methyladenosine (m6A) epigenetic modification on an mRNA from the tissue biopsy, wherein the mRNA is encoded by a gene from at least one pathway selected from mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, regulation of cAMP- dependent protein kinase activity (PKA) pathway, epidermal growth factor receptor 3 (ERBB3) signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway, cardiac ventricle morphogenesis pathway, regulation of receptor recycling pathway, protein mannosylation pathway, protein O-linked mannosylation pathway, presynaptic active zone membrane pathway, presynaptic active zone pathway, regulation of cell population proliferation pathway, regulation of mitochondrial translation pathway, regulation of bone morphogenetic protein (BMP) signaling pathway, histone kinase activity pathway, oogenesis pathway, nuclear receptor activity pathway, ligand-activated transcription factor activity pathway, regulation of T cell cytokine production pathway, translational initiation pathway, structural constituent of ribosome pathway, protein folding pathway, ribosome pathway, signal-recognition particle (SRP)- dependent cotranslational protein targeting to membrane pathway, cytoplasmic translation pathway, large ribosomal subunit pathway, protein folding chaperone pathway, or protein targeting pathway, diagnosing the subject with either the progressive glioma, the pseudoprogressive glioma, or combination of pathways, and treating the subject with a chemotherapy, an immunotherapy, a palliative therapy, or combinations thereof if diagnosed with the progressive glioma or treating with a steroid if diagnosed with the pseudoprogressive glioma.
In some aspects, disclosed herein is a method of identifying, diagnosing, treating, preventing, ameliorating, and/or decreasing either a progressive glioma or a pseudoprogressive glioma in a subject, the method comprising isolating a tissue biopsy from the subject, detecting an altered Nb-Methyladenosine (m6A) epigenetic modification on an mRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, or more pathways.
In some embodiments, disclosed herein is a method of treating either a progressive glioma or a pseudoprogressive glioma in a subject, the method comprising isolating a tissue biopsy from the subject, detecting an altered N6 -Methyl adenosine (m6A) epigenetic modification on an rnRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from at least one pathway selected from Table 1, Table 2, Table 3, and/or Table 4, diagnosing the subject with either the progressive glioma or the pseudoprogressive glioma, and treating the subject with a chemotherapy, an immunotherapy, or a palliative therapy if diagnosed with the progressive glioma or treating with a steroid if diagnosed with the pseudoprogressive glioma.
In one aspect, disclosed herein are methods of any preceding aspect, wherein any of the genes of Table 1 comprise the altered m6A epigenetic modification. In one aspect, disclosed herein are methods of any preceding aspect, wherein any of the genes of Table 2 comprise the altered m6A epigenetic modification. In one aspect, disclosed herein are methods of any preceding aspect, wherein any of the genes of Table 3 comprise the altered m6A epigenetic modification. In one aspect, disclosed herein are methods of any preceding aspect, wherein any of the genes of Table 4 comprise the altered m6A epigenetic modification.
In some embodiments, the method detects the altered m6A epigenetic modification in 5 or more genes from the at least one pathway. In some embodiments, the method detects the altered m6A epigenetic modification in 10 or more genes from the at least one pathway. In some embodiments, the method detects the altered m6A epigenetic modification in 50 or more genes from the at least one pathway. In some embodiments, the method detects the altered m6A epigenetic modification in 100 or more genes from the at least one pathway. In some embodiments, the method detects the altered m6A epigenetic modification in 250 or more genes from the at least one pathway. In some embodiments, the method detects the altered m6A epigenetic modification in 500 or more genes from the at least one pathway.
In some embodiments, the method detects the altered m6A epigenetic modification in 5 or more genes from Table 1. In some embodiments, the method detects the altered m6A epigenetic modification in 10 or more genes from Table 1. In some embodiments, the method detects the altered m6A epigenetic modification in 50 or more genes from Table 1. In some embodiments, the method detects the altered m6A epigenetic modification in 100 or more genes from Table 1. In some embodiments, the method detects the altered m6A epigenetic modification in 250 or more genes from Table 1. In some embodiments, the method detects the altered m6A epigenetic modification in 500 or more genes from Table 1.
In some embodiments, the method detects the altered m6A epigenetic modification in 5 or more genes from Table 2. In some embodiments, the method detects the altered m6A epigenetic modification in 10 or more genes from Table 2. In some embodiments, the method detects the altered m6A epigenetic modification in 50 or more genes from Table 2. In some embodiments, the method detects the altered m6A epigenetic modification in 100 or more genes from Table 2. In some embodiments, the method detects the altered m6A epigenetic modification in 250 or more genes from Table 2. In some embodiments, the method detects the altered m6A epigenetic modification in 500 or more genes from Table 2.
In some embodiments, the method detects the altered m6A epigenetic modification in 5 or more genes from Table 3. In some embodiments, the method detects the altered m6A epigenetic modification in 10 or more genes from Table 3. In some embodiments, the method detects the altered m6A epigenetic modification in 50 or more genes from Table 3. In some embodiments, the method detects the altered m6A epigenetic modification in 100 or more genes from Table 3. In some embodiments, the method detects the altered m6A epigenetic modification in 250 or more genes from Table 3. In some embodiments, the method detects the altered m6A epigenetic modification in 500 or more genes from Table 3.
In some embodiments, the method detects the altered m6A epigenetic modification in 5 or more genes from Table 4. In some embodiments, the method detects the altered m6A epigenetic modification in 10 or more genes from Table 4. In some embodiments, the method detects the altered m6A epigenetic modification in 50 or more genes from Table 4. In some embodiments, the method detects the altered m6A epigenetic modification in 100 or more genes from Table 4. In some embodiments, the method detects the altered m6A epigenetic modification in 250 or more genes from Table 4. In some embodiments, the method detects the altered m6A epigenetic modification in 500 or more genes from Table 4. In some embodiments, the method detects the altered m6A epigenetic modification in a gene sequence. In some embodiments, the method detects the altered m6A epigenetic modification in one or more of 1997 gene sequences. In some embodiments, the method detects the altered m6A epigenetic modification in two or more of 1997 gene sequences. In some embodiments, the method detects the altered m6A epigenetic modification in five or more of 1997 gene sequences. In some embodiments, the method detects the altered m6A epigenetic modification in ten or more of 1997 gene sequences. In some embodiments, the method detects the altered m6A epigenetic modification in 50 or more of 1997 gene sequences. In some embodiments, the method detects the altered m6A epigenetic modification in 100 or more of 1997 gene sequences. In some embodiments, the method detects the altered m6A epigenetic modification in 250 or more of 1997 gene sequences. In some embodiments, the method detects the altered m6A epigenetic modification in 500 or more of 1997 gene sequences. In some embodiments, the method detects the altered m6A epigenetic modification in 750 or more of 1997 gene sequences. In some embodiments, the method detects the altered m6A epigenetic modification in 1000 or more of 1997 gene sequences. In some embodiments, the method detects the altered m6A epigenetic modification in 1500 or more of 1997 gene sequences.
