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WO2024257013A1 - Biomarqueurs et méthodes de traitement avec des inhibiteurs de nae - Google Patents

Biomarqueurs et méthodes de traitement avec des inhibiteurs de nae Download PDF

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WO2024257013A1
WO2024257013A1 PCT/IB2024/055802 IB2024055802W WO2024257013A1 WO 2024257013 A1 WO2024257013 A1 WO 2024257013A1 IB 2024055802 W IB2024055802 W IB 2024055802W WO 2024257013 A1 WO2024257013 A1 WO 2024257013A1
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Prior art keywords
gene
marker
idh1
mutation
idh2
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Tammie YEH
Radha RAMESH
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Takeda Pharmaceutical Co Ltd
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Takeda Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Hematological cancers of the hematopoietic and lymphoid tissues have a variety of treatment options, such as chemotherapy, radiation therapy, immunotherapy and stem cell transplantation. Depending on the origin, location or severity of the cancer, the chemotherapy might comprise treatment with antimetabolites, antimitotics, alkylating agents, histone deacetylase inhibitors, hypomethylating agents, proteasome inhibitors, kinase inhibitors, immunomodulators, and/or other agents, such as new agents acting in recently studied pathways.
  • One set of recently studied pathways relates to E1 enzyme activity.
  • Ubiquitin and other ubiquitin-like molecules are activated by a specific enzyme (an E1 enzyme, an ATP-dependent activating enzyme) which catalyzes the formation of an acyl-adenylate intermediate with the C-terminal glycine of the ubl.
  • the activated ubl is then transferred to a - 1 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 catalytic cysteine residue within the E1 enzyme through formation of a thioester bond intermediate.
  • the ubl is then conjugated to the target protein, through isopeptide bond formation with the amino group of a lysine side chain in the target protein.
  • Inhibiting E1 enzymatic activity means reducing the ability of an E1 enzyme to activate ubiquitin like (ubl) conjugation to a substrate peptide or protein (e.g., ubiquitination, neddylation, sumoylation).
  • Neural precursor cell-Expressed Developmentally Downregulated 8 (NEDD8) is activated by the heterodimer NEDD8-activating enzyme (NAE, also known as APPBP1-UBA3, UBE1C (ubiquitin-activating enzyme E1C)) and is transferred to one of two E2 conjugating enzymes (ubiquitin carrier protein 12 (UBC12) and UBC17), ultimately resulting in ligation of NEDD8 to cullin proteins by the cullin-RING subtype of ubiquitin ligases.
  • a function of neddylation is the activation of cullin-based ubiquitin ligases involved in the turnover of many cell cycle and cell signaling proteins, including p27 and I- ⁇ B.
  • the present disclosure relates to methods and kits for treatment of cancer, e.g., a hematological cancer in patients, e.g., human patients, who are characterized by marker analysis for favorable outcome of treatment. Marker analysis can include measurement of the amount, presence or changes of markers provided herein.
  • the markers are predictive of whether there will be an improved outcome (e.g., improvement in good response, long time-to- progression, negative minimal residual disease, long progression-free survival and/or long term survival) after treatment with a regimen comprising a NEDD8-activating enzyme (NAE) inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof, compared with similar treatment lacking an NAE inhibitor.
  • NAE NEDD8-activating enzyme
  • Other benefits may include reduced side effects, a shorter course of treatment, or other benefits related to patient quality of life that may not - 2 - 56340669.1
  • Attorney Docket No.223266-549182 / PAT27156PCT01 directly reflect benefits in disease outcome.
  • a treatment regimen comprising pevonedistat treatment can further comprise treatment with one or more additional agent(s), such as a BCL2 inhibitor, such as venetoclax (ABT-199); and/or a hypomethylating agent, e.g., azacitidine or a pharmaceutically acceptable salt thereof.
  • a treatment regimen that incorporates administration to a hematological cancer patient with a combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199) and a hypomethylating agent to the patient reduces one or more symptoms of the hematological cancer, or delays or terminates the progression of the hematological cancer.
  • a biological sample e.g., a sample obtained from a patient, e.g., a sample comprising tumor cells or contents or products thereof, e.g., in vitro, to detect or measure the presence, amounts or changes of genetic or phenotypic markers, e.g., the mutational status of at least one marker gene, identifies particular patients whose outcomes are expected to benefit from including an NAE inhibitor in their treatment regimen.
  • test results identify those patients who are expected to gain additional benefit from including an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof, in the treatment. Conversely, the test results also identify those patients who are not expected to gain an additional benefit from including an NAE inhibitor in the treatment.
  • a particular treatment e.g., treatment with venetoclax (ABT-199) and a hypomethylating agent such as azacitidine or a pharmaceutically acceptable salt thereof
  • NAE inhibitor such as pevonedistat or a pharmaceutically acceptable salt thereof
  • the invention provides compositions, such as kits, or methods useful in detecting or measuring characteristics, e.g., amounts, presence or changes, of markers in a biological sample, e.g., a sample obtained from a cancer patient, e.g., a human patient. Such compositions and methods can determine the mutational status of marker genes described herein.
  • the invention provides disease, e.g., cancer, management strategies.
  • the characteristic e.g., size, sequence, composition or amount of marker, e.g., nucleic acid or protein, in a biological sample comprising tumor cells, e.g., hematological tumor cells, or contents or products thereof is measured.
  • the cancer is a hematological cancer, such as leukemia, lymphoma or myeloma.
  • the hematological cancer is acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) or chronic myelomonocytic leukemia (CMML).
  • AML acute myelogenous leukemia
  • MDS myelodysplastic syndrome
  • CMML chronic myelomonocytic leukemia
  • the MDS is high risk MDS.
  • the AML is low-blast acute AML.
  • the marker characteristic e.g., size, sequence, composition or amount of DNA, the size, sequence, composition or amount of RNA and/or the size, sequence, composition or amount of protein corresponding to a marker gene, either wild - 3 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 type or with one or more genotypic or phenotypic alteration, e.g., a polymorphism or mutation, e.g., somatic mutation, described herein is measured. In some embodiments, the measurement indicates that a gene is underexpressed or overexpressed. In some embodiments, the measurement indicates that a gene is amplified, deleted or translocated.
  • the measurement indicates that the gene is misexpressed or the altered gene product alters a signaling or functional pathway contributing to the cancer state.
  • Disease management strategy is undertaken when assay results reveal information about a marker gene or marker genes, e.g., whether a gene is altered, or not, the identity of the alteration, and/or whether the RNA or protein amount of an altered marker gene or marker genes indicates favorable outcome to therapy comprising NAE inhibition, e.g., pevonedistat therapy.
  • NAE inhibition e.g., pevonedistat therapy.
  • a therapeutic regimen which is likely to benefit a particular patient or type of patient, e.g., whether a particular regimen should be started or avoided, continued, discontinued or altered.
  • analyses can be made on a patient-by-patient basis, e.g., identifying and/or selecting for treatment a cancer patient who is expected to demonstrate a favorable outcome upon administration of a therapeutic regimen, e.g., a therapeutic regimen comprising an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof.
  • a marker gene useful for analysis in methods or kits described herein is a cancer marker gene, e.g., a marker gene selected from the group consisting of isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2).
  • IDH1 isocitrate dehydrogenase 1
  • IDH2 isocitrate dehydrogenase 2
  • a result with mutational status, such as wild type status or alterations in DNA or effects, e.g., on marker RNA and/or protein characteristics, e.g., amounts, size, sequence or composition provides a selection, treatment or disease management of cancer patients.
  • a gene or a mutant or genetically altered form thereof is useful, has a DNA, an RNA and/or protein characteristic, e.g., size, sequence, composition or amount, e.g., in a biological sample comprising tumor cells, e.g., hematological tumor cells, or contents or products thereof, if it is different than a normal DNA, RNA and/or protein.
  • a biological sample comprising tumor cells, e.g., hematological tumor cells, or contents or products thereof, if it is different than a normal DNA, RNA and/or protein.
  • markers genes whose analysis can provide such results.
  • therapeutic methods include the step of beginning, continuing, or commencing a therapy accordingly where the presence of an alteration in a marker gene or the characteristic, e.g., size, sequence, composition or amount of a patient’s marker or markers, e.g., in a first and/or subsequent biological sample, e.g., a sample obtained from the patient, e.g., comprising tumor cells, e.g., hematological tumor cells, or contents or products - 4 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 thereof, indicates that the patient is expected to have a favorable outcome with the therapy, e.g., the NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof therapeutic regimen.
  • the NAE inhibitor such as a pevonedistat or a pharmaceutically acceptable salt thereof therapeutic regimen.
  • the methods include therapeutic methods which further include the step of stopping, discontinuing, altering or halting a therapy accordingly where the presence of an alteration in a marker gene or the characteristic, e.g., size, sequence, composition or amount of a patient’s marker, e.g., in a second sample obtained from the patient, indicates that the patient is expected to demonstrate an unfavorable outcome with the treatment, e.g., with the NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof regimen, e.g., as compared to a patient identified as being expected to have a favorable outcome receiving the same therapeutic regimen.
  • the NAE inhibitor such as a pevonedistat or a pharmaceutically acceptable salt thereof regimen
  • methods for analysis and treatment of a patient not yet being treated with a therapy, e.g., an NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof therapy and identification and report of predicted outcome of the treatment based upon the presence of an alteration in a marker gene or characteristic, e.g., size, sequence, composition or amount of one or more of a patient’s marker described herein.
  • a therapy e.g., an NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof therapy
  • Such methods can include not being treated with the therapy, being treated with the therapy, being treated with the therapy in combination with one or more additional therapies, or being treated with a more aggressive dosing and/or administration regimen, e.g., a more frequent dosing regimen or higher dose with the NAE inhibitor therapy, as compared to the dosing and/or administration regimen of a patient identified as having a favorable outcome to standard treatment with an NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof, optionally in combination with one or more additional therapies.
  • an NAE inhibitor such as a pevonedistat or a pharmaceutically acceptable salt thereof
  • the provided methods of the invention can eliminate ineffective or inappropriate use of therapy, e.g., NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof therapy regimens.
  • Additional methods include methods to identify new therapeutic combinations. Such methods include methods to identify an agent, or more than one agent, as useful in combination with an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof, for treating a cancer, e.g., a hematological cancer (e.g., myeloma, leukemias, lymphoma, etc.), based on its ability to affect the presence of a genotypic or phenotypic alteration in a marker gene or characteristic, e.g., size, sequence, composition or amount of a marker or markers of the invention.
  • a cancer e.g., a hematological cancer (e.g., myeloma, leukemias, lymphoma, etc.), based on its ability to affect the presence of a genotypic or phenotypic alteration in a marker gene or characteristic, e.g., size, sequence, composition or amount of a marker or markers of the invention.
  • an agent or agents which in combination with an NAE inhibitor decreases or increases the presence of an alteration in a marker gene or characteristic, e.g., size, sequence, composition or amount of a marker or markers provided herein in a manner that indicates favorable outcome of a - 5 - 56340669.1
  • a marker gene or characteristic e.g., size, sequence, composition or amount of a marker or markers provided herein in a manner that indicates favorable outcome of a - 5 - 56340669.1
  • Attorney Docket No.223266-549182 / PAT27156PCT01 patient having a cancer, e.g., a hematological cancer would be a candidate agent for the combination.
  • an agent or agents which in combination with an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof, is able to decrease the viability of a tumor cell comprising a marker indicative of a favorable outcome would be a candidate agent for the combination.
  • the present invention is also directed to methods of treating a cancer patient, e.g., a human patient, with a therapeutic regimen, e.g., with an NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof therapy regimen (e.g., alone, or in combination with one or more additional agent(s) such as a chemotherapeutic agent, e.g., a proteasome inhibitor, an alkylating agent, a BCL2 inhibitor, a hypomethylating agent, an antibiotic or an antimetabolite), which includes the step of selecting for treatment a patient whose marker characteristic, e.g., size, sequence, composition or amount, indicates that the patient is expected to have a favorable outcome with the therapeutic regimen, and treating the patient with the therapy, e.g., NAE inhibition, by an inhibitor such as pevonedistat or a pharmaceutically acceptable salt thereof therapy, with or without one or more additional agent(s).
  • an NAE inhibitor such as a pevonedistat or a
  • the method can include the step of administering therapy to a patient whose cancer is characterized by marker characteristic, e.g., size, sequence, composition or amount or amounts indicative of a favorable outcome.
  • marker characteristic e.g., size, sequence, composition or amount or amounts indicative of a favorable outcome.
  • a favorable outcome in the methods includes the use of pevonedistat or a pharmaceutically acceptable salt thereof, in combination with one or more additional agent(s), for treating a patient characterized as having tumor cells, e.g., hematological tumor cells, wherein IDH1 gene and/or IDH2 gene has a genetic alteration.
  • a favorable outcome in the methods include the use of pevonedistat or a pharmaceutically acceptable salt thereof, in combination with one or more additional agent(s), for treating a patient characterized as having tumor cells, e.g., hematological tumor cells, wherein has a genetic alteration.
  • a favorable outcome in the methods further includes the use of a BCL2 inhibitor, e.g. venetoclax (ABT-199) and/or a hypomethylating agent, e.g. azacitidine.
  • FIG. 1 A bar graph is depicted representing the statistical significance of response rate (CR/CRi) of PEV+VEN+AZA versus VEN+AZA treatment in AML patients with - 6 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 IDH1 mutated, IDH1/IDH2 mutated, IDH2 mutated, and wild-type IDH1/2 genes , in clinical trial NCT04266795. [0018] Figure 2A.
  • FIG. 1 Kaplan-Meier curves for patients with EFS data, showing the statistical significance between PEV+VEN+AZA versus VEN+AZA in patients with mutated IDH1 or IDH2 in accordance with clinical trial NCT04266795.
  • Figure 2B Kaplan-Meier curves for patients with EFS data, showing no statistical significance between PEV+VEN+AZA versus VEN+AZA in patients with no mutated IDH1 nor IDH2 in accordance with clinical trial NCT04266795.
  • Figure 3 represents a bar chart illustrating 11 genes, from subjects who were response evaluable and had at least one mutation during the phase II study, that were considered for further analysis due to prevalence or interest.
  • cancer patients e.g., hematological cancer patients
  • particular cancer therapies e.g., hematological cancer patients
  • cancer patients who are expected to have a favorable outcome when administered particular cancer therapies as well as particular cancer patients who may have a favorable outcome using more aggressive and/or alternative cancer therapies, e.g., alternative to previous cancer therapies administered to the patient.
  • cancer patients including, e.g., hematological cancer patients (e.g., patients suffering from leukemia, lymphoma, or myeloma) who would benefit from particular cancer treatment therapies as well as those who would benefit from a more aggressive and/or alternative cancer treatment therapy, e.g., alternative to a cancer therapy or therapies the patient has received, thus resulting in appropriate preventative and/or therapeutic measures.
  • hematological cancer patients e.g., patients suffering from leukemia, lymphoma, or myeloma
  • cancer treatment therapies e.g., a more aggressive and/or alternative cancer treatment therapy, e.g., alternative to a cancer therapy or therapies the patient has received, thus resulting in appropriate preventative and/or therapeutic measures.
  • the present invention is based, in part, on the mutational status of a marker gene in samples from cancer patients, e.g.
  • the regimen comprising an NAE inhibitor further comprises a hypomethylating agent, e.g., azacitidine in combination with pevonedistat or a pharmaceutically acceptable salt thereof.
  • the regimen comprising an NAE inhibitor further comprises a BCL2 inhibitor, e.g., venetoclax (ABT-199), and/or a hypomethylating agent, e.g., azacitidine, in combination with pevonedistat or a pharmaceutically acceptable salt thereof.
  • the marker gene e.g., the hematological cancer marker gene, encodes a metabolic enzyme, e.g., is a gene whose encoded protein is an enzyme involved in a cellular metabolic pathway. Examples of cancer marker genes include IDH1 and IDH2.
  • marker genes are described in WO 2019/109016 (e.g., TET2, RUNX1, NRAS, KRAS, DNMT3A, TP53, IDH2, EZH2, IDH1, NPM1, and PHF6), which is incorporated by reference.
  • Such marker genes can be found with genetic alterations, e.g., mutations or truncations, in hematological cancers, such as AML, MDS, or CMML.
  • at least one marker gene e.g., a hematological cancer marker gene, selected from the group consisting of IDH1 and IDH2 has a genetic alteration.
  • Marker genes can be wild type or exhibit genotypic or phenotypic alteration, e.g., a copy number change, a polymorphism or mutation, e.g., somatic mutation, whose presence can affect expression or activity of the encoded gene product.
  • there can be marker gene alterations in cells which have alterations in additional genes, including alterations that can lead to tumorigenesis, but the additional altered genes may not be marker genes as considered herein.
  • the alteration is an inactivating mutation. In some embodiments, the alteration is an activating mutation. In other embodiments, the alteration affects the expression of the marker gene. In other embodiments, a genotypic alteration can result in an altered interaction of the encoded gene product with a cellular binding partner. [0024] In some embodiments, alterations in a marker gene indicates a favorable outcome to treatment with a regimen comprising pevonedistat or a pharmaceutically acceptable salt thereof. In one marker gene alteration embodiment, IDH1 is altered in a sample from a patient, e.g., a human patient.
  • a patient with an alteration in IDH1 may respond more favorably to a therapeutic regimen comprising an NAE inhibitor, e.g., pevonedistat or a pharmaceutically acceptable salt thereof, in combination with a BCL2 inhibitor, such as venetoclax (ABT-199), and/or a hypomethylating agent, e.g. azacitidine.
  • alterations in a marker gene indicates a favorable outcome to treatment with a regimen comprising pevonedistat or a pharmaceutically acceptable - 8 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 salt thereof.
  • IDH2 is altered in a sample from a patient, e.g., a human patient.
  • a patient with an alteration in IDH2 may respond more favorably to a therapeutic regimen comprising an NAE inhibitor, e.g., pevonedistat or a pharmaceutically acceptable salt thereof, in combination with a BCL2 inhibitor, such as venetoclax (ABT-199), and/or a hypomethylating agent, e.g. azacitidine.
  • alterations in a combination of marker genes indicates a favorable outcome to treatment with a regimen comprising pevonedistat or a pharmaceutically acceptable salt thereof.
  • both IDH1 and IDH2 are altered in a sample from a patient, e.g., a human patient.
  • a patient with an alteration in both IDH1 and IDH2 may respond more favorably to a therapeutic regimen comprising pevonedistat or a pharmaceutically acceptable salt, such as pevonedistat or a pharmaceutically acceptable salt thereof, in combination with a BCL2 inhibitor, such as venetoclax (ABT-199), and/or a hypomethylating agent, e.g. azacitidine.
  • a favorable outcome to treatment with a regimen comprising pevonedistat or a pharmaceutically acceptable salt thereof in combination with one or more additional agent(s) is indicated from an altered marker gene combination where IDH1 is altered and IDH2 is wild type.
  • a favorable outcome to treatment with a regimen comprising pevonedistat or a pharmaceutically acceptable salt thereof in combination with one or more additional agent(s) is indicated from an altered marker gene combination where IDH2 is altered and IDH1 is wild type.
  • the mutational status result e.g., identification, detection and/or measurement of the wild type gene or a mutation in a marker gene can be used to determine whether a favorable outcome can be expected by treatment of a hematological cancer, e.g., with a regimen comprising an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof, or whether an alternative therapy to and/or a more aggressive therapy with, e.g., an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof may enhance expected survival time.
  • an NAE inhibitor such as pevonedistat or a pharmaceutically acceptable salt thereof
  • compositions and methods provided herein can be used to determine whether a patient is expected to have a favorable outcome to treatment with an NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof therapeutic agent or to an NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof dosing or administration regimen.
  • an NAE inhibitor such as a pevonedistat or a pharmaceutically acceptable salt thereof therapeutic agent
  • an NAE inhibitor such as a pevonedistat or a pharmaceutically acceptable salt thereof dosing or administration regimen.
  • mutation in marker genes described herein is associated with sensitivity to or favorable outcome of treatment with a regimen comprising an NAE inhibitor.
  • marker genes which can function as a cancer marker gene e.g.
  • Described herein are, without limitation: 1) methods and compositions for determining whether a treatment regimen comprising an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof will or will not be effective to achieve a favorable outcome and/or manage the cancer; 2) methods and compositions for monitoring the effectiveness of an NAE inhibitor, such as a pevonedistat or a pharmaceutically acceptable salt thereof therapy (alone or in a combination of agents) and dosing and administrations used for the treatment of cancer e.g., a hematological cancer; 3) methods and compositions for treatment of cancer e.g., a hematological cancer comprising administering an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof; 4) methods and compositions for identifying specific combinations of therapeutic agents as well as dosing and administration regimens that are effective for the treatment of cancer, e.g., a hematological cancer in specific patients; and 5) methods and compositions for treatment of cancer, e.
  • NAE inhibitor refers to an inhibitor of the NEDD8-activating enzyme (NAE) heterodimer.
  • NAE inhibitors include pevonedistat (formerly known as MLN4924 and TAK-924) or a pharmaceutically acceptable salt thereof.
  • NAE inhibitors do not inhibit, or are very poor at inhibiting, other (non- NAE) E1 enzymes.
  • the compounds are useful for inhibiting NAE activity in vitro and in vivo and are useful for the treatment of disorders of cell proliferation, e.g., cancer, and other disorders associated with NAE activity, such as pathogenic infections and neurodegenerative disorders.
  • disorders of cell proliferation e.g., cancer
  • disorders associated with NAE activity such as pathogenic infections and neurodegenerative disorders.
  • One class of compounds described in Langston et al. are 4–substituted ((1S, 2S, 4R)-2-hydroxy- 4- ⁇ 7H-pyrrolo[2,3-d]pyrimidin-7-yl ⁇ cyclopentyl)methyl sulfamates.
  • Pevonedistat ((1S,2S,4R)-4- ⁇ 4-(1S)-2,3-dihydro-1H-inden-1-ylamino-7H- pyrrolo2,3-dpyrimidin-7-yl ⁇ -2-hydroxycyclopentyl)methyl sulfamate) is an NAE-specific E1 inhibitor which disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumor cells by perturbation of cellular protein homeostasis (Soucy et al. (2009) Nature 458:732-736).
  • pevonedistat (MLN4924) in cellular and tumor xenograft studies has revealed two distinct mechanisms of action.
  • the first is the induction of DNA re- replication, DNA damage and cell death through MLN4924-mediated dysregulation of the - 10 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 CRL1 SKP2 and CRL4 DDB1 substrate Cdt-1 (Milhollen et al. (2011) Cancer Res.71:3042-3051). It has been shown that p53 status does not impact the induction of DNA re-replication but may make cells more prone to undergo apoptosis or senescence depending on the appropriate genetic background (Milhollen et al. (2011) supra, Lin et al. (2010) Nature 464:374-379 and Lin et al.
  • the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.
  • Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • the pharmaceutically acceptable salt is a hydrochloride salt form.
  • - 11 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01
  • hypomethylating agent refers to compounds which inhibit DNA methyltransferase. Examples of hypomethylating agents include azacitidine (also known as azacytidine) and decitabine.
  • BCL2 inhibitor refers to compounds which inhibit BCL2. Examples of BCL2 inhibitors include venetoclax (ABT-199) and navitoclax.
  • “combination” therapy refers to simultaneous, separate or sequential administration of two or more therapies (e.g., two or more different medications).
  • “combination” refers to simultaneous administration of two or more different medications, where the two or more different medications are present in a single dosage form.
  • “combination” refers to separate administration of two or more different medications, where the two or more different medications are found in different dosage forms (e.g., one medication in an oral tablet form and another in, e.g., a different oral tablet form, an oral capsule form or an injectable form).
  • “combination” refers to sequential administration of two or more different medications, where the two or more different medications are given at different points in time.
  • a “marker gene” refers to a gene which can have an alteration such that its marker nucleic acid (e.g., DNA or RNA) and/or its marker protein has a characteristic, e.g., size, sequence, composition or amount(s) which, alone or in combination with one or more marker genes, indicate outcome or prognosis upon treatment.
  • Marker genes described herein as linked to outcome after NAE inhibitor treatment are examples of genes within chromosome locus markers and are provided in Table 1 and further described in subsequent paragraphs.
  • a marker gene listed in Table 1 can have isoforms which are either ubiquitous or have restricted expression.
  • Wild-Type IDH1 gene encoded protein from human cells has the following primary amino acid sequence: MSKKISGGSV VEMQGDEMTR IIWELIKEKL IFPYVELDLH SYDLGIENRD ATNDQVTKDA AEAIKKHNVG VKCATITPDE KRVEEFKLKQ MWKSPNGTIR NILGGTVFRE AIICKNIPRL VSGWVKPIII GRHAYGDQYR ATDFVVPGPG KVEITYTPSD GTQKVTYLVH NFEEGGGVAM GMYNQDKSIE DFAHSSFQMA LSKGWPLYLS TKNTILKKYD GRFKDIFQEI YDKQYKSQFE AQKIWYEHRL IDDMVAQAMK SEGGFIWACK NYDGDVQSDS VAQGYGSLGM MTSVLVCPDG KTVEAEAAHG TVTRHYRMYQ KGQETSTNPI ASIFAWTRGL AHRAKLDNNK ELAFFANALE EVSI
  • Wild-Type IDH2 gene encoded protein from human cells has the following primary amino acid sequence: MAGYLRVVRS LCRASGSRPA WAPAALTAPT SQEQPRRHYA DKRIKVAKPV VEMDGDEMTR IIWQFIKEKL ILPHVDIQLK YFDLGLPNRD QTDDQVTIDS ALATQKYSVA VKCATITPDE ARVEEFKLKK MWKSPNGTIR NILGGTVFRE PIICKNIPRL VPGWTKPITI GRHAHGDQYK ATDFVADRAG TFKMVFTPKD GSGVKEWEVY NFPAGGVGMG MYNTDESISG FAHSCFQYAI QKKWPLYMST KNTILKAYDG RFKDIFQEIF DKHYKTDFDK NKIWYEHRLI DDMVAQVLKS SGGF
  • the mutation in IDH1 is a mutation of arginine 132 to cysteine, glycine, histidine, or serine (R132C, R132G, R132H, or R132S).
  • the mutation is a substitution in a codon of the IDH1 gene and the mutant codon is nucleotides 689-691 of SEQ ID NO:3 or 394-396 of SEQ ID NO:4.
  • the mutant nucleotide is base 689 and/or 690 of SEQ ID NO:3 or 394 and/or 395 of SEQ ID NO:4.
  • the mutation in IDH2 is a mutation of leucine 86 to proline (L86P).
  • the mutation is a substitution in a codon of the IDH2 gene and the mutant codon is nucleotides 420-422 of SEQ ID NO:5 or 256-258 of SEQ ID NO:6.
  • the mutant nucleotide is base 421 of SEQ ID NO:5 or 257 of SEQ ID NO:6.
  • the mutation in IDH2 is a mutation of glutamine 316 to leucine (Q316L).
  • the mutation is a substitution in a codon of the IDH2 gene and the mutant codon is nucleotides 1110-1112 of SEQ ID NO:5 or 946-948 of SEQ ID NO:6. - 15 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01
  • the mutant nucleotide is base 1111 of SEQ ID NO:5 or 947 of SEQ ID NO:6.
  • the mutation in IDH2 is a mutation of arginine 140 to glycine, or glutamine (R140G or R140Q).
  • the mutation is a substitution in a codon of the IDH2 gene and the mutant codon is nucleotides 582-584 of SEQ ID NO:5 or 418-420 of SEQ ID NO:6.