In some embodiments, the method detects the altered m6A epigenetic modification in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106,
107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,
127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,
147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,
167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,
187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,
207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,
227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,
247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,
267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286,
287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306,
307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,
327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,
347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,
367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,
407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,
427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,
447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,
467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506,
507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521 , 522, 523, 524, 525, 526,
527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546,
547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566,
567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586,
587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606,
607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626,
627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646,
647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666,
667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686,
687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706,
707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726,
727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746,
747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766,
767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781 , 782, 783, 784, 785, 786,
787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806,
807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826,
827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846,
847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866,
867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886,
887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906,
907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926,
927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946,
947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961 , 962, 963, 964, 965, 966,
967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986,
987, 988, 989, 990, 991 , 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001 , 1002, 1003, 1004,
1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020,
1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036,
1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068,
1069, 1070, 1071 , 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084,
1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100,
1101, 1102, 1 103, 1104, 1105, 1 106, 1107, 1108, 1 109, 1110, 111 1, 1 112, 1113, 1114, 11 15, 1116,
1117, 11 18, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1 131, 1132,
1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148,
1 149, 1150, 1151 , 1 152, 1153, 1154, 1 155, 1156, 1157, 1 158, 1159, 1160, 1 161, 1162, 1163, 1 164,
1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180,
1181 , 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1 195, 1196,
1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212,
1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221 , 1222, 1223, 1224, 1225, 1226, 1227, 1228,
1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244,
1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260,
1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276,
1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292,
1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308,
1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324,
1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340,
1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356,
1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371 , 1372,
1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388,
1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404,
1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420,
1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436,
1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452,
1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468,
1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484,
1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500,
1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516,
1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532,
1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548,
1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564,
1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580,
1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, 1603, 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612,
1613, 1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621 , 1622, 1623, 1624, 1625, 1626, 1627, 1628,
1629, 1630, 1631, 1632, 1633, 1634, 1635, 1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644,
1645, 1646, 1647, 1648, 1649, 1650, 1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660,
1661 , 1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674, 1675, 1676,
1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692,
1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701 , 1702, 1703, 1704, 1705, 1706, 1707, 1708,
1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1721, 1722, 1723, 1724,
1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735, 1736, 1737, 1738, 1739, 1740,
1741, 1742, 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756,
1757, 1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768, 1769, 1770, 1771, 1772,
1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780, 1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788,
1789, 1790, 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804,
1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820,
1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836,
1837, 1838, 1839, 1840, 1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852,
1853, 1854, 1855, 1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1867, 1868,
1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884,
1885, 1886, 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900,
1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913, 1914, 1915, 1916,
1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932,
1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948,
1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961, 1962, 1963, 1964,
1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980,
1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996,
1997, or more genes from at least one pathway of any preceding aspect, Table 1 , Table 2, Table 3, and/or Table 4.
In one aspect, disclosed herein are methods of any preceding aspect, wherein the altered m6A epigenetic modification is either increased or decreased in the progressive glioma compared to the pseudoprogressive glioma. Definitions of “increase” and “decrease” are found above.
In some embodiments, the mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, or regulation of cAMP-dependent PKA pathway comprises at least one gene selected from ABCD4, AHSA1, APBA2, ATP6AP1, ATXN7L3B, AUNIP, BACH L BICD1, CABLES!, CALR. CCAR2, CDCA5, CDCA8, CDK4, CDR2, CLCN7, CNNM4, DNMBP, DOHH, DXO, EIF2S3, ELF1, EPAS1, EXOC7, FEM1A, FKBP1A, FOSB, FUNDCI, GCNT4, HMG20A, HR, HIT, C6ORF106, INTS11, JPT1, KLAA0930, KXDI, LETM1, LOC100129034, LYAR. MEX3D, MIIP, MKRN1, MMADHC, MRPL54, MTRNR2L2, NLRX1, N0TCH1, NRBF2, PAK4, PAXIP1-AS1, PCDH17, PDGFB, PIGO, PPP2R1A, PRKARIA, PRKCQ, PTK7, PTPN18, PTPN7, RAB39B, RABL6, RAC1, RAPGEF1, RAVER1, RBBP5, RND1, RNF14, RNF167, SEC23IP, SERTAD1, SIK2, SLC25A19, SLC43A2, SLC7A6, SNRNP25, SNTA1, SOCS3, SPIN4, STAG1, SYNDIG1, SYNGR2, TBCID9, TP53, TRIM65, TSC22DI, TTC28, TTYH3, TUBB6, TULP3, UBE2L3, UBE3B, WAPL, ZBED3, ZBTB48. ZSWIM1, LOC 100507437, or combinations thereof.
In some embodiments, the ERBB3 signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway, cardiac ventricle morphogenesis pathway, regulation of receptor recycling pathway, protein mannosylation pathway, or protein O-linked mannosylation pathway comprises at least one gene selected from ACTN4, AP2A2, ARHGEF25, ATP10A, BTN3A1, BUB3, C14ORF93, C2ORF42, CAPZA1, CASP8, CDCA2, COL6A1, CPSF7, DDR1, DOK1, DPM3, DSP, DUSP8, ECE1, EIF3G, ETV3, FAM83G, FBL, FUK, FTSJ3, GATA2, HIP1, INAFM2, JMJD4, JTB, KHNYN, KIF3B, KLHDC10, KNOP1, LARP4, LRP12, MAP3K10, MEGF6, MPHOSPHIO, MRTFB, NADK, NCKAP5L, NCLN, NR2F2, NRG1, NSF, OGFOD3, OS9, PLEKHH3, PMVK, PNPO, POMGNT2, QSOX2, RAB15, RAB1 A, RHOC, RIN3, RNF43, RTN4, SALL1 , SDF2, SOX4, SP140, SPTLC2, SRRM1, SYT17, RABI, TBC1D22A, TBC1D8, TERF2, THRAP3, TIAL1, TOX, TPD52L2, TPRA1, TRABD, TRIP6, USP40, ZBTB26, ZBTB9, ZNF318, ZNF503, ZNF668, ZNF827, or ZNF865.