  • the mutant nucleotide is base 582, or 583 of SEQ ID NO:5 or 418, or 419 of SEQ ID NO:6.
  • the mutation in IDH2 is a mutation of arginine 172 to lysine, (R172K).
  • the mutation is a substitution in a codon of the IDH2 gene and the mutant codon is nucleotides 678-680 of SEQ ID NO:5 or 514-516 of SEQ ID NO:6.
  • the mutant nucleotide is base 679 of SEQ ID NO:5 or 515 of SEQ ID NO:6.
  • the mutation in IDH2 is a deletion of leucine 143, glycine 144, glycine 145 and threonine 146.
  • the mutation is an in-frame deletion of codons of the IDH2 gene, deletion of nucleotides 591-602 of SEQ ID NO:5 or 427-438 of SEQ ID NO:6.
  • the amino acid sequence comprises a new NIVF motif (SEQ ID NO:17, residues 141, 142, 147, and 148 of SEQ ID NO:2) resulting from the deletion of amino acids 143-146 of SEQ ID NO:2.
  • Table 2 Exemplary codon mutations leading to IDH1 and IDH2 mutations used in embodiments of the disclosure.
  • a gene is defined as “genetically altered” or “mutated” “mutant” or as having a “genetic alteration” or a “mutation” if a change from wild type, e.g., a cancer-driving alteration, e.g., not normal allelic variation, is detected by sequencing a nucleic acid marker corresponding to a marker gene.
  • the sequencing method is Next Generation Sequencing (NGS).
  • a “truncating mutation” as used herein refers to a sequence change selected from the group consisting of a frameshift insertion, a frameshift deletion, an in- frame deletion, a nonsense mutation, and a splice site mutation in a codon such that the marker nucleic acid encodes, e.g., its expression results in, a shortened, sometimes nonfunctional, altered version of a protein corresponding to the marker gene.
  • a “single amino acid change” as used herein in the context of cancer results from a substituted nucleotide in a codon that encodes, e.g., its expression results in, a non-reference, cancer driving amino acid in an altered version of a protein corresponding to the marker gene.
  • IDH1 or “isocitrate dehydrogenase (NADP(+)) 1, cytosolic” refers to Gene ID 3417, the gene corresponding to at least three expressed mRNA variants, one of which is the mRNA described in GenBank Accession No. NM_005896, encoding GenPept Accession No. NP_005887.
  • the open reading frame of the IDH1 gene is nucleotides 296 to 1540 of SEQ ID NO: 3.
  • Other names for IDH1 include epididymis luminal protein-216 and oxalosuccinate decarboxylase.
  • IDH1 catalyzes the oxidative decarboxylation of isocitrate to 2- oxoglutarate in the cytoplasm and peroxisome and can play a role in NADPH production.
  • the IDH1 gene can be found on chromosome 2 (2q34).
  • IDH2 or “isocitrate dehydrogenase (NADP(+)) 2” refers to Gene ID 3418, the gene corresponding to at least three expressed mRNA variants, one of which is the mRNA described in GenBank Accession No. NM_002168, encoding GenPept Accession No. NP_002159.
  • IDH2 The open reading frame of the IDH2 gene is nucleotides 165 to 1523 of SEQ ID NO: 5.
  • Other names for IDH2 include isocitrate dehydrogenase-mitochondrial (ICD-M). IDH2 catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate and can play a role in metabolism. The IDH2 gene can be found on chromosome 15 (15q26). [0054] Mutations in IDH1 and IDH2 can permit tumors to utilize alternative energy pathways. For example, the structures of IDH1 and IDH2 have arginines (R100 and R132 for IDH1 and R140 and R172 for IDH2) in their active sites.
  • Mutations of, or near, these arginines can confer the ability of these enzymes to catalyze the reverse reaction and generate R(-)-2- hydroxyglutarate in tumors.
  • non- limiting examples of mutations found in AML tumor samples include IDH1 R132L and IDH2 R140L and R140W. - 17 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 [0055] There has been interest in public cataloging alterations associated with cancers.
  • Examples of public databases which include information about alterations associated with cancers are the Database of Genotypes and Phenotypes (dbGaP) maintained by the National Center for Biotechnology Information (Bethesda, MD) and Catalogue of Somatic Mutations in Cancer (COSMIC) database maintained by the Wellcome Trust Sanger Institute (Cambridge, UK).
  • dbGaP Database of Genotypes and Phenotypes
  • COSMIC Catalogue of Somatic Mutations in Cancer
  • markers and marker genes described herein were identified based on genetic profiles of samples from patients in clinical trials, such as a trial identified on the clinical trials website maintained by the U.S. National Library of Medicine, NCT04266795.
  • the patients in this trial were suffering from acute myelogenous leukemia (AML).
  • AML acute myelogenous leukemia
  • MDS myelodysplastic syndrome
  • CMML chronic myelomonocytic leukemia
  • GenBank or GenPept accession numbers and useful nucleic acid and peptide sequences can be found at the website maintained by the National Center for Biotechnology Information, Bethesda, MD. Ensemble Annotation Transcripts are from the GRCH37 release 94 available at the website maintained by the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI).
  • EBL-EBI European Molecular Biology Laboratory-European Bioinformatics Institute
  • a “favorable” outcome or prognosis refers to complete remission (CR), incomplete remission (CRi), long term survival, long progression-free survival (PFS), long time-to-progression (TTP), a negative minimal residual disease (MRD), e.g., at a 10- 5 threshold, and/or good response.
  • an “unfavorable” prognosis refers to short term survival, short time-to-progression (TTP), short progression-free survival, a positive minimal residual disease and/or poor response.
  • a “marker” as used herein includes a material corresponding to a marker gene whose mutational status has been identified in a biological sample, e.g., tumor cells or contents or products thereof of a patient and furthermore that status is characteristic of a patient whose outcome is favorable or unfavorable with treatment e.g., treatment comprising an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof.
  • a marker examples include a material, e.g., marker nucleic acid or marker protein, e.g., a chromosome locus, DNA for a gene, RNA for a gene or protein for or corresponding to a gene. Outcome can be determined using each single marker individually as a marker; or alternatively can include one or more, or all of the characteristics collectively when reference is made to “markers” or “marker sets.” Marker sets can be combinations of chromosome locus, DNA, RNA or protein from more than one marker gene, combinations of chromosome locus, DNA, RNA or protein from a single gene, - 19 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 or any combination of the foregoing.
  • a marker DNA, marker RNA or marker protein can correspond to base pairs on a chromosome locus marker.
  • a marker DNA can include genomic DNA from a chromosome locus marker
  • marker RNA can include a polynucleotide transcribed from a locus marker
  • a marker protein can include a polypeptide resulting from expression at a chromosome locus marker in a biological sample, e.g., comprising tumor cells or contents or products thereof.
  • a “marker nucleic acid” is a nucleic acid (e.g., genomic DNA, RNA, cDNA) encoded by or corresponding to a marker gene of the invention.
  • marker nucleic acids include DNA, e.g., sense and anti-sense strands of genomic DNA (e.g., including any introns occurring therein), comprising the entire or a partial sequence, e.g., one or more of the exons of the genomic DNA, up to and including the open reading frame of any of the marker genes or the complement of such a sequence.
  • the marker nucleic acids also include RNA comprising the entire or a partial sequence of any marker or the complement of such a sequence, wherein all thymidine residues are replaced with uridine residues, mRNA generated by transcription of genomic DNA (i.e. prior to splicing), and mRNA generated by splicing of RNA transcribed from genomic DNA.
  • a “marker nucleic acid” may also include a cDNA made by reverse transcription of an RNA generated by transcription of genomic DNA (including spliced RNA).
  • a marker nucleic acid also includes sequences which differ from the wild type nucleotide sequence, e.g., as listed in Table 1, due to degeneracy of the genetic code, and encode wild type protein or protein whose sequence alteration does not affect the healthy state of the subject, e.g., is not a cancer-associated change. It will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation.
  • allelic variant refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence. Such naturally occurring allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene.
  • Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals, e.g., in cells, e.g., germline cells, of individuals without cancer. Such changes are compiled in the ExAc database and can be readily identified by using hybridization probes to identify the same genetic locus in a variety of individuals.
  • a “marker protein” is a protein encoded by a marker nucleic acid or corresponding to a marker, e.g., a mutant nucleic acid, of the invention.
  • a marker protein can be generated by translation of mRNA, e.g., mature or spliced RNA, and includes proteins both before and after cleavage of normally cleaved regions such as transmembrane signal sequences and pro-sequences.
  • mRNA e.g., mature or spliced RNA
  • proteins both before and after cleavage of normally cleaved regions such as transmembrane signal sequences and pro-sequences.
  • protein and “polypeptide” are used interchangeably.
  • a protein marker specifically can be referred to by its name or amino acid sequence, e.g., SEQ ID NO:1 or SEQ ID NO:2.
  • a “characteristic” of a marker includes a size, sequence, composition or amount whose value or difference is correlated with prognosis or outcome.
  • the characteristic, e.g., size, sequence, composition or amount of a marker can be obtained by analyzing either nucleic acid, e.g., DNA or RNA, or protein corresponding to the marker gene.
  • a characteristic size of a marker is length or molecular weight.
  • a characteristic sequence of a marker is a nucleic acid sequence or protein sequence.
  • a characteristic composition of a marker is nucleotide base or amino acid composition or peptide digest or gene fragment pattern.
  • a characteristic amount of a marker is copy number and/or expression level.
  • a characteristic of a marker e.g., in a sample from a patient, can indicate outcome of treatment if it is different than the characteristic of the wild type or allelic variant of the marker gene. In some embodiments, a characteristic of a marker can indicate outcome if it is wild type.
  • an amount can indicate outcome if it is greater than or less than a reference amount by a degree greater than the standard error of the assay employed to assess expression.
  • the relative expression level of a marker can be determined upon statistical correlation of the measured expression level and the outcome, e.g., response, time-to-progression, progression-free survival, minimal residual disease or overall survival. The result of the statistical analysis can establish a threshold for selecting markers or marker sets to use in the methods described herein.
  • a marker e.g., a chromosome locus marker, or a marker gene that has differential characteristic, e.g., size, sequence, composition or amount will have typical ranges that are predictive of outcome, depending on whether the characteristic, e.g., size, sequence, composition or amount falls within the range determined for the outcome.
  • a set of markers may indicate outcome if the - 21 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 combination of their characteristics, e.g., sizes, sequences, compositions or amounts either meets or is above or below a pre-determined score as determined by methods provided herein.
  • Genetic alterations including, but not limited to, gene translocation, transcript splice variation, deletion and truncation are examples of alterations which can change marker size, sequence or composition, in addition to point mutations, for example, substitutions, which can change marker sequence or composition.
  • Measurement of only one characteristic of a marker gene e.g., of a marker nucleic acid (i.e., DNA, RNA) or protein can provide a prognosis, i.e., indicate likely outcome.
  • Measurement of more than one characteristic of a marker gene can provide a prognosis, i.e., indicate outcome when the amounts of the two characteristics are consistent with each other, e.g., the biologies of the results are not contradictory.
  • Examples of consistent results from measurement of multiple characteristics of a marker gene can be identification of a nonsense alteration in a DNA or RNA and a low amount or low molecular weight of encoded protein, or an alteration in a region which encodes a binding pocket or active site of a protein and low activity of the encoded protein.
  • a different example can occur when a protein is in a pathway with a feedback loop controlling its synthesis based on its activity level.
  • a low amount or activity of protein can be associated with a high amount of its altered mRNA as a tissue, due to the marker gene alteration, thus is starved for the protein activity and repeatedly signals the production of the protein.
  • gene deletion refers to an amount of DNA copy number less than 2 and “amplification” refers to an amount of DNA copy number greater than 2.
  • a “diploid” amount refers to a copy number equal to 2.
  • the term “diploid or amplification” can be interpreted as “not deletion” of a gene copy.
  • the term “diploid or deletion” can be interpreted as “not amplification” of copy number.
  • sequence deletion can occur within a gene as a result of marker gene alteration and can result in absence of transcribed protein or a shortened mRNA or protein. Such a deletion may not affect copy number.
  • long term survival refers to the length of time after receiving a first dose of treatment that a cancer patient is predicted to live.
  • a “long term survivor” refers to a patient expected have a slower rate of progression or later death from the tumor than those patients identified as short term survivors.
  • Enhanced survival or “a slower rate of death” are estimated life span determinations based upon characteristic, e.g., size, sequence, composition or amount of one or more of marker genes described herein, e.g., as compared to a reference standard such that 70%, 80%, 90% or more of the population will be alive a sufficient time period after receiving a first dose of treatment.
  • a “faster rate of death” or “shorter survival time” refer to estimated life span - 22 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 determinations based upon characteristic, e.g., size, sequence, composition or amount of one or more of marker genes described herein, e.g., as compared to a reference standard such that 50%, 40%, 30%, 20%, 10% or fewer of the population will not live a sufficient time period after receiving a first dose of treatment.
  • the sufficient time period is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, 12, 18, 24 or 30 months, or 3 years, 4 years or 5 years as measured from the first day of receiving a cancer therapy.
  • a cancer is “responsive” to a therapeutic agent, or combination of therapeutic agents, or there is a “good response” to a therapeutic regimen if its rate of growth is inhibited as a result of contact with a therapeutic agent, or combination of therapeutic agents compared to its growth in the absence of contact with the therapeutic agent, or combination of therapeutic agents, or one or more symptoms of the cancer are ameliorated.
  • Growth of a cancer can be measured in a variety of ways, for example, the size of a tumor or the expression of tumor markers appropriate for that tumor type may be measured.
  • International Working Groups convene periodically to set, update and publish disease and response criteria for various types of cancers. Such published reports can be followed to support the identification of markers of the subject tumors and their response to NAE inhibitors.