In some embodiments, the presynaptic active zone membrane pathway, presynaptic active zone pathway, regulation of cell population proliferation pathway, regulation of mitochondrial translation pathway, regulation of BMP signaling pathway, histone kinase activity pathway, oogenesis pathway, nuclear receptor activity pathway, ligand-activated transcription factor activity pathway, or regulation of T cell cytokine production pathway comprises at least one gene selected from ABHD5, ACBD3, ACSL3, ACVR1B, ADI1, ADIPOR2, AFG3L2, AHSA2P, AK2, AKIRIN1, AKTIP, ALDH1B1, AMIG02, ANKRD50, ANKRD54, APOL6, APPL2, AR, ARHGAP26, ATP5F1A, ATP5IF1, AVIN, AZI2, B4GALT2, BCL2, BICRAL, BIRC3, BRWD1, BTBD2, BTG1, BTN2A1, C16ORF72, C3ORF18, C3ORF38, C5ORF15, C8ORF33, CACNB1, CCDC71L, CCNT2, CCT7, CDCA4, CDK2, CDKN1B, CDS2, CEACAM6, CEP78, CHST11, CHST12, CHTOP, CLN5, CL.PB, CNOT6, CO A3, COLEC12, COQ5, CRIM1 CTCF, CTR9, CWF19L1, DARS2, DCAF1, DCAF5, DCUN1D2, DDX19B, DDX20, DDX24, DDX42, DENND4A, DESK, WDR92, DNAJA1, DNMT1, DTL, DYR.K3, EBLN3P, EBP, ECPAS, EFL .1 , EIF1 , EIF2A, EIF4G1 , ELOF1, EMC1, ENG, EOMES, EPC2, EPS 15, ERCC6, FAM107B, FAM193A, FAM83D, FBXL5, FBXO30, FBXO34, FBXO5, FDFT1, FGL2, FNBP1L, FOXN2, FRMD8, FRS2, FSTL1, FZD8, GATA3, GFOD1, GJC1 , GOLGA4, GOLPH3, GPATCH2, GPATCH2L, GVQW3, HASPIN, HALTS3, HALTS6, HDAC6, HIPK1, HKDC1, HL A- A, HNF4A, HNRNPUL2, HOTAIR, H0XB7, H0XC13, HTRA1, IDI1, IER5L, INIP, INO80C, INTS10, ITPRIP, IVD, KANSL3, KAT2A, KCNG1, KCTD3, KIAA0232, KLHL13, KLHL15, KLHL24, KLHL9, L ARP IB, LLPH, LNPK, LOC107985939, LRFN3, LRRN2, LYRM2, LYST, MAD2L1BP, MAGEF1, MAML3, MAN1A1, MANBA, MAP3K7, MARF1, MARK 1, MDM4, MDN1 , MED13L, Ml A3. MINPP1, MIS18BP1, MLLT3, MOB3B, MRPL17, MRPS26, MTCH1, MTDH, M ITR E MTSS1L, MYD88, NC ALD. NCR3LG1, NDUFV3, NECT1N1, NETO2, NGRN, FAM129A, NKX3-1 NNT-AS1, NOL9, NOP14-AS1, NOTCH2, NR1D2, NR2C2, NSL1, NTN3, NXT1, ORC2, OTUB2, PARPI4, PBRM1, PCMTD1, PCOLCE, PDGFRL, PDXP, PGP, PGRMC2, PHB, PI4KB, PIK3R1, PLCL2, PLEKHM1, KDELC2, POLE, POLE3, PPARA, PPM1A, PPPIR9B, PPP2CB, PRR14L, PRSS23, PTGER4, PTGFRN, PTP4A2, PUS7, PVR, QSER1, RAB23, RABEP1, RAE1, RANBP3, RANBP6, RBBP8, RBM12B, RBM25, RCOR1, RLF, RNASEH1, RNF2I2, RPIA, RPS6KA1, RPUSD4, RRP12, RSL1D1, SAALI, SARI A, SATB2, SEPHS1, SETD2, SF1, SFMBT1, SGMS2, SHQ1, SLC25A20, SLC35G1, SLC39A6, SLC41A1, SMAD6, SMARCA2, SMIM15, SMIM26, SNED1, S0CS5, S0S1 , SOXI2, SRBD1, ST3GAL2, STARD10, STX2, SYDE1, SYMPK, SYNM, SYTL2, TAF1, TBC1DI0A, TBCC, TCOFI, THBS1, TIMM13, TLE3, TLR2, TMED8, TMEM131L, TMEMI 86, TMEMI98B, TMEM44, TP73-AS1, TPCN2, TPM2, TRABD2A, TRAF2, TRANK1, TRMT10C, TSPAN17, TTI1, TUT4, TUT7, TXNDC11, UBAP1, UBAP2L, UBQLN2, USF3, USP54, VANGL1, VIRMA, WDR24, WDR6, WDR89, WSCD1, YWHAE, ZBTB14, LINC01137, ZC3H12C, ZC3H18, ZDHHC8, ZFP91, ZNF107, ZNF184, ZNF189, ZNF202, ZNF419, ZNF514, ZNF518A, ZNF527, ZNF567, ZNF627, ZNF629, ZNF64I, ZNF669, ZNF701, ZNF703, ZNF740, ZNF84I, ZNF862, ZSCAN29, ZSWIM4, ZYGI IB, or combinations thereof..