  • AML Acute Myelogenous Leukemia
  • CML chronic myelomonocytic leukemia
  • CML Swerdlow et al. eds. (2008) in WHO Classification of Tumours of Haemotopoietic and Lymphoid Tissues (4th edition, IARC Press
  • lymphomas e.g., non-Hodgkin’s and Hodgkin’s lymphoma (Cheson et al. (2007) J.Clin. Oncol.25:579-596).
  • the percentage of blasts in the bone marrow or the presence of blasts with Auer rods can be a measure of response to treatment. Criteria can take into account analysis methods such as Positron Emission Tomography (PET), e.g., for identifying sites with measurable altered metabolic activity (e.g., at tumor sites) or to trace specific markers into tumors in vivo, immunohistochemistry, e.g., to identify tumor cells by detecting binding of antibodies to specific tumor markers, and flow cytometry, e.g., to characterize cell types by differential markers and fluorescent stains, in addition to traditional methods such as histology to identify cell composition (e.g., blast counts in a blood smear or a bone marrow biopsy, presence and number of mitotic figures) or tissue structure (e.g., disordered tissue architecture or cell infiltration of basement membrane).
  • PET Positron Emission Tomography
  • tissue structure e.g., disordered tissue architecture or cell infiltration of basement membrane
  • the quality of being responsive to a therapy comprising an NAE inhibitor, such as pevonedistat or a pharmaceutically acceptable salt thereof can be a variable one, with different cancers exhibiting different levels of “responsiveness” to a given therapeutic agent, under different conditions. Still further, measures - 23 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 of responsiveness can be assessed using additional criteria beyond growth size of a tumor, including, but not limited to, patient quality of life, degree of metastases.
  • a cancer is “non-responsive” or has a “poor response” to a therapeutic agent or therapeutic regimen if its rate of growth is not inhibited, or inhibited to a very low degree, as a result of contact with the therapeutic agent when compared to its growth in the absence of contact with the therapeutic agent.
  • growth of a cancer can be measured in a variety of ways, for instance, the size of a tumor or the expression of tumor markers appropriate for that tumor type may be measured.
  • the response definitions used to support the identification of markers associated with non- response of tumors to therapeutic agents, guidelines such as those described above can be used.
  • the quality of being non-responsive to a therapeutic agent can be a highly variable one, with different cancers exhibiting different levels of “non-responsiveness” to a given therapeutic agent, under different conditions. Still further, measures of non-responsiveness can be assessed using additional criteria beyond growth size of a tumor, including, but not limited to, patient quality of life, degree of metastases. [0070] As used herein, “long time-to-progression, “long TTP” and “short time-to- progression,” “short TTP” refer to the amount of time until when the stable disease brought by treatment converts into an active disease.
  • a treatment results in stable disease which is neither a good nor a poor response, e.g., MR, the disease merely does not get worse, e.g., become a progressive disease, for a period of time.
  • This period of time can be at least 4-8 weeks, at least 3-6 months or more than 6 months.
  • progression free survival or “PFS” refers to the time elapsed between treatment initiation and tumor progression or death from any cause.
  • complete response or “CR” refers to the fraction of patients in a treated population who experience a complete response following treatment.
  • CRi complete response with incomplete bone marrow recovery
  • CRi complete response with incomplete bone marrow recovery following treatment.
  • minimal residual disease or “MRD” refers to the result of an assay to detect residual malignant cancer or tumor cells in a patient, e.g., after at least some treatment with a therapeutic regimen.
  • MRD negative refers to a result when no residual tumor cells can be found in a sample from the patient.
  • MRD positive refers to a result when a small number of tumor cells can be found in a sample from the patient.
  • MRD can be qualified by an - 24 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 assay threshold, e.g., a 10-4, a 10-5 or a 10-6 threshold, i.e., related to a limit of detection of the assay, such as flow cytometry for detecting tumor cells.
  • "Treatment” as used herein in the context of cancer shall mean the use of a therapy to prevent or inhibit further tumor growth, to cause shrinkage of a tumor, alleviate tumor burden, and/or to provide longer survival times. Treatment is also intended to include prevention of metastasis of tumor.
  • a tumor is "inhibited” or “treated” (e.g., as determined by responsiveness, time to progression, progression-free survival, minimal residual disease or indicators known in the art and described herein) if at least one symptom of the cancer or tumor is alleviated, terminated, slowed, minimized, or prevented. Any amelioration of any symptom, physical or otherwise, of a tumor pursuant to treatment using a therapeutic regimen (e.g., comprising an NAE inhibitor, such as comprising pevonedistat or a pharmaceutically acceptable salt thereof) as further described herein, is within the scope of the invention.
  • a therapeutic regimen e.g., comprising an NAE inhibitor, such as comprising pevonedistat or a pharmaceutically acceptable salt thereof
  • the term “agent” is defined broadly as anything that cancer cells, including tumor cells, may be exposed to in a therapeutic regimen.
  • probe refers to any molecule, e.g., an isolated molecule, which is capable of selectively binding to a specifically intended target molecule, for example a marker of the invention. Probes can be either synthesized by one skilled in the art or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein.
  • a “normal” or “reference” characteristic, e.g., size, sequence, composition or amount of a marker may refer to the characteristic, e.g., size, sequence, composition or amount in a “reference sample.”
  • a reference sample can be a matched normal or control, e.g., germline, sample from the same patient from whom the cancer sample is derived.
  • a reference sample can be a sample from a healthy subject not having the marker-associated disease or having a reference characteristic e.g., the average characteristic, e.g., size, sequence, composition or amount of the wild type marker in several healthy subjects.
  • a reference sample characteristic e.g., size, sequence, composition or amount may be comprised of a characteristic, e.g., size, sequence, composition or amount of one or more markers from a reference database. - 25 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01
  • a “normal” characteristic e.g., size, sequence, composition or amount of a marker is the characteristic, e.g., size, sequence, composition or amount of the marker, e.g., marker gene in non-tumor cells in a similar environment or response situation from the same patient from whom the tumor is derived.
  • the normal amount of DNA copy number is 2 or diploid, with the exception of X-linked genes in males, where the normal DNA copy number is 1.
  • "Over-expression” or “upregulation” and “under-expression” or “downregulation” of a marker gene refer to expression of the marker gene of a patient at a greater or lesser level (e.g. more than three-halves-fold, at least two-fold, at least three-fold, greater or lesser level etc.), respectively, than normal level of expression of the marker gene, e.g., as measured by mRNA or protein, in a test sample that is greater than the standard error of the assay employed to assess expression.
  • a “significant” expression level may refer to a level which either meets or is above or below a pre-determined score for a marker gene set as determined by methods provided herein.
  • "Complementary" in the context of nucleic acids refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil.
  • a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, at least about 75%, at least about 90%, or at least about 95% or all of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • "Homologous" as used herein refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position.
  • a first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide - 26 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 residue positions of the two regions that are occupied by the same nucleotide residue (i.e., by percent identity).
  • a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share homology with 50% identity.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. In an embodiment of 100% identity, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
  • antibody broadly encompass naturally-occurring forms of antibodies, e.g., polyclonal antibodies (e.g., IgG, IgA, IgM, IgE) and monoclonal and recombinant antibodies such as IgG, single-chain antibodies, two-chain and multi-chain proteins, chimeric, CDR-grafted, human and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments (e.g., dAbs, scFv, Fab, F(ab)′2, Fab′) and derivatives having at least an antigenic binding site.
  • polyclonal antibodies e.g., IgG, IgA, IgM, IgE
  • monoclonal and recombinant antibodies such as IgG, single-chain antibodies, two-chain and multi-chain proteins, chimeric, CDR-grafted, human and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the
  • Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
  • the term “antibody” also includes synthetic and genetically engineered variants.
  • a “kit” is any article of manufacture (e.g., a package or container) comprising at least one reagent, e.g. a probe, for specifically detecting a marker or marker set of the invention.
  • the article of manufacture may be promoted, distributed, sold or offered for sale as a unit for performing, e.g., in vitro, the methods of the present invention, e.g., on a sample having been obtained from a patient, e.g., a human patient.
  • kits can comprise at least one probe, such as a nucleic acid probe and, optionally, one or more primers and/or at least one antibody probe, for use in detecting marker characteristics, e.g., size, sequence composition or amount, e.g., expression.
  • a kit of the present invention can contain instructions which describe a suitable detection assay.
  • kit can be conveniently used, e.g., in a clinical or a contract testing setting, to generate results, e.g., on characteristic, e.g., size, sequence, composition or amount of one or more marker, to be recorded, stored, transmitted or received to allow for diagnosis, evaluation or treatment of patients exhibiting symptoms of cancer, in particular patients exhibiting the possible presence of a cancer capable of treatment with a regimen comprising NAE inhibition therapy, including, e.g., hematological cancers e.g., myelomas (e.g., multiple myeloma), lymphomas (e.g., non-Hodgkin’s lymphoma), leukemias (e.g., acute myelogenous leukemia, chronic myelomonocytic leukemia, myelodysplastic syndrome), or solid tumors (e.g., tumors of skin, lung, breast, ovary).
  • hematological cancers e.g., myelomas (e.
  • the present methods and compositions are designed for use in diagnostics and therapeutics for a patient suffering from cancer.
  • a cancer or tumor is treated or diagnosed according to the present methods.
  • “Cancer” or “tumor” is intended to include any neoplastic growth in a patient, including an initial tumor and any metastases.
  • the cancer can be of the hematological or solid tumor type.
  • Hematological tumors include tumors of hematological origin, including, e.g., myelomas (e.g., monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma, smoldering myeloma, multiple myeloma), leukemias (e.g., Waldenstrom’s syndrome, chronic lymphocytic leukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia, chronic myelomonocytic leukemia, myelodysplastic syndrome, other leukemias), lymphomas (e.g., B-cell lymphomas, e.g., diffuse large B-cell lymphoma, non- Hodgkin’s lymphoma) and myelodysplastic syndrome.
  • myelomas e.g., monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma, smoldering myeloma, multiple
  • the MDS is high risk MDS, typically characterized by more than 5% of the bone marrow comprised of immature blast cells.
  • the AML is low-blast acute AML, de novo AML or secondary AML.
  • the AML patient can have blasts that can express CD33, CD123 and/or CLEC12a.
  • Solid tumors can originate in organs or can metastasize from other tumors, and include cancers such as, but not limited to, in skin, lung, brain, breast, prostate, ovary, colon, kidney, pancreas, liver, esophagus, stomach, intestine, bladder, uterus, cervix, head and neck, central nervous system, bone, testis, and adrenal gland.
  • the cancer can comprise a cell in which a marker gene has an alteration.
  • cancer cells including tumor cells, refer to cells that divide at an abnormal (increased) rate or whose control of growth or survival is different than for cells in the same tissue where the cancer cell arises or lives.
  • Cancer cells include, but are not limited to, cells in carcinomas, such as squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosar
  • noninvasive refers to a procedure which inflicts minimal harm to a subject.
  • a noninvasive sampling procedure can be performed quickly, e.g., in a walk-in setting, typically without anaesthesia and/or without surgical implements or suturing.
  • noninvasive samples include, but are not limited to, blood, serum, saliva, urine, buccal swabs, throat cultures, stool samples and cervical smears.
  • Noninvasive diagnostic analyses include x-rays, magnetic resonance imaging, positron emission tomography.
  • a cancer e.g., a hematological cancer, e.g., lymphoma, leukemia, e.g., AML, CMML, MDS, myeloma (e.g., multiple myeloma) can employ bone marrow biopsy to collect tissue for genotype or phenotype, e.g., histological analysis.
  • the invention provides methods for determining, assessing, advising or providing an appropriate therapy regimen for treating a tumor or managing cancer in a patient.
  • biological sample is intended to include a material, e.g., tissue, cells, biological fluids and isolates thereof, obtained, e.g., isolated or collected, from a subject, e.g., a human, such as a patient or a normal subject.
  • a tumor sample from a cancer patient can comprise tumor cells or contents or products thereof.
  • a sample for primary analysis of the tumor can be a bone marrow sample.
  • some tumor cells e.g., clonotypic tumor cells, circulating endothelial cells
  • G-CSF granulocyte-colony stimulating factor
  • noninvasive samples e.g., for in vitro - 29 - 56340669.1
  • Attorney Docket No.223266-549182 / PAT27156PCT01 measurement of markers to determine outcome of treatment can include peripheral blood samples. Accordingly, cells within peripheral blood can be tested for marker characteristics.
  • a control, reference sample for normal characteristic e.g., size, sequence, composition or amount can be obtained from skin or a buccal swab of the patient.
  • a typical tumor sample is a biopsy of the tumor and thus comprises solid tumor cells.
  • a sample of tumor cells shed or scraped from the tumor site can be collected noninvasively, such as, but not limited to, in blood, sputum, a nipple aspirate, urine, stool, cervical smear.
  • nucleic acid markers described herein can be identified or measured in cell-free portions of blood. For example, nucleic acids released from tumor cells can become circulating tumor DNA.
  • Circulating tumor DNA can be analyzed in samples of plasma or serum.
  • a control reference sample for normal characteristic e.g., size, sequence, composition or amount can be obtained from blood of the patient.
  • Blood or bone marrow collection containers can comprise an anti-coagulant, e.g., heparin or ethylene-diaminetetraacetic acid (EDTA), sodium citrate or citrate solutions with additives to preserve blood integrity, such as dextrose or albumin or buffers, e.g., phosphate.
  • a DNA stabilizer e.g., an agent that inhibits DNase, can be added to the sample.
  • an RNA stabilizer e.g., an agent that inhibits RNase
  • a protein stabilizer e.g., an agent that inhibits proteases
  • An example of a blood collection container is PAXGENE® tubes (PREANALYTIX, Valencia, CA), useful for RNA stabilization upon blood collection.