In some embodiments, the translational initiation pathway, structural constituent of ribosome pathway, protein folding pathway, ribosome pathway, SRP-dependent cotranslational protein targeting to membrane pathway, cytoplasmic translation pathway, large ribosomal subunit pathway, protein folding chaperone pathway, or protein targeting pathway comprises at least one gene selected from ALMS1, ANTXR2, AUTS2, B3GNT10, KIAA1109, BOD1L1, C1ORF174, C5ORF24, C8ORF58, CRYBG1, Cl. E L DIAPH1 , EIF3FI, EIF4E2, ELM01, ETSI, FAMI 56B, FNDC3A, GGA2, HERC1, HMGN4, HSD17B12, HSP90AA1, H SPA IL, HSPA8, KIDINS220, LI .MD2. LURAP1L, MAML2, MAPK4, MARCH7.. MARK3, MCA T, MFSD6, MPHOSPH8, MRPL12, MRPL41, MTMR1, MY09A, NCAPD3, NCK1, NT5DC3, PLK4, POLG2, PRPF38B, QRICH1, RBBP6, RGP1, RPL35A, RPL37A, RPLPO, RPP38, S100A10, SFPQ, SLFN12L, SPARE, SPOP, TPX2, TRAIP, TR1M2, IJBALD1, USP15, USP9X, UTP3, ZDHHC16, ZKSCAN1, ZNF232, ZNF398, ZNF747, ZZZ3, or combinations thereof.
It should be noted that the genes of any preceding aspects or any preceding embodiments can be grouped into a group of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 , 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109 , 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 , 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 31 1, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 41 1, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, or more groups. In some embodiments, the m6A epigenetic modification occurs at any group of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 genes. In some embodiments, the m6A epigenetic modification occurs at any group of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or 1900 genes. In some embodiments, the specific m6A epigenetic modification occurs on any one of 2590 nucleotide sites. In some embodiments, the specific m6A epigenetic modification occurs at any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,
1 10, 111, 112, 113, 1 14, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,
130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,
150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,
170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,
190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,
210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,
250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,
290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309,
310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329,
330, 331 , 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349,
350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,
370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,
390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,
410, 411 , 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,
430, 431 , 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469,
470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489,
490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509,
510, 51 1 , 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529,
530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549,
550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569,
570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651 , 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671 , 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751 , 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831 , 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931 , 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091 , 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1 102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1 120, 1121, 1122, 1 123, 1124, 1125, 1 126, 1127, 1128, 1 129, 1130, 1131, 1 132, 1133, 1134, 1 135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1 166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1 184, 1185, 1186, 1 187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202. 1203, 1204, 1205. 1206, 1207, 1208. 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381 , 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491 , 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, 1603, 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627, 1628, 1629, 1630, 1631, 1632, 1633, 1634, 1635, 1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648, 1649, 1650, 1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674, 1675, 1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701, 1702, 1703, 1704, 1705, 1706, 1707, 1708, 1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1721, 1722, 1723, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756, 1757, 1758, 1759, 660, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768, 1769, 1770, 1771, 1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780, 1781 , 1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804, 1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840, 1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854, 1855, 1856, 1857, 1858, 1859, 1860, 1861 , 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, 1889, 1890, 1891 , 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104, 2105, 2106, 2107, 2108, 2109, 21 10, 2111, 2112, 2113, 2114, 2115, 2116, 2117, 2118, 2119, 2120, 2121, 2122, 2123, 2124, 2125, 2126, 2127, 2128, 2129, 2130, 2131, 2132, 2133, 2134, 2135, 2136, 2137, 2138, 2139, 2140, 2141, 2142, 2143, 2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151, 2152, 2153, 2154, 2155, 2156, 2157, 2158, 2159, 2160, 2161, 2162, 2163, 2164, 2165, 2166, 2167, 2168, 2169, 2170, 2171, 2172, 2173, 2174, 2175, 2176, 2177, 2178, 2179, 2180, 2181, 2182, 2183, 2184, 2185, 2186, 2187, 2188, 2189, 2190, 2191, 2192, 2193, 2194, 2195, 2196, 2197, 2198, 2199, 2200, 2201, 2202, 2203, 2204, 2205, 2206, 2207, 2208, 2209, 2210, 2211, 2212, 2213, 2214, 2215, 2216, 2217, 2218, 2219, 2220, 2221, 2222, 2223, 2224, 2225, 2226, 2227, 2228, 2229, 2230, 2231, 2232, 2233, 2234, 2235, 2236, 2237, 2238, 2239, 2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247, 2248, 2249, 2250, 2251, 2252, 2253, 2254, 2255, 2256, 2257, 2258, 2259, 2260, 2261, 2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272, 2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301, 2302, 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2310, 2311, 2312, 2313, 2314, 2315, 2316, 2317, 2318, 2319, 2320, 2321, 2322, 2323, 2324, 2325, 2326, 2327, 2328, 2329, 2330, 2331, 2332, 2333, 2334, 2335, 2336, 2337, 2338, 2339, 2340, 2341, 2342, 2343, 2344, 2345, 2346, 2347, 2348, 2349, 2350, 2351, 2352, 2353, 2354, 2355, 2356, 2357, 2358, 2359, 2360, 2361, 2362, 2363, 2364, 2365, 2366, 2367, 2368, 2369, 2370, 2371 , 2372, 2373, 2374, 2375, 2376, 2377, 2378, 2379, 2380, 2381, 2382, 2383, 2384, 2385, 2386, 2387, 2388, 2389, 2390, 2391, 2392, 2393, 2394, 2395, 2396, 2397, 2398, 2399, 2400, 2401, 2402, 2403, 2404, 2405, 2406, 2407, 2408, 2409, 2410, 241 1, 2412, 2413, 2414, 2415, 2416, 2417, 2418, 2419, 2420, 2421, 2422, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443, 2444, 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452, 2453, 2454, 2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464, 2465, 2466, 2467, 2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475, 2476, 2477, 2478, 2479, 2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487, 2488, 2489, 2490, 2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500, 2501, 2502, 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512, 2513, 2514, 2515, 2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524, 2525, 2526, 2527, 2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535, 2536, 2537, 2538, 2539, 2540, 2541, 2542, 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581 , 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, or 2590 sites, or any combination thereof. In some embodiments, the m6A epigenetic modification occurs at any group of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 sites.
In some embodiments, the gene comprises the altered m6A epigenetic modification.
In some embodiments, the progressive glioma comprises an increased alteration of m6A epigenetic modifications relative to the pseudoprogressive glioma. In some embodiments, the progressive glioma comprises a decreased alteration of m6A epigenetic modifications relative to the pseudoprogressive glioma.
In some embodiments, the pseudoprogressive glioma comprises an inflammatory response to a cancer treatment given after the tissue biopsy. In some embodiments, the inflammatory response is treated with a steroid. In some embodiments, the steroid is selected from prednisolone, methylprednisolone, dexamethasone, hydrocortisone, or combinations thereof.