  • Peripheral blood samples can be modified, e.g., fractionated, clotted for serum, centrifuged for plasma, sorted or concentrated (e.g., to result in samples enriched with tumor or depleted of tumor (e.g., for a reference sample)).
  • modified samples include clonotypic myeloma cells, which can be collected by e.g., negative selection, e.g., separation of white blood cells from red blood cells (e.g., differential centrifugation through a dense sugar or polymer solution (e.g., FICOLL® solution (Amersham Biosciences division of GE healthcare, Piscataway, NJ) or HISTOPAQUE®-1077 solution, Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co., St.
  • negative selection e.g., separation of white blood cells from red blood cells (e.g., differential centrifugation through a dense sugar or polymer solution (e.g., FICOLL® solution (Amersham Biosciences division of GE healthcare, Piscataway, NJ) or HISTOPAQUE®-1077 solution, Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co., St.
  • FICOLL® solution Amersham Biosciences division of GE healthcare, Piscat
  • a selection agent e.g., a reagent which binds to a tumor cell or myeloid progenitor marker, such as CD34, CD38, CD138, or CD133, for direct isolation (e.g., the application of a magnetic field to - 30 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 solutions of cells comprising magnetic beads (e.g., from Miltenyi Biotec, Auburn, CA) which bind to the B cell markers) or fluorescent-activated cell sorting).
  • a selection agent e.g., a reagent which binds to a tumor cell or myeloid progenitor marker, such as CD34, CD38, CD138, or CD133
  • direct isolation e.g., the application of a magnetic field to - 30 - 56340669.1
  • a tumor cell line e.g., OCI-Ly3, OCI-Ly10 cell (Alizadeh et al. (2000) Nature 403:503-511), a RPMI 6666 cell, a SUP-B15 cell, a KG-1 cell, a CCRF-SB cell, an 8ES cell, a Kasumi-1 cell, a Kasumi-3 cell, a BDCM cell, an HL-60 cell, a Mo-B cell, a JM1 cell, a GA-10 cell or a B-cell lymphoma (e.g., BC-3) or a cell line or a collection of tumor cell lines (see e.g., McDermott et al.
  • mutational status of a marker gene e.g., whether wild type or comprising a gene alteration in a marker
  • a nucleic acid e.g., a DNA, RNA, cDNA or a protein correlated with the marker gene.
  • a nucleic acid primer can be designed to bind to a region comprising a potential alteration site or can be designed to complement the altered sequence rather than the wild type sequence.
  • SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, and/or 16 can be a primer or probe or be used to design a primer to detect a mutation in an IDH1 and/or IDH2 nucleic acid marker.
  • Primer pairs can be designed to bracket a region comprising a potential alteration in a marker gene.
  • a primer or primer pair can be used for sequencing one or both strands of DNA corresponding to the marker gene.
  • a primer can be used in conjunction with a probe, e.g., a nucleic acid probe, e.g., a hybridization probe, to amplify a region of interest prior to sequencing to boost sequence amounts for detection of an alteration in a marker gene.
  • regions which can be sequenced include an entire gene, transcripts of the gene and a fragment of the gene or the transcript, e.g., one or more of exons or - 31 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 untranslated regions.
  • Examples of alterations to target for primer selection and sequence or composition analysis can be found in public databases which collect alteration information, such as COSMIC and dbGaP.
  • Some alterations of marker genes such as IDH1 and IDH2 can be associated with sensitivity to an NAE inhibitor, e.g., pevonedistat or a pharmaceutically acceptable salt thereof.
  • alterations in IDH1 and IDH2 markers are listed in the above disclosure, in Table 2 and the Examples.
  • Sequencing methods are known to one skilled in the art. Examples of methods include the Sanger method, the SEQUENOMTM method and Next Generation Sequencing (NGS) methods.
  • the Sanger method comprising the use of electrophoresis, e.g., capillary electrophoresis to separate primer-elongated labeled DNA fragments, can be automated for high- throughput applications.
  • the primer extension sequencing can be performed after PCR amplification of regions of interest. Software can assist with sequence base calling and with alteration identification.
  • SEQUENOMTM MASSARRAY® sequencing analysis is a mass-spectrometry method which compares actual mass to expected mass of particular fragments of interest to identify alterations.
  • NGS technology also called “massively parallel sequencing” and “second generation sequencing” in general provides for much higher throughput than previous methods and uses a variety of approaches (reviewed in Zhang et al. (2011) J. Genet. Genomics 38:95-109 and Shendure and Hanlee (2008) Nature Biotech.26:1135- 1145).
  • NGS methods can identify low frequency alterations in a marker in a sample.
  • Some NGS methods see, e.g., GS-FLX Genome Sequencer (Roche Applied Science, Branford, CT), Genome analyzer (Illumina, Inc.
  • cyclic array sequencing with or without clonal amplification of PCR products spatially separated in a flow cell and various schemes to detect the labeled modified nucleotide that is incorporated by the sequencing enzyme (e.g., polymerase or ligase).
  • the sequencing enzyme e.g., polymerase or ligase.
  • primer pairs can be used in PCR reactions to amplify regions of interest. Amplified regions can be ligated into a concatenated product.
  • Clonal libraries are generated in the flow cell from the PCR or ligated products and further amplified (“bridge” or “cluster” PCR) for single-end sequencing as the polymerase adds a labeled, reversibly terminated base that is imaged in one of four channels, depending on the identity of the labeled base and then removed for the next cycle.
  • Software can aid in the comparison to genomic sequences to identify alterations.
  • Composition of proteins and nucleic acids can be determined by many ways known in the art, such as by treating them in ways that cleave, degrade or digest them and then - 32 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 analyzing the components.
  • Mass spectrometry, electrophoresis and chromatography can separate and define components for comparison. Alterations which cause deletions or insertions can be identified by size or charge differences in these methods. Protein digestion or restriction enzyme nucleic acid digestion can reveal different fragment patterns after some alterations. Antibodies that recognize particular mutant amino acids, e.g., the mutant amino acids (IDH1132C, 132G, 132H, or 132S; IDH286P, 316L, 140G, 140Q, 172K or SEQ ID NO:17) listed in the above disclosure, in Table 2 and the Examples in their structural contexts, can identify and detect these alterations in samples.
  • the mutant amino acids IDH1132C, 132G, 132H, or 132S; IDH286P, 316L, 140G, 140Q, 172K or SEQ ID NO:17
  • DNA e.g., genomic DNA corresponding to the wild type or altered marker gene
  • DNA can be analyzed both by in situ and by in vitro formats in a biological sample using methods known in the art.
  • DNA can be directly isolated from the sample or isolated after isolating another cellular component, e.g., RNA or protein. Kits are available for DNA isolation, e.g., QIAAMP® DNA Micro Kit (Qiagen, Valencia, CA). DNA also can be amplified using such kits.
  • mRNA corresponding to the marker gene can be analyzed both by in situ and by in vitro formats in a biological sample using methods known in the art. An example of a method for measuring expression level is included in the Examples.
  • a nucleic acid probe can be used to hybridize to a marker and the amount of probe hybridized can be measured.
  • Many expression detection methods use isolated RNA.
  • any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from tumor cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999).
  • large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Patent No.4,843,155).
  • RNA can be isolated using standard procedures (see e.g., Chomczynski and Sacchi (1987) Anal. Biochem.162:156-159), solutions (e.g., trizol, TRI REAGENT® (Molecular Research Center, Inc., Cincinnati, OH; see U.S. Patent No.5,346,994) or kits (e.g., a QIAGEN® Group RNEASY® isolation kit (Valencia, CA) or LEUKOLOCKTM Total RNA Isolation System, Ambion division of Applied Biosystems, Austin, TX). [0097] Additional steps may be employed to remove DNA from RNA samples.
  • RNA is extracted from cells of the various types of interest using guanidinium thiocyanate lysis followed by CsCl centrifugation to separate the - 33 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 RNA from DNA (Chirgwin et al. (1979) Biochemistry 18:5294-99).
  • separation of RNA from DNA can be accomplished by organic extraction, for example, with hot phenol or phenol/chloroform/isoamyl alcohol.
  • RNAse inhibitors may be added to the lysis buffer.
  • mRNAs transfer RNA (tRNA) and ribosomal RNA (rRNA).
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • Most mRNAs contain a poly(A) tail at their 3' end. This allows them to be enriched by affinity chromatography, for example, using oligo(dT) or poly(U) coupled to a solid support, such as cellulose or SEPHADEX.RTM medium (see Ausubel et al. (1994) Current Protocols In Molecular Biology, vol.2, Current Protocols Publishing, New York).
  • poly(A)+mRNA is eluted from the affinity column using 2 mM EDTA/0.1% SDS. (see Sambrook et al. (1989) Molecular Cloning--A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
  • a biological sample e.g., a bone marrow sample, a whole blood sample, a plasma sample, a serum sample, a tumor biopsy, a sample comprising tumor cells, tumor cell products or tumor cell components, e.g., cytoplasm or mitochondria, or a reference sample
  • a biological sample e.g., a bone marrow sample, a whole blood sample, a plasma sample, a serum sample, a tumor biopsy, a sample comprising tumor cells, tumor cell products or tumor cell components, e.g., cytoplasm or mitochondria, or a reference sample
  • a nucleic acid e.g., RNA, mRNA, genomic DNA, or cDNA
  • Non-limiting examples of such methods include immunological methods for detection of secreted, cell-surface, cytoplasmic, mitochondrial or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.
  • These methods include gene array/chip technology, RT- PCR, TAQMAN® gene expression assays (Applied Biosystems, Foster City, CA), e.g., under GLP approved laboratory conditions, in situ hybridization, immunohistochemistry, immunoblotting, FISH (fluorescence in situ hybridization), FACS analyses, northern blot, southern blot, INFINIUM® DNA analysis Bead Chips (Illumina, Inc., San Diego, CA), quantitative PCR, bacterial artificial chromosome arrays, single nucleotide polymorphism (SNP) arrays (Affymetrix, Santa Clara, CA) or cytogenetic analyses.
  • SNP single nucleotide polymorphism
  • the detection methods of the invention can thus be used to detect mutational status in RNA, mRNA, protein, cDNA, ctDNA, or genomic DNA, for example, in a biological sample in vitro as well as in vivo.
  • in vivo techniques for detection of a polypeptide or nucleic acid corresponding to a marker of the invention include introducing into a subject a labeled probe to detect the biomarker, e.g., a nucleic acid complementary to the transcript of a biomarker or a labeled antibody, Fc receptor or - 34 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 antigen directed against the polypeptide, e.g., wild type or mutant marker.
  • the antibody can be labeled with a radioactive isotope whose presence and location in a subject can be detected by standard imaging techniques.
  • these assays can be conducted in a variety of ways. A skilled artisan can select from these or other appropriate and available methods based on the nature of the marker(s), tissue sample and alteration in question. Different methods or combinations of methods could be appropriate in different cases or, for instance in different types of tumors or patient populations.
  • detection assays involve preparing a sample or reaction mixture that may contain a marker, and a probe, under appropriate conditions and for a time sufficient to allow the marker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture.
  • assays can be conducted in a variety of ways.
  • one method to conduct such an assay would involve anchoring the marker or probe onto a solid phase support, also referred to as a substrate, and detecting target marker/probe complexes anchored on the solid phase at the end of the reaction.
  • a sample from a subject which is to be assayed for presence and/or concentration of marker, can be anchored onto a carrier or solid phase support.
  • the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component of the assay.
  • One example of such an embodiment includes use of an array or chip which contains a marker or marker set anchored for expression analysis of the sample.
  • an array or chip which contains a marker or marker set anchored for expression analysis of the sample.
  • assay components e.g., glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • marker or probe molecules which are immobilized through conjugation of biotin and streptavidin.
  • biotinylated assay components can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Protein Biology, Thermo Scientific), and immobilized in the wells of streptavidin-coated 96 well plates.
  • the surfaces with immobilized assay components can be prepared in advance and stored. [00101] In order to conduct assays with the above-mentioned approaches, the non- immobilized component is added to the solid phase upon which the second component is anchored.
  • uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the - 35 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 solid phase.
  • the detection of marker/probe complexes anchored to the solid phase can be accomplished in a number of methods outlined herein.
  • the probe when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art.
  • labeled with regard to the probe (e.g., nucleic acid or antibody), is intended to encompass direct labeling of the probe by coupling (i.e., physically linking) a detectable substance to the probe, as well as indirect labeling of the probe by reactivity with another reagent that is directly labeled.
  • the label can be a radioisotope, a fluorescent compound, an enzyme, an enzyme co- factor, a hapten, a sequence tag, a protein or an antibody.
  • An example of indirect labeling includes detection of a primary antibody using a fluorescently labeled secondary antibody.
  • marker/probe complex formation without further manipulation or labeling of either component (marker or probe), for example by utilizing the technique of fluorescence energy transfer (FET, see, for example, Lakowicz et al., U.S. Patent No.5,631,169; Stavrianopoulos, et al., U.S. Patent No.4,868,103).
  • FET fluorescence energy transfer
  • determination of the ability of a probe to recognize a marker can be accomplished without labeling either assay component (probe or marker) by utilizing a technology such as real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal.
  • BIOA Biomolecular Interaction Analysis
  • BIOA or “surface plasmon resonance” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIACORETM). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.
  • SPR surface plasmon resonance
  • analogous detection assays can be conducted with marker and probe as solutes in a liquid phase.
  • the complexed marker and probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.
  • Nucleic acid probes of the invention may be prepared by chemical synthesis using any suitable methodology known in the art, may be produced by recombinant technology, or may be derived from a biological sample, for example, by restriction digestion.
  • the nucleic - 36 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 acids can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone.
  • An example of a nucleic acid label is incorporated using SUPERTM Modified Base Technology (Nanogen, Bothell, WA, see U.S. Patent No.7,045,610).
  • the level of expression can be measured as general nucleic acid levels, e.g., after measuring the amplified DNA levels (e.g.