A glioma is a type of tumor that occurs in the brain and spinal cord that begin in the glial cells that surround nerve cells. Similarly, a progressive glioma is a tumor that occurs in the brain wherein the tumor growth worsens over time, thus resulting in decline of general overall health. Gliomas are classified into four grades (I, II, III, or IV), wherein the treatment and prognosis depend upon the glioma grade. In particular, grade IV gliomas are also referred to as high-grade gliomas. In some embodiments, the progressive glioma comprises a high-grade glioma. In some embodiments, the high-grade glioma is a glioblastoma. As used herein, a glioblastoma refers to an aggressive, fast growing type of glioma that occurs in the brain and spinal cord.
In some embodiments, chemotherapy and/or immunotherapy is given as a treatment for progressive glioma. Such chemotherapy can be selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, topoisomerase inhibitors, or corticosteroids. In other embodiments, the immunotherapy is either a PD1 antibody therapy or a PD-L1 antibody therapy. For example, standard first-line chemotherapy can consist of temozolomide (75 mg/m2 daily) during radiotherapy followed by a further six cycles of temozolomide (150-200 mg/m2 on days 1- 5 every 28 days). Lomustine, carmustine, and rechallenge with temozolomide are all potential options for treatment of progressive glioma as well. Further options include further surgical resection, reirradiation, systemic therapies such as lomustine or bevacizumab, combined approaches, or supportive care alone. Other options include radiotherapy, surgery, or palliative care.
In a specific example, single-agent bevacizumab can be used for the treatment of progressive glioma. Bevacizumab in combination with lomustine improves PFS compared with lomustine alone. Typically, the combination of bevacizumab with chemotherapy is recommended after failure on bevacizumab alone, which is different than the treatment for pseudoprogressive glioma. Importantly, those with progressive glioma should not be treated the same as those with pseudoprogressive glioma, particularly concerning bevacizumab. This is due to its effect on inflammatory and radio chemotherapy-induced changes in GBM vasculature.
As discussed above, the term “pseudoprogression” is used to describe the phenomenon of subacute radio chemotherapy treatment-related sequelae in CNS tumors presenting as increasing lesion volume or new contrast enhancement on MRI suggestive of tumor progression. However, these patients often recover or stabilize spontaneously, usually without any change in treatment paradigm. (Brandsma D, Stalpers L, Taal W, Sminia P, van den Bent MJ. Clinical features, mechanisms, and management of pseudoprogression in malignant gliomas. Lancet Oncol 2008;9:453-461). Brandsma discloses that in patients managed with temozolomide chemoradiotherapy who have clinically asymptomatic progressive lesions at the end of treatment, adjuvant temozolomide should be continued; in clinically symptomatic patients, surgery' should be considered. If mainly necrosis is noted during surgery, continuation of adjuvant temozolomide is logical. Trials on the treatment of recurrent malignant glioma should exclude patients with progression within the first 3 months after temozolomide chemoradiotherapy unless histological confirmation of tumor recurrence is available.
A pseudoprogressive disease refers to an occurrence in which an initial increase in tumor size is observed or new lesions appear, followed by a decrease in tumor burden following the subject receiving a cancer therapy. Similarly, a pseudoprogressive glioma refers to a transient imaging pattern that mimics tumor progression but not presenting with the accompanied decline of health. The pseudo-progression of brain tumors is attributed to damage from a cancer treatment leading to local inflammation and edema. In some embodiments, the pseudoprogressive glioma is an inflammatory response to a cancer treatment given after the tissue biopsy. In other embodiments, the inflammatory response is treated with a steroid. In other embodiments, the steroid is selected from the group consisting of prednisolone, methylprednisolone, dexamethasone, hydrocortisone, or any combination thereof. Further treatments for pseudoprogression include, but are not limited to, Apatinib and bevacizumab (at different doses than those given for progressive glioma). (Wang et al., Apatinib treatment for symptomatic pseudoprogression after standard treatment for glioblastoma multiforme: a case report, APM Vol. 8, No, 5, Nov. 2019, herein incorporated by reference for its teaching concerning treating pseudo progression).
The etiology of pseudoprogression is thought to be due to vascular and oligodendroglia cell injury, leading to inflammation and increased BBB permeability. Because the enhancement seen in pseudoprogression can be mistaken for actual tumor progression, patients are often routed to bevacizumab as second-line therapy for recurrence. Bevacizumab has been observed to decrease the permeability of not only tumor-related leaky vasculature but also of radiation-induced leaky vasculature, thereby “curing” biopsy-proven pseudoprogression (Weinstein JS, Varallyay CG, Dosa E, et al. Superparamagnetic iron oxide nanoparticles: diagnostic magnetic resonance imaging and potential therapeutic applications in neurooncology and central nervous system inflammatory pathologies, a review. J Cereb Blood Flow Metab 2010;30: 15-35). This is particularly worrisome as pseudoprogression has been shown to be significantly associated with methylated MGMT promoter status and increased survival. The presence of a methylated MGMT promoter is associated with a significant survival benefit. The presence of a methylated MGMT promoter is associated with a significant survival benefit. The MGMT protein removes alkyl groups from the O6 position of guanine. Silencing of the MGMT promoter by methylation is clinically important because cancer cells produce less MGMT protein and cannot repair DNA alkylation by agents such as temozolomide.46 Bevacizumab may decrease the correlation between MGMT status and pseudoprogression (Thompson EM, Frenkel EP, Neuwelt EA. The paradoxical effect of bevacizumab in the therapy of malignant gliomas. Neurology. 2011 Jan 4;76(1):87-93.) For treating progressive glioma, a range of chemotherapy partners, including lomustine, carmustine, and temozolomide, can also be used in tandem with chemotherapy. Immunotherapy is also an option to be used alone or in combination with a chemotherapy and/or a palliative therapy. Immune checkpoint inhibitors, predominantly targeting PD-1/PD-L1 and/or CTLA-4, can be used. Chimeric antigen receptor (CAR) T-cell therapy with genetically modified T cells can also be used. Examples of treatment types can be found in Tan et al. (Tan, AC, Ashley, DM, Lopez, GY, Malinzak, M, Friedman, HS, Khasraw, M. Management of glioblastoma: State of the art and future directions. CA Cancer J Clin. 2020: 70: 299- 312), herein incorporated by reference in its entirety for its teaching concerning treatment of glioma.
In one embodiment, the tissue biopsy is a brain tissue. In other embodiments, the tissue biopsy is a spinal cord tissue. In one embodiment, the subject is a human.
A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.
EXAMPLES
The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.
Example 1: Rationale for epitranscriptomic analysis in recurrent glioma
RNA modification on mRNA by methylation of adenosine on position 6 (m6A) shows high prevalence, with classical studies indicating that on average each mRNA transcript containing three m6A modifications with some transcripts containing over twenty m6A sites which, when located in clusters of less than 100 nucleotides, are deemed “clustered m6A” sites. Several biological functions are modified by m6A modification of RNA. Single and clustered m6A sites promote noncanonical cap-independent translation, modulate translation-elongation dynamics, promote IncRNA decay in dendritic cell migration, and promote tumor cell proliferation. In summary, m6A modification and the machinery that promotes the “writing” and “reading” of this RNA modification have been implicated in the initiation, progression, maintenance, and drug resistance of various types of disease. Many cancers immune-evasion processes have been shown to be modified by ni6A RNA modification. Herein, m6A RNA modifications are mapped out in primary glioblastoma, so as to predict from the first, biopsy which patients would ultimately undergo pseudoprogression and which would undergo progression.
Example 2: Methods
Formalin fixed paraffin embedded tissue from the first resection of thirty-six glioblastoma patients were obtained. These thirty-six patients were chosen because they had undergone a second resection later in the course of their disease at the point of identifying a new contrast enhancing lesion by the MRI. 18 of these patients experienced progressive disease, and 18 pseudoprogressive disease that was confirmed in a second biopsy. The analysis was performed on the epitranscriptomic m6A array by. raystar. Using unbiased hierarchical clustering, eighteen patients were clustered into two distinct groups (FIG. 2 and 3). Using WGCNA, clusters of gene expression modules were found to show statistically significant correlation with the clinical phenotype (FIG. 4). The pathways of genes in these modules (Tables 1-4) are the biomarkers to be used for development of predictive molecular assays.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention. TABLES
Table 1. Gene expression module 1 listing genes involved in the following pathways: mesangial cell development, glomerular mesangial cell development, positive regulation of phagocytosis, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation, cell cycle, regulation of platelet activation, regulation of cohesion loading, pericyte cell differentiation, and regulation of c AMP-dependent protein kinase activity.
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Table 2, Gene expression module 2 listing genes involved in the following pathways: ER.BB3 signaling pathway, receptor metabolic process, positive regulation of ERBB signaling pathway, regulation of secretion, regulation of exocytosis, cardiac chamber development, cardiac ventricle morphogenesis, positive regulation of receptor recycling, protein mannosylation, and protein O- linked mannosylation.
Figure imgf000039_0002
Figure imgf000040_0001
Figure imgf000041_0001
Table 3. Gene expression module 3 listing genes involved in the following pathways: presynaptic active zone membrane, presynaptic active zone, negative regulation of cell population proliferation, positive regulation of mitochondrial translation, regulation of BMP signaling pathway, histone kinase activity, oogenesis, nuclear receptor activity, ligand-activated transcription factor activity, and regulation of T cell cytokine production. f
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Table 4. Gene expression module 1 listing genes involved in the following pathways: translational initiation, structural constituent of ribosome, protein folding, ribosome, SRP-dependent cotranslational protein targeting to membrane, cytoplasmic translation, large ribosomal subunit, protein folding chaperone, protein targeting, cotranslational protein targeting to membrane.
Figure imgf000052_0002
Figure imgf000053_0001
Figure imgf000054_0001
Table 5. Calculation of m6a site abundance
Figure imgf000054_0002

Claims

CLAIMS What is claimed is:
1. A method of identifying whether a subj ect has a progressive glioma or a pseudoprogressive glioma, the method comprising: a. isolating a tissue biopsy from the subject; b. detecting an altered N6-Methyladenosine (m6A) epigenetic modification on an mRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from at least one pathway selected from mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, regulation of c AMP- dependent protein kinase activity (PKA) pathway, epidermal growth factor receptor 3 (ERBB3) signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway, cardiac ventricle morphogenesis pathway, regulation of receptor recycling pathway, protein mannosylation pathway, protein O-linked mannosylation pathway, presynaptic active zone membrane pathway, presynaptic active zone pathway, regulation of cell population proliferation pathway, regulation of mitochondrial translation pathway, regulation of bone morphogenetic protein (BMP) signaling pathway, histone kinase activity pathway, oogenesis pathway, nuclear receptor activity pathway, ligand-activated transcription factor activity pathway, regulation of T cell cytokine production pathway, translational initiation pathway, structural constituent of ribosome pathway, protein folding pathway, ribosome pathway, signal-recognition particle (SRP)- dependent cotranslational protein targeting to membrane pathway, cytoplasmic translation pathway, large ribosomal subunit pathway, protein folding chaperone pathway, or protein targeting pathway; and c. diagnosing the subject with either the progressive glioma or the pseudoprogressive glioma.
2. A method of treating either a progressive glioma or a pseudoprogressive glioma in a subject, the method comprising: a. isolating a tissue biopsy from the subject; b. detecting an altered N6 -Methyladenosine (m6A) epigenetic modification on an mRNA from the tissue biopsy; wherein the mRNA is encoded by a gene from at least one pathway selected from mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, regulation of cAMP-dependent protein kinase activity (PKA) pathway, epidermal growth factor receptor 3 (ERBB3) signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway, cardiac ventricle morphogenesis pathway, regulation of receptor recycling pathway, protein mannosylation pathway, protein O-linked mannosylation pathway, presynaptic active zone membrane pathway, presynaptic active zone pathway, regulation of cell population proliferation pathway, regulation of mitochondrial translation pathway, regulation of bone morphogenetic protein (BMP) signaling pathway, histone kinase activity pathway, oogenesis pathway, nuclear receptor activity pathway, ligand-activated transcription factor activity pathway, regulation of T cell cytokine production pathway, translational initiation pathway, structural constituent of ribosome pathway, protein folding pathway, ribosome pathway, signal-recognition particle (SRP)-dependent cotranslational protein targeting to membrane pathway, cytoplasmic translation pathway, large ribosomal subunit pathway, protein folding chaperone pathway, or protein targeting pathway; c. diagnosing the subject with either the progressive glioma or the pseudoprogressive glioma; and d. treating the subj ect with a chemotherapy, an immunotherapy , or a palli ative therapy if diagnosed with the progressive glioma or treating with a steroid if diagnosed with the pseudoprogressive glioma.