  • Hybridization of an RNA, ctDNA or a cDNA with the nucleic acid probe can indicate that the marker in question is being expressed.
  • hybridizes is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other.
  • the conditions are such that sequences at least about 70%, at least about 80%, at least about 85%, 90% or 95% identical to each other remain hybridized to each other for subsequent amplification and/or detection.
  • Stringent conditions vary according to the length of the involved nucleotide sequence but are known to those skilled in the art and can be found or determined based on teachings in Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additional stringent conditions and formulas for determining such conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), chapters 7, 9 and 11.
  • a non-limiting example of stringent hybridization conditions for hybrids that are at least 10 basepairs in length includes hybridization in 4X sodium chloride/sodium citrate (SSC), at about 65-70°C (or hybridization in 4X SSC plus 50% formamide at about 42-50°C) followed by one or more washes in 1X SSC, at about 65-70°C.
  • a non-limiting example of highly stringent hybridization conditions for such hybrids includes hybridization in 1X SSC, at about 65-70°C (or hybridization in 1X SSC plus 50% formamide at about 42-50°C) followed by one or more washes in 0.3X SSC, at about 65-70°C.
  • a non-limiting example of reduced stringency hybridization conditions for such hybrids includes hybridization in 4X SSC, at about 50-60°C (or alternatively hybridization in 6X SSC plus 50% formamide at about 40-45°C) followed by one or more washes in 2X SSC, at about 50-60°C.
  • nucleic acid probes of the invention can refer to - 37 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 nucleic acids which hybridize to the region of interest and which are not further extended.
  • the RNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated RNA on an agarose gel and transferring the RNA from the gel to a membrane, such as nitrocellulose.
  • the nucleic acid probe(s) are immobilized on a solid surface and the RNA is contacted with the probe(s), for example, in an AFFYMETRIX® gene chip array or a SNP chip (Santa Clara, CA) or customized array using a marker set comprising at least one marker indicative of treatment outcome.
  • a skilled artisan can readily adapt known RNA and DNA detection methods for use in detecting the amount of the markers of the present invention.
  • the high density microarray or branched DNA assay can benefit from a higher concentration of tumor cell in the sample, such as a sample which had been modified to isolate tumor cells as described in earlier sections.
  • a mixture of transcribed polynucleotides obtained from the sample is contacted with a substrate having fixed thereto a polynucleotide complementary to or homologous with at least a portion (e.g., at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or more nucleotide residues) of a marker nucleic acid.
  • polynucleotides complementary to or homologous with the marker are differentially detectable on the substrate (e.g., detectable using different chromophores or fluorophores, or fixed to different selected positions), then the levels of expression of a plurality of markers can be assessed simultaneously using a single substrate (e.g., a "gene chip" microarray of polynucleotides fixed at selected positions).
  • a method of assessing marker expression is used which involves hybridization of one nucleic acid with another, the hybridization can be performed under stringent hybridization conditions.
  • a bait set can have a higher relative concentration for more specifically desired sequences of interest that comprise one or more IDH1 and/or IDH2 gene mutations as provided herein.
  • Such bait sets are combined with a sample under conditions that allow hybridization of the target molecules with the baits. Then, captured molecules are isolated using the capture moiety.
  • a biotin capture moiety is bead-based streptavidin.
  • RNA does not need to be isolated from the cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known - 38 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 histological methods.
  • RNA that encodes the marker is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to RNA that encodes the marker.
  • a support typically a glass slide
  • a probe that can hybridize to RNA that encodes the marker.
  • In vitro techniques for detection of a polypeptide corresponding to a marker of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, protein array, immunoprecipitations and immunofluorescence.
  • ELISAs enzyme linked immunosorbent assays
  • Western blots Western blots
  • protein array protein array
  • immunoprecipitations immunofluorescence
  • expression of a marker is assessed using an antibody (e.g., a radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain) which binds specifically with a marker protein or fragment thereof, e.g., a protein or fragment comprising a region which can be altered, e.g., comprising an active site amino acid (such as R100 or R132 of IDH1 or R140 or R172 of IDH2) or a portion comprising an altered, e.g., mutant amino acid, sequence, or an altered residue in its structural context, including a marker protein which has undergone all or a portion of its normal post-translational
  • An antibody can detect a marker gene protein described herein, e.g., a protein corresponding to IDH1 or IDH2.
  • an antibody can detect a genetically altered IDH1 or IDH2 protein, e.g., a protein with an amino acid sequence comprising a mutant of SEQ ID NO:1 or 2.
  • IDH1 or IDH2 alterations that can be detected by antibodies include the mutations described above as they pertain to IDH1 and IDH2 (IDH1132C, 132G, 132H, or 132S; IDH286P, 316L, 140G, 140Q, 172K or SEQ ID NO:17) listed in Table 2 and in the Examples.
  • Residues translated from altered codons can be prepared in immunogenic compositions for generation of antibodies that will specifically recognize and bind to the mutant residues.
  • Another method can employ pairs of antibodies, wherein one of the pair would bind a marker protein upstream, i.e., N-terminal to the region of expected alteration, e.g., nonsense or deletion and the other of the pair would bind the protein downstream. Wild type protein would bind both antibodies of the pair, but a protein with a nonsense or deletion alteration would bind only the N-terminal antibody of the pair.
  • indirect methods for determining the amount or functionality of a protein marker also include measurement of the activity of the protein. For example, a sample, or a protein isolated from the sample or expressed from nucleic acid isolated, cloned or amplified from the sample can be assessed for marker protein activity.
  • intact proteins or peptides can be analyzed from a biological sample, e.g., a bone marrow sample, a blood sample, a lymph sample or other sample, containing one or more polypeptide markers.
  • the method can further include treating the sample to lower the amounts of abundant proteins, e.g., serum albumin, to increase the sensitivity of the method.
  • liquid chromatography can be used to fractionate the sample so portions of the sample can be analyzed separately by mass spectrometry. The steps can be performed in separate systems or in a combined liquid chromatography/mass spectrometry system (LC/MS, see for example, Liao, et al.
  • the mass spectrometry system also can be in tandem (MS/MS) mode.
  • the charge state distribution of the protein or peptide mixture can be acquired over one or multiple scans and analyzed by statistical methods, e.g. using the retention time and mass-to- charge ratio (m/z) in the LC/MS system, to identify proteins expressed at statistically significant levels differentially in samples from patients responsive or non-responsive to NAE inhibition therapy.
  • mass spectrometers which can be used are an ion trap system (ThermoFinnigan, San Jose, CA) or a quadrupole time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA).
  • the method can further include the step of peptide mass fingerprinting, e.g. in a matrix-assisted laser desorption ionization with time-of-flight (MALDI- TOF) mass spectrometry method.
  • the method can further include the step of sequencing one or more of the tryptic peptides. Results of this method can be used to identify proteins from primary sequence databases, e.g., maintained by the National Center for Biotechnology Information, Bethesda, MD, or the Swiss Institute for Bioinformatics, Geneva, Switzerland, and based on mass spectrometry tryptic peptide m/z base peaks.
  • expression of a marker is assessed by preparing mRNA/cDNA (i.e., a transcribed polynucleotide) from cells in a biological sample, and by hybridizing the mRNA/cDNA with a reference polynucleotide, e.g., an isolated nucleic acid probe, e.g., a hybridization probe, which is a complement of a marker nucleic acid, or a fragment thereof.
  • cDNA can, optionally, be amplified using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide. Expression of one or more markers likewise can be detected using quantitative PCR to assess the level of expression of the marker(s).
  • An example of the use of measuring mRNA levels is that an inactivating alteration in a marker gene can result in an altered level of mRNA in a cell.
  • the level can be upregulated due - 40 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 to feedback signaling protein production in view of nonfunctional or absent protein or downregulated due to instability of an altered mRNA sequence.
  • any of the many known methods of detecting alterations e.g. single nucleotide polymorphisms, deletions, discussed above
  • a marker of the invention may be used to detect occurrence of an alteration in a marker gene in a patient.
  • direct measurement of nucleic acid amount is quantification of transcripts.
  • the level or amount of expression refers to the absolute amount of expression of an mRNA encoded by the marker or the absolute amount of expression of the protein encoded by the marker.
  • determinations may be based on normalized expression amounts.
  • Expression amount can be normalized by correcting the absolute expression level of a marker upon comparing its expression to the expression of a control marker that is not a marker, e.g., in a housekeeping role that is constitutively expressed. Suitable markers for normalization also include housekeeping genes, such as the actin gene or beta-2 microglobulin.
  • Reference markers for data normalization purposes include markers which are ubiquitously expressed and/or whose expression is not regulated by oncogenes. Constitutively expressed genes are known in the art and can be identified and selected according to the relevant tissue and/or situation of the patient and the analysis methods. Such normalization allows one to compare the expression level in one biological sample, to another biological sample, e.g., between biological samples from different times or different subjects. Further, the expression level can be provided as a relative expression level.
  • the baseline of a genomic DNA sample e.g., diploid copy number, can be determined by measuring amounts in cells from subjects without a tumor or in non-tumor cells from the patient.
  • the amount of the marker or marker set is determined for at least 1, or 2, 3, 4, 5, or more samples, e.g., 7, 10, 15, 20 or 50 or more samples in order to establish a baseline, prior to the determination of the expression level for the sample in question.
  • the mean amount or level of each of the markers or marker sets assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker or marker sets in question.
  • the amount of the marker or marker set determined for the test sample (e.g., absolute level of expression) is then divided by the baseline value obtained for that marker or marker set. This provides a relative amount and aids in identifying abnormal levels of marker protein activity.
  • Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences corresponding to one or more markers of the - 41 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 invention.
  • the probe can comprise a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Probes can be used as part of a diagnostic test kit for identifying cells or tissues which express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been altered.
  • Primers or nucleic acid probes comprise a nucleotide sequence complementary to a specific marker or an altered region thereof and are of sufficient length to selectively bind or hybridize with a marker gene or nucleic acid associated with a marker gene, e.g., they can bind to the nucleic acid with base sequence specificity and remain bound, after washing.
  • the primer or nucleic acid probe e.g., a substantially purified oligonucleotide, an isolated nucleic acid, comprises a region having a nucleotide sequence which binds, e.g., hybridizes, e.g., under stringent conditions, to about 5 to 15, 10 to 25, 15 to 50, 20 to 100, 50 to 350 or 500 or more consecutive nucleotides of a marker gene or a region comprising an alteration in a marker gene or transcript therefrom, e.g., a mutated codon described in the disclosure above, in Table 2, and the Examples, or a complement thereof.
  • the primer or nucleic acid probe is capable of hybridizing to a marker nucleic acid corresponding to a marker gene described herein, e.g., IDH1 or IDH2, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 3, 4, 5 or 6, or a complement of any of the foregoing.
  • a primer or nucleic acid probe comprising a nucleotide sequence of at least about 10 consecutive nucleotides, at least about 15 consecutive nucleotides, about 10 to 25 consecutive nucleotides, about 20 to 40 consecutive nucleotides, about 30 to 60 consecutive nucleotides, or having from about 15 to about 30 nucleotides set forth in any of SEQ ID NOS: 3 or 5, an open reading frame of IDH1 or IDH2, e.g. SEQ ID NOs:4 or 6, or a complement of any of the foregoing are provided by the invention.
  • Primers or nucleic acid probes having a sequence of more than about 25, 40 or 50 nucleotides of SEQ ID NOs:3, 4, 5 or 6, e.g., targeting a IDH1 or IDH2 sequence portion comprising a mutated codon described in the disclosure above, in Table 2, and in the Examples, or having a sequence comprising SEQ ID NOs:7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or a complement of any of the foregoing are also within the scope of the invention.
  • a primer or nucleic acid probe can have a sequence at least 70%, at least 75%, 80% or 85%, or at least, 90%, 95% or 97% identical to the nucleotide sequence of IDH1, IDH2, or a complement of any of the foregoing.
  • Nucleic acid analogs can be used as binding sites for hybridization.
  • An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Egholm et al., Nature 363:566568 (1993); U.S. Pat. No.5,539,083).
  • a nucleic acid probe can be designed to bind to the wild type sequence, so the presence of an alteration in that region can cause a decrease, e.g., measurable decrease, in binding or hybridization by that probe.
  • a nucleic acid probe can be designed to bind to a mutant sequence, e.g., a mutated codon described in the disclosure above, in Table 2, and in the Examples, so the presence of an alteration in that region can cause an increase in binding or hybridization by that probe.
  • a probe and primer set or a primer pair can be designed to bracket a region in a marker that can have an alteration so amplification based on that set or pair can result in nucleic acids which can be sequenced to identify the alteration, as described above.
  • Primers or nucleic acid probes can be selected using an algorithm that takes into account binding energies, base composition, sequence complexity, cross-hybridization binding energies, and secondary structure (see Friend et al., International Patent Publication WO 01/05935, published Jan.25, 2001; Hughes et al., Nat. Biotech.19:342-7 (2001).
  • Useful primers or nucleic acid probes of the invention bind sequences which are unique for each transcript, e.g., target altered regions and can be used in PCR for amplifying, detecting and sequencing only that particular nucleic acid, e.g., transcript or altered transcript.
  • Some marker genes e.g., IDH1 and/or IDH2, which may be altered in cancer, e.g., hematological cancer, e.g., AML, CMML or MDS, are found in Table 1.
  • Examples of alterations of IDH1 and/or IDH2 associated with favorable outcome to treatment comprising an NAE inhibitor, e.g., pevonedistat or a pharmaceutically acceptable salt thereof, are described in the Examples and disclosure above, and Table 2.
  • an oligonucleotide primer may have a non-complementary fragment at its 5' end, with the remainder of the primer being complementary to the target region.
  • non-complementary nucleotides may be interspersed into the nucleic acid probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
  • An indication of treatment outcome can be assessed by studying a characteristic of 1 marker, characteristics of markers in a marker set comprising 2 markers, 3 markers or 4 markers, or more, e.g., 5, 6, 7, 8, 9, 10, 15, 20, or 25 markers, or altered portions thereof e.g., marker genes which interact with DNA, signaling pathways or are involved in AML tumorigenesis.
  • the marker set includes a plurality of chromosome loci, a plurality of marker genes, and/or a plurality of markers of one or more marker genes (e.g., nucleic acid and protein, genomic DNA and mRNA, or various combinations of markers described herein).
  • Markers can be studied in combination with another measure of treatment outcome, e.g., biochemical markers (e.g., M protein level in myeloma, kidney health marker such as proteinuria, serum levels of C-reactive protein or cytokeratin 19, cytokeratin fragment 21-1 (CYFRA21-1) for NSCLC, urine levels of fibrinogen/fibrinogen degradation products for bladder cancer, urine or blood levels of catecholamines for neuroblastoma, serum levels of carbohydrate antigen 19-9 (CA 19-9) or metabolic profiling for pancreatic cancer or blood levels of soluble mesothelin-related peptides (SMRP) in mesothelioma) or histology assessment (e.g., fewer than 5% blast cells in the bone marrow of a leukemia or myeloma patient, number of mitotic figures per unit area, depth measurement of invasion of melanoma tumors, esophageal tumors or bladder tumors).
  • biochemical markers
  • Statistical methods can assist in the determination of treatment outcome upon measurement of a characteristic such as an amount of a marker, e.g., measurement of DNA, RNA or protein.
  • the amount of one marker can be measured at multiple timepoints, e.g., before treatment, during treatment, after treatment with an agent, e.g., an NAE inhibitor.
  • an agent e.g., an NAE inhibitor.
  • the expression results can be analyzed by a repeated measures linear regression model (Littell, Miliken, Stroup, Wolfinger, Schariberger (2006) SAS for Mixed Models, 2nd edition.
  • Equation 1 Y ijk ⁇ Y ij0 ⁇ Y ij0 ⁇ treatmen i t ⁇ day k ⁇ (treatmen*t day ) ik ⁇ ⁇ ijk
  • Y ijk is the log2 transformed expression (normalized to the housekeeping genes) on the kth day of the jth animal in the ith treatment
  • Yij0 is the defined baseline log2 transformed expression (normalized to the housekeeping genes) of the j th animal in the i th treatment
  • day k is treated as a - 44 - 56340669.1
  • ⁇ ijk is the residual error term.
  • a covariance matrix (e.g., first-order autoregressive, compound symmetry, spatial power law) can be specified to model the repeated measurements on each animal over time. Furthermore, each treatment time point can be compared back to the same time point in the vehicle group to test whether the treatment value was significantly different from vehicle. [00121]
  • a number of other methods can be used to analyze the data. For instance, the relative expression values could be analyzed instead of the cycle number. These values could be examined as either a fold change or as an absolute difference from baseline. Additionally, a repeated-measures analysis of variance (ANOVA) could be used if the variances are equal across all groups and time points.
  • ANOVA repeated-measures analysis of variance
  • a difference in amount from one timepoint to the next or from the tumor sample to the normal sample can indicate prognosis or treatment outcome.
  • a baseline level can be determined by measuring expression at 1, 2, 3, 4, or more times prior to treatment, e.g., at time zero, one day, three days, one week and/or two weeks or more before treatment.
  • a baseline level can be determined from a number of subjects, e.g., normal subjects or patients with the same health status or disorder, who do not undergo or have not yet undergone the treatment, as discussed above.
  • GEO Gene Expression Omnibus
  • NBI National Center for Biotechnology Information
  • a dataset of SNP genotype profiling of paired tumor and normal samples from AML patients has the GEO Accession number GSE61323 and can be used to query additional patient populations from the tumor sample data or to establish reference characteristics from the normal sample data.
  • the expression of the marker can be measured at any time or multiple times after some treatment, e.g., after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months and/or 6 or more months of treatment.
  • the amount of a marker can be measured once after some treatment, or at multiple - 45 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 intervals, e.g., 1-week, 2-week, 4-week or 2-month, 3-month or longer intervals during treatment.
  • the measurement of a marker during treatment can be compared to the same marker measurement at baseline. In other embodiments, the measurement of a marker during treatment can be compared to the same marker measurement at an earlier timepoint.
  • the amount of the marker can be measured at any time or multiple times after, e.g., 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months and/or 6 or more months after the last treatment.
  • the measurement of a marker after treatment can be compared to the same marker measurement at the end of treatment.
  • One of skill in the art would determine the timepoint or timepoints to assess the amount of the marker depending on various factors, e.g., the pharmacokinetics of the treatment, the treatment duration, pharmacodynamics of the treatment, age of the patient, the nature of the disorder or mechanism of action of the treatment.
  • a trend toward an unfavorable outcome relative to baseline or a pre-determined standard of expression of a marker of sensitivity to NAE inhibition therapy can indicate a decrease in response of the tumor to the therapy, e.g., increase in resistance.
  • a trend toward a favorable outcome relative to the baseline or a pre-determined standard of expression of a marker of treatment outcome indicates usefulness of the therapeutic regimen or continued benefit of the therapy, and accordingly, treatment with the therapy would be continued.
  • Any marker e.g., nucleic acid or protein corresponding to a marker gene or combination of markers of the invention, or alterations thereof as well as any known markers in combination with the markers of the invention, may be used in the compositions, kits, and methods of the present invention.
  • markers are selected for as great as possible ability to judge mutational status of a marker gene to predict outcome of treatment with a therapeutic regimen comprising an NAE inhibitor, more preferably a combination comprising an NAE inhibitor for example, pevonedistat, or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and a hypomethylating agent, for example azacitidine.
  • an NAE inhibitor for example, pevonedistat, or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199)
  • a hypomethylating agent for example azacitidine.
  • the choice of markers are selected for as great as possible difference between the characteristic, e.g., size, sequence, composition or amount of the marker in samples comprising tumor cells and the characteristic, e.g., size, sequence, composition or amount of the same marker in control cells.
  • RNA or protein amount can be at least 1.5-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 100-, 500-, 1000-fold or greater.
  • RNA or protein amount can be that expression relative to the overall mean across tumor samples (e.g., from a hematological tumor, e.g., leukemia or myeloma) is low.
  • amount of DNA e.g., copy number
  • the amount is 0, 1, 2, 3, 4, 5, 6, or more copies.
  • a deletion causes the copy number to be 0 or 1; an amplification causes the copy number to be greater than 2.
  • the difference can be qualified by a confidence level, e.g., p ⁇ 0.05, p ⁇ 0.02, p ⁇ 0.01 or lower p-value.
  • Selected marker sets can be assembled from the markers provided herein or selected from among markers using methods provided herein and analogous methods known in the art.
  • a way to qualify a new marker for use in an assay of the invention is to correlate a characteristic, e.g., size, sequence, composition or amount in a sample comprising tumor cells with differences in characteristic, e.g., size, sequence, composition or amount (e.g., fold-change from baseline) of a marker, e.g., a marker gene.
  • a useful way to judge the relationship is to calculate the coefficient of determination r2, after solving for r, the Pearson product moment correlation coefficient and/or preparing a least squares plot, using standard statistical methods.
  • a correlation can analyze DNA copy number versus the level of expression of marker, e.g., a marker gene.
  • a gene product can be selected as a marker if the result of the correlation (r2, e.g., the linear slope of the data in this analysis), is at least 0.1- 0.2, at least 0.3- 0.5, or at least 0.6-0.8 or more.
  • Markers can vary with a positive correlation to response, PFS or survival (i.e., change expression levels in the same manner as copy number, e.g., decrease when copy number is decreased). Markers which vary with a negative correlation to copy number (i.e., change expression levels in the opposite manner as copy number levels, e.g., increase when copy number is decreased) provide inconsistent determination of outcome.
  • marker set can be prepared using a scoring method known in the art (e.g., weighted voting, combination of threshold features (CTF), Cox proportional hazards analysis, principal components scoring, linear predictive score, K-nearest neighbor, etc.), e.g., using expression values deposited with the Gene Expression Omnibus (GEO) program at the National Center for Biotechnology Information (NCBI, Bethesda, MD).
  • GEO Gene Expression Omnibus
  • the compositions, kits, and methods of the invention are used for characterizing treatment outcome in a patient, the marker or set of markers is selected such that a significant result is obtained in at least about 20%, at least about 40%, 60%, or 80%, or in substantially all patients treated with the test agent.
  • the marker or set of markers can be selected such that a positive predictive value (PPV) of greater than about 10% is obtained for the general population and additional confidence in a marker can be inferred when the PPV is coupled with an assay specificity greater than 80%.
  • PPV positive predictive value
  • treatment with a regimen comprising an NAE inhibitor, e.g., pevonedistat or a pharmaceutically acceptable salt thereof comprises determining the presence or absence of mutations in IDH1 and/or IDH2 in cancer cells, e.g., one or more mutations described in the disclosure above, in Table 2, and in the Examples.
  • Evaluation of the mutation status of IDH1 and/or IDH2 is then used to determine whether the patient is expected to have a favorable outcome and would benefit from treatment, e.g., treatment with a NAE inhibitor , for example, pevonedistat, or a pharmaceutically acceptable salt thereof, a BCL2 inhibitor, such as venetoclax (ABT-199), and a hypomethylating agent, e.g., azacitidine; or treatment with a BCL2 inhibitor, a hypomethylating agent, and the NAE inhibitor; or other agent(s) or treatments expected to have a similar effect on survival.
  • a NAE inhibitor for example, pevonedistat, or a pharmaceutically acceptable salt thereof
  • BCL2 inhibitor such as venetoclax (ABT-199)
  • a hypomethylating agent e.g., azacitidine
  • treatment with a BCL2 inhibitor, a hypomethylating agent, and the NAE inhibitor or other agent(s) or treatments expected to have a similar effect on survival.
  • Evaluation of the mutation status of IDH1 and/or IDH2 can also be used to determine whether a patient is expected to have an unfavorable outcome and would benefit from a cancer therapy other than NAE inhibition therapy or would benefit from an altered NAE inhibition therapy regimen.
  • a patient whose cancer is characterized as having a marker profile indicative of favorable outcome to NAE inhibition therapy e.g., pevonedistat or a pharmaceutically acceptable salt thereof, will undergo treatment with the therapy and a more aggressive therapy regimen will be identified for a patient with an expected unfavorable outcome.
  • Some illustrative methods of treating a hematological cancer, or hematological cancer patient may include, without limitation, a) measuring at least one characteristic of at least one marker corresponding to the IDH1 gene or the IDH2 gene in a biological sample obtained from the patient; b) identifying at least one mutation in the IDH1 gene and/or the IDH2 gene in the biological sample from the at least one characteristic measured in step a); and c) administering the combination to the patient, wherein the combination comprises treatment with a NAE inhibitor , for example, pevonedistat, or a pharmaceutically acceptable salt thereof, a BCL2 inhibitor, such as venetoclax (ABT-199), and a hypomethylating agent, e.g., azacitidine.
  • a NAE inhibitor for example, pevonedistat, or a pharmaceutically acceptable salt thereof
  • BCL2 inhibitor such as venetoclax (ABT-199)
  • a hypomethylating agent e.g., azacitidine.
  • Some illustrative methods of preventing progression or treating a hematological cancer, or hematological cancer patient may include, administering a therapeutically effective amount of a combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and azacitidine to the hematological cancer patient.
  • the patient has been identified as having a gain of function mutation in the IDH1 gene and/or the IDH2 gene, for example, in at least a portion of the hematological cancer.
  • the mutation in the IDH1 gene results in the mutation of the IDH1 protein, wherein the mutation is position R132, of SEQ ID NO: 1, and the mutation in the IDH2 gene results in the mutation of the IDH2 protein, selected from the group consisting of position - 48 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 L86, position R140, positions L143-T146, position R172 and position Q316, of SEQ ID NO: 2. In some embodiments, one of the nucleotides at a position selected from the group consisting of 689, 690, and 691of SEQ ID NO:3 is mutated.
  • one of the nucleotides at a position selected from the group consisting of 394, 395, and 396 of SEQ ID NO:4 is mutated.
  • one of the nucleotides at a position selected from the group consisting of 420, 421, 422, 582, 583, 584, 678, 679, 680, 1110, 1111, 1112, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601 and 602 of SEQ ID NO:5 is mutated.
  • one of the nucleotides at a position selected from the group consisting of 256, 257, 258, 418, 419, 420, 514, 515, 516, 946, 947,948, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437 and 438 of SEQ ID NO:6 is mutated.
  • a deletion begins at nucleotide 591, 592 or 593 of SEQ ID NO:5.
  • a deletion begins at nucleotide 427, 428 or 429 of SEQ ID NO:6.
  • the hematological tumor cancer is selected from the group consisting of leukemia, lymphoma, myeloma, acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML).
  • the method also includes the steps of: [00129] a) measuring at least one characteristic of at least one marker corresponding to IDH1 or IDH2 in a biological sample obtained from the patient; [00130] b) identifying at least one characteristic selected from: (i) a mutation of the IDH1 gene, or the IDH2 gene, (ii) a mutation of an IDH1 nucleotide selected from base 689, 690, and 691of SEQ ID NO:3 or selected from base 394, 395, and 396, of SEQ ID NO:4, (iii) an IDH1 protein having an amino acid mutation selected from R132C, R132G, R132H, or R132S or (iv) a mutation of an IDH2 nucleotide selected from base 420, 421, 422, 582, 583, 584, 678, 679, 680, 1110, 1111, and 1112 of SEQ ID NO:5 or selected from base 256, 257, 258, 418, 419
  • the at least one marker is a nucleic acid or a protein, for example, the at least one marker is a nucleic acid, and also wherein the at least one characteristic is protein or nucleic acid sequence.