3. The method of claim 1 or 2, wherein said method detects the altered m6A epigenetic modification in 5 or more genes from the at least one pathway.
4. The method of claim 1 or 2, wherein said method detects the altered m6A epigenetic modification in 10 or more genes from the at least one pathway.
5. The method of claim 1 or 2, wherein said method detects the altered m6A epigenetic modification in 50 or more genes from the at least one pathway.
6. The method of claim 1 or 2, wherein said method detects the altered m6A epigenetic modification in 100 or more genes from the at least one pathway.
7. The method of claim 1 or 2, wherein said method detects the altered m6A epigenetic modification in 250 or more genes from the at least one pathway.
8. The method of claim 1 or 2, wherein said method detects the altered m6A epigenetic modification in 500 or more genes from the at least one pathway.
9. The method of any one of claims 1-8, wherein the mesangial cell development pathway, glomerular mesangial cell development pathway, regulation of phagocytosis pathway, regulation of insulin receptor signaling pathway, regulation of fibroblast proliferation pathway, cell cycle pathway, regulation of platelet activation pathway, regulation of cohesion loading pathway, pericyte cell differentiation pathway, or regulation of cAMP-dependent PKA pathway comprises at least one gene selected from ABCD4, AHSA1, APBA2, ATP6AP1, ATXN7L3B, AUNIP, BACH1, BICD1, CABLES 1, CALR, CCAR2, CDCA5, CDCA8, CDK4, CDR2, CLCN7, CNNM4, DNMBP, DOH H , DXO, EEF2S3, ELF 1, EPAS1, EXOC7, FEM1A, FKBP1A, FOSB, FUNDCI, GCNT4, HMG20A, HR, HIT, C6ORF106, 1NTS11, JPT1, KIAA0930, KXD1, LETM 1, LOC100129034, LYAR, MEX3D, MIIP, MKRN1, MMADHC, MRPL54, MTRNR2L2, NLRX1, NOTCH ! . NRBF2, PAK4. PAXIP1 -AS1, PCDH17, PDGFB, PIGO, PPP2R1A, PRKAR1A, PRKCQ, PTK7, PTPN18, PTPN7, RAB39B, RABL6, RAC1, RAPGEF1, RAVER1, RBBP5, RND1, RNF14, RNF167, SEC23IP, SERTAD1, S1K2, SLC25A19, SLC43A2, SLC7A6, SNRNP25, SNTA1, SOCS3, SPIN4, STAG!, SYNDIG1, SYNGR2, TBC1D9, TP53, TRIM65, TSC22D1, TTC28, TTYH3, TUBB6, TUL.P3, UBE2L3, UBE3B, WAPL, ZBED3, ZBTB48, ZSWIMl, or LOC100507437.
10. The method of any one of claims 1-8, wherein the ERBB3 signaling pathway, receptor metabolic process pathway, regulation of ERBB signaling pathway, regulation of secretion pathway, regulation of exocytosis pathway, cardiac chamber development pathway, cardiac ventricle morphogenesis pathway, regulation of receptor recycling pathway, protein mannosylation pathway, or protein O-linked mannosylation pathway comprises at least one gene selected from ACTN4, AP2A2, ARHGEF25, ATP10A, BTN3A 1, BUB3, C14ORF93, C2ORF42, CAPZA1, CASP8, CDCA2, COL6A1, CPSF7, DDR1, DORE, DPM3, DSP, DUSP8, ECEI, EIF3G, ETV3, FAM83G, FBL, FUK, FTSJ3, GATA2, HIP1, INAFM2, JMJD4, JTB, KHNYN, KIF3B, KLFEDC10, KNOP1, LARP4, LRP12, MAP3K10, MEGF6, MPHOSPHIO, MRTFB, NADK, NCKAP5L, NCLN, NR2F2, NRGI, NSF, OGFOD3, OS9, PLEKHH3, PMVK, PNPO, P0MGNT2, QS0X2, RAB15, RAB1A, RHOC, RIN3, RNF43, RTN4, SALL1, SDF2, SOX4, SP140, SPTLC2, SRRM1, SYT17, TAB1, TBC1D22A, TBC1D8, TERF2, THRAP3, TIAL1, TOX, TPD52L2, TPRA1, TRABD, TRIP6, USP40, ZBTB26, ZBTB9, ZNF318, ZNF503, ZNF668, ZNF827, orZNF865.
11. The method of any one of claims 1-8, wherein the presynaptic active zone membrane pathway, presynaptic active zone pathway, regulation of cell population proliferation pathway, regulation of mitochondrial translation pathway, regulation of BMP signaling pathway, histone kinase activity pathway, oogenesis pathway, nuclear receptor activity pathway, ligand-activated transcription factor activity pathway, or regulation of I' cell cytokine production pathway comprises at least one gene selected from ABHD5, ACBD3, ACSL3, ACVR1B, ADI1, ADIPOR2, AFG3L2, AHSA2P, AK2, AKIRIN1, AKTIP, ALDH1B1, AMIG02, ANKRD50, ANKRD54, APOL6, APPL2, AR, ARHGAP26, ATP5F1A, ATP5IF1, AVL9, AZI2, B4GAL T2, BCL2, BICRAL, BIRC3, BRWD1, BTBD2, BTG1, BTN2A1, C16ORF72, C3ORF18, C3ORF38, C5ORFT5, C8ORF33, CACNB1, CCDC71L, CCNT2, CCT7, CDCA4, CDK2, CDKN1B, CDS2, CEACAM6, CEP78, CHST11, CHST12, CHTOP, CLN5, CLPB, CNOT6, COA3, COLEC12, COQ5, CRIME CTCF, CTR9, CWF19L1, DARS2, DCAF1, DCAF5, DCCN1D2, DDX19B, DDX20, DDX24, DDX42, DENND4A, DESI2, WDR92, DNAJA1, DNMT1, DTE, DYRK3, EBLN3P, EBP, ECPAS, EFL1, LU- 1. EIF2A, EIF4G1, ELOF1, EMC1, ENG, EOMES, EPC2, EPS15, ERCC6, FAM107B, FAM193A, FAM83D, FBXL5, FBXO30, FBXO34, FBXO5, FDFT1, FGL2, FNBP1L, FOXN2, FRMD8, FRS2, FSTL1, FZD8, GATA3, GFOD1, GJC1, GOLGA4, GOLPH3, GPATCH2, GPATCH2L, GVQW3, HASPIN, HAUS3, HAUS6, HDAC6, HIPK1, HKDC1, HLA-A, HNF4A, HNRNPUL2, HOTAIR, H0XB7, HOXC13, HTRAl, IDI1, IER5L, INIP, INO80C, INTS10, ITPRIP, IVD, KANSL3, KAT2A, KCNG1, KCTD3, KIAA0232, KLHL13, Kl.i n.15, KLHL24, KLHL9, LARP1B, LLPH, LNPK, LOC107985939, LRFN3, LRRN2, LYRAE, LYST, MAD2L1BP, MAGEFI, MAML3, MANI Al, MANBA, MAP3K7, MARF1, MARK1, MDM4, MDN1, MED13L, MIA3, MINPP1, MIS18BP1, MLLT3, M0B3B, MRPL17, MRPS26, MTCH1, MTDH, MTFR1, MTSS1L, MYD88, NCALD, NCR3LG1, NDUFV3, NECTIN1, NETO2, NGRN, FAM129A, NKX3-1, NNT-AS1, NOL9, NOP14-AS1, NOTCH2, NRID2, NR2C2, NSL1, NTN3, NXT1, ORC2, OTUB2, PARP14, PBRM1, PCMTDI, PCOLCE, PDGFRL, PDXP, PGP, PGR.MC2, PHB, PI4KB, PIK3R1, PLCL2, PLEKHM1, KDELC2, POLE, POLE3, PPARA, PPMIA, PPP1R9B, PPP2CB, PRR14L, PRSS23, PTGER4, PTGFRN, PTP4A2, PUS7, PVR, QSER1, RAB23, RABEPI, RAE1, RANBP3, RANBP6,
RBBP8, RBM12B, RBM25, RCOR1, RLF, RNASEH1, RNF212, RPIA, RPS6KA1, RPUSD4 RRP12, RSL1D1, SAAL1, SAR1A, SATB2, SEPHS1, SETD2, SF1, SFMBT1, SGMS2. SHQ1, SLC25A20, SLC35G1, SLC39A6, SLC41A1, SMAD6, SMARCA2, SMIM15, SMIM26, SNED1, S0CS5, S0S1, S0X12, SRBD1, ST3GAL2, STARD10, STX2, SYDE1 , SYMPK, SY.WL SYT1.2. TAF1, TBC1D10A, TBCC, TCOF1, THBSI, TIMM13, TLE3, TLR2, TMED8, TMEM131L, TMEM186, TMEM198B, TMEM44, TP73-AS1, TPCN2, TPM2, TRABD2A, TRAF2, TRANK 1, TRMT10C, TSPAN17, TTI1, TUT4, TUT7, TXNDCl l, UBAP1, UBAP2L, UBQLN2, USF3, USP54, VANGL1, VIRMA, WDR24, WDR6, WDR89, WSCD1 , YWHAE, ZBTB14, LINC01137, ZC3H12C, ZC3H18, ZDHHC8, ZFP91, ZNF107, ZNF184, ZNF189, ZNF202, ZNF419, ZNF514, ZNF518A, ZNF527, ZNF567, ZNF627, ZNF629, ZNF641, ZNF669, ZNF701, ZNF703, ZNF740, ZNF841, ZNF862, ZSCAN29, ZSWIM4, or ZYGI IB.
12. The method of any one of claims 1-8, wherein the translational initiation pathway, structural constituent of ribosome pathway, protein folding pathway, ribosome pathway, SRP- dependent cotranslational protein targeting to membrane pathway, cytoplasmic translation pathway, large ribosomal subunit pathway, protein folding chaperone pathway, or protein targeting pathway comprises at least one gene selected from ALMS1, ANTXR2, AUTS2, B3GNT10, KIAA1109, BOD1L1, C1ORF174, C5ORF24, C8ORF58, CRYBG1, CUL1, DIAPH1, EIF3H, EIF4E2, ELMO1, ETS1, FAM156B, FNDC3A, GGA2, HERC1, HMGN4, HSD17B12, HSP90AA1, HSPA1L, HSPA8, KIDINS220, LEMD2, LURAP1L, MAML2, MAPK4 , MARCH7, MARK3, MCAT, MFSD6, MPHOSPH8, MRPL12, MRPL41, MTMR1, MY09A, NCAPD3, NCK1, NT5DC3, PLK4, POLG2, PRPF38B, QRICH1, RBBP6, RGP1, RPL35A, RPL37A, RPLPO, RPP38, SI00A10, SFPQ, SLFN12L, SPART, SPOP, TPX2, TRAIP, TRIM2, UBALD1, USP15, USP9X, UTP3, ZDHHC16, ZKSCAN1, ZNF232, ZNF398, ZNF747, or ZZZ3.
13. The method of any one of claims 1-12, wherein the gene comprises the altered m6A epigeneti c mo dif ic atio n.
14. The method of any one of claims 1-13, wherein the progressive glioma comprises an increased alteration of m6A epigenetic modifications relative to the pseudoprogressive glioma.
15. The method of any one of claims 1-13, wherein the progressive glioma comprises a decreased alteration of m6A epigenetic modifications relative to the pseudoprogressive glioma.
16. The method of any one of claims 1-15, wherein the progressive glioma comprises a highgrade glioma.
17. The method of claim 16, wherein the high-grade glioma comprises a glioblastoma.
18. The method of any one of claims 2-17, wherein the chemotherapy is selected from an alkylating agent, an antimetabolite, an anthracycline, a topoi somerase inhibitor, or a corticosteroid.
19. The method of any one of claims 2-18, wherein the immunotherapy comprises a PD1 antibody therapy, a PD-L1 antibody therapy, or combinations thereof.
20. The method of any one of claims 1-19, wherein the pseudoprogressive glioma comprises an inflammatory response to a cancer treatment given after the tissue biopsy.
21. The method of claim 20, wherein the inflammatory response is treated with a steroid.
22. The method of claim 21, wherein the steroid is selected from prednisolone, methylprednisolone, dexamethasone, hydrocortisone, or combinations thereof.
23. The method of any one of claims 1 -22, wherein the tissue biopsy comprises a brain tissue.
24. The method of any one of claims 1-23, wherein the subject comprises a human.
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