  • the at least one characteristic sequence related to the marker genes - 49 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 IDH1 and/or IDH2 is a nucleic acid sequence of the subject’s tumor, ctDNA or cancer cell IDH1 gene or IDH2 gene, which may be determined by any method known to a person skilled in the art, for example, performed using Sanger sequencing or next-generation sequencing (NGS).
  • the hematological cancer comprises mutations in the IDH1 protein having substitutions selected from one or more of: R132C, R132G, R132H, and R132S relative to SEQ. ID NO: 1.
  • the hematological cancer comprises mutations in the IDH1 protein having substitutions selected from the R132C, R132G, R132H, and R132S relative to SEQ. ID NO: 1, and the hematological tumor is a cancer selected from leukemia, lymphoma and myeloma.
  • the hematological cancer comprises mutations in the IDH2 protein having substitutions selected from one or more of L86P, Q316L, R140G, R140Q, and R172K, or an in-frame deletion of L143-T146 relative to SEQ. ID NO: 2.
  • the hematological cancer comprises mutations in the IDH2 protein having substitutions selected from the L86P, Q316L, R140G, R140Q, R172K, or an in-frame deletion of L143-T146 relative to SEQ ID NO: 2.
  • the hematological tumor is a cancer selected from leukemia, lymphoma and myeloma.
  • the present disclosure provides a method for continuing to treat hematological cancer in a patient with a therapeutic regimen comprising a therapeutically effective amount of a combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and a hypomethylating agent, the method comprising: a) obtaining a first biological sample from the patient and a second biological sample from the patient, wherein both samples comprise hematological cancer cells or ctDNA, and wherein the first sample is obtained prior to the second sample and the patient is treated with the therapeutic regimen prior to the second sample; b) measuring at least one characteristic of at least one marker corresponding to an IDH1 gene and/or an IDH2 gene in the two samples; c) comparing the results of the measurements in b); and d) continuing treatment with the therapeutic regimen if the comparison indicates that the hematological cancer cells or ctDNA in the second sample comprises at least one marker gene whose mutational status indicates a favorable outcome
  • the at least one characteristic is selected from the group consisting of size, sequence, composition and amount, - 50 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 and wherein the mutational status of the at least one marker is mutant.
  • the at least one marker is selected from the group consisting of nucleic acid and protein corresponding to the at least one marker, wherein in some embodiments, the at least one characteristic is sequence of the at least one marker.
  • the at least one marker is selected from the group consisting of nucleic acid and protein.
  • the nucleic acid is selected from the group consisting of DNA, ctDNA, mRNA and cDNA and any portion of any of the foregoing, wherein the nucleic acid corresponds to at least one mutation site of the at least one marker gene.
  • the present application provides a use of a combination comprising pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax (ABT-199), and a hypomethylating agent, for example azacitidine, for the manufacture of a medicament for treating a hematological cancer patient, said treating comprises a) using a mutational status of at least one marker gene in a patient sample comprising hematological cancer cells or ctDNA to select a hematological cancer patient who is expected to have a favorable outcome of treatment with the combination, wherein the mutational status is indicative of the favorable outcome, wherein the at least one marker gene is an IDH1 gene and/or an IDH2 gene; and b) treating the hematological cancer patient expected to have the favorable outcome with the combination.
  • a hypomethylating agent for example azacitidine
  • the mutational status is determined by measuring at least one characteristic of at least one marker associated with the at least one marker gene.
  • the at least one characteristic is selected from the group consisting of size, sequence, composition, and amount.
  • the at least one marker is selected from the group consisting of a nucleic acid, and a protein associated with the at least one marker gene, for example, the nucleic acid is genomic DNA, RNA, mRNA, ctDNA or cDNA of the at least one marker gene.
  • the at least one characteristic is a sequence and the at least one marker is a nucleic acid.
  • the present methods encompass determination of the mutational status of the at least one marker gene, wherein the marker gene is mutant.
  • the use described above can be further executed using the following steps: a) measuring at least one characteristic of at least one marker corresponding to the IDH1 gene and/or the IDH2 gene in a biological sample obtained from the patient; b) identifying at least one mutation in the IDH1 gene and/or the IDH2 gene in the biological sample from the at least one characteristic measured in step a); and - 51 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 c) administering the combination to the patient.
  • the at least one marker is selected from the group consisting of nucleic acid and protein corresponding to the IDH1 gene and/or the IDH2 gene.
  • the at least one marker is nucleic acid, such that, the at least one characteristic is selected from the group consisting of: size, sequence, composition, activity and amount of the nucleic acid or protein.
  • the at least one characteristic is sequence, and when the sequence is a sequence of nucleic acid, the nucleic acid is selected from the group consisting of DNA, ctDNA, mRNA and cDNA and a portion of any of the foregoing, and wherein the nucleic acid comprises at least one mutation of the IDH1 gene and/or the IDH2 gene as exemplified in the tables and examples disclosed herein.
  • exemplary mutant marker genes for use in the present methods can include a mutant marker gene that results in the mutation of the codon encoding amino acid R132 of the IDH1 gene.
  • the mutations to the IDH1 gene can include a mutation of the codon relating to R132, wherein the mutated sequence of the IDH1 results in one or more of the following mutations: R132C, R132G, R132H, and R132S.
  • Exemplary mutant marker genes for use in the present methods can include a mutant marker gene that results in the mutation of the codon encoding amino acid L86, R140, L143-T146, R172, or Q316 of the IDH2 gene.
  • the mutations to the IDH2 gene can include a mutation of the codon relating to L86, R140, L143-T146, R172, or Q316, wherein the mutated sequence of the IDH2 results in one or more of the following mutations: L86P, Q316L, R140G, R140Q, and R172K,.
  • the methods of use and treatment of a hematological cancer patient contemplates the treatment of a hematological cancer selected from the group consisting of leukemia, lymphoma, myeloma, acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML).
  • the markers and marker sets described herein assess the likelihood of favorable outcome of therapy (e.g., sensitivity to a therapeutic agent) in patients, e.g., patients having cancer, e.g., hematological cancer, such as leukemia, lymphoma or myeloma (e.g., acute myelogenous leukemia, myelodysplastic syndrome or chronic myelomonocytic leukemia), based on a characteristic, e.g., size, sequence, composition or amount of a marker or markers of the invention. Using this prediction, the patient can be treated with a therapy regimen best suitable for a favorable outcome.
  • hematological cancer such as leukemia, lymphoma or myeloma
  • myeloma e.g., acute myelogenous leukemia, myelodysplastic syndrome or chronic myelomonocytic leukemia
  • the methods characterize patient cancer treatment outcome with a regimen comprising an NAE inhibitor as described in earlier sections.
  • the agents provided in the - 52 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 present methods can be a single agent or a combination of agents.
  • the methods include combination of NAE inhibition therapy, for example, pevonedistat, or a pharmaceutically acceptable salt thereof, with a BCL2 inhibitor, for example, venetoclax (ABT-199), and a hypomethylating agent, for example, azacitidine.
  • the present methods can be used to determine whether a hematological cancer (e.g., AML, MDS, or CMML) is more likely to benefit from treatment with (a) a regimen that includes a BCL2 inhibitor, a hypomethylating agent, and an NAE inhibitor; or (b) a regimen that includes a BCL2 inhibitor and a hypomethylating agent, but does not include an NAE inhibitor.
  • a hematological cancer e.g., AML, MDS, or CMML
  • Useful combinations can include agents that have other mechanisms of action, e.g., an anti-mitotic agent, an alkylating agent, an antimetabolite, or a proteasome inhibitor.
  • the methods provide for a therapeutic regimen comprising pevonedistat or a pharmaceutically acceptable salt thereof, a BCL2 inhibitor, e.g. venetoclax (ABT-199), and a hypomethylating agent, e.g. azacitidine.
  • the methods provide for a therapeutic regimen comprising pevonedistat or a pharmaceutically salt thereof, venetoclax (ABT-199), and azacitidine.
  • Illustrative hematological cancers treatable using the methods of the present disclosure may include, acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML).
  • AML acute myelogenous leukemia
  • MDS myelodysplastic syndrome
  • CMML chronic myelomonocytic leukemia
  • AML acute myelogenous leukemia
  • MDS myelodysplastic syndrome
  • CMML chronic myelomonocytic leukemia
  • proteasome inhibitor refers to any substance which directly inhibits enzymatic activity of the 20S or 26S proteasome in vitro or in vivo.
  • proteasome inhibitors are bortezomib, carfilzomib, ixazomib, disulfiram, epigallocatechin-3- gallate, salinosporamid A, ONX0912, CEP-18770, and epoxomicin.
  • Other therapeutic agents for use in combination with NAE inhibition therapy include chemotherapeutic agents.
  • a "chemotherapeutic agent” is intended to include chemical reagents which inhibit the growth of proliferating cells or tissues wherein the growth of such cells or tissues is undesirable.
  • Chemotherapeutic agents used in the treatment of hematological cancer such as leukemia, e.g., AML, MDS or CMML include antibiotics, e.g., daunorubicin, adriamycin or idarubicin, or antimetabolites, e.g., pyrimidine analogs, e.g., cytarabine or gemcitabine, and are well known in the art (see e.g., Gilman A.G., et al., The Pharmacological - 53 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 Basis of Therapeutics, 8th Ed., Sec 12:1202-1263 (1990)), and are typically used to treat neoplastic diseases.
  • the patient with cancer e.g., a hematological cancer, for example, acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML)
  • a hematological cancer for example, acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML)
  • ABT-199 venetoclax
  • a hypomethylating agent for example, azacitidine
  • patients with AML are ineligible for intensive chemotherapy due to pre-existing comorbidities, such as severe cardiac or pulmonary disorder or disorders identified by clinical laboratory values that make them ineligible for standard-of-care, and/or older age, e.g., ⁇ 75 years old.
  • pevonedistat or a pharmaceutically acceptable salt thereof is administered on each of days 1, 3, and 5 of a 28 day cycle.
  • the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is less than or equal to 50 mg/m2.
  • the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 37 mg/m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 25 mg/m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 20 mg/m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 15 mg/m2.
  • the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 10 mg/m2 to about 30 mg/m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 15 mg/m2 to about 30 mg/m2. [00147] In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day cycle is about 20 mg/m2, which can be given as a 60-minute intravenous infusion.
  • pevonedistat or a pharmaceutically acceptable salt thereof is administered on each of days 1, 3, and 5 of a 21 day cycle.
  • the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each - 54 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 of days 1, 3, and 5 of a 21 day cycle is less than or equal to 50 mg/m2.
  • the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 21 day cycle is about 37 mg/m2.
  • the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 21 day cycle is about 25 mg/m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 21 day cycle is about 20 mg/m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 21 day cycle is about 15 mg/m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 21 day cycle is about 10 mg/m2 to about 30 mg/m2.
  • the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 21 day cycle is about 15 mg/m2 to about 35 mg/m2.
  • pevonedistat or a pharmaceutically acceptable salt thereof is administered on each of days 1, 8, and 15 of a 28 day cycle.
  • the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 8, and 15 of a 28 day cycle is less than or equal to 100 mg/m2.
  • the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 8, and 15 of a 28 day cycle is about 25 mg/m2.
  • the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 8, and 15 of a 28 day cycle is about 20 mg/m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 8, and 15 of a 28 day cycle is about 15 mg/m2. In some embodiments, the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 8, and 15 of a 28 day cycle is about 15 mg/m2 to about 40 mg/m2.
  • the number of cycles of pevonedistat or a pharmaceutically acceptable salt thereof administered to patients is at least two cycles, at least three cycles, at least four cycles, at least five cycles, at least six cycles, at least seven cycles, at least eight cycles, at least nine cycles or at least ten cycles.
  • a patient is administered two to twenty-five cycles of pevonedistat or a pharmaceutically acceptable salt thereof.
  • a patient is administered four to twelve cycles of pevonedistat or a pharmaceutically acceptable salt thereof.
  • pevonedistat or a pharmaceutically acceptable salt thereof is administered on each of days 1, 3, and 5 of a 28 day cycle at about 20 mg/m2 for at least six cycles.
  • the agents disclosed herein may be administered by any route, including intradermally, subcutaneously, orally, intraarterially or intravenously. In one embodiment, administration will be by the intravenous route. Parenteral administration can be provided in a bolus or by infusion. [00152] In some embodiments, pevonedistat or a pharmaceutically acceptable salt thereof is administered in combination with venetoclax.
  • a 100 mg venetoclax tablet is administered orally on day 1; a 200 mg venetoclax tablet is administered orally on day 2; and thereafter a 400 mg venetoclax tablet is administered daily on days 3-28 in a first 28-day cycle; and in subsequent cycle(s), a 400 mg venetoclax tablet is administered daily on days 1-28, if tolerated.
  • pevonedistat or a pharmaceutically acceptable salt thereof, and venetoclax is administered in combination with azacitidine.
  • azacitidine is administered intravenously or subcutaneously at about 75 mg/m2 on days 1 through 7 of a 28-day cycle; or, at about 75 mg/m2 on days 1 through 5, day 8, and day 9 of a 28-day cycle. In some embodiments, azacitidine is administered intravenously or subcutaneously at about 75 mg/m2 on days 1 through 5, day 8, and day 9 of a 28-day cycle. [00154] In some embodiments, a patient with a confirmed mutation in IDH1 and/or IDH2 is treated with a combination of pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax, and azacitidine.
  • the amount of pevonedistat or a pharmaceutically acceptable salt thereof that is administered on each of days 1, 3, and 5 of a 28 day schedule is about 20 mg/m2.
  • a 100 mg venetoclax tablet is administered orally on day 1; a 200 mg venetoclax tablet is administered orally on day 2; and thereafter a 400 mg venetoclax tablet is administered daily on days 3-28 in a first 28-day cycle; and in subsequent cycle(s), a 400 mg venetoclax tablet is administered daily on days 1-28, if tolerated.
  • azacitidine is administered intravenously or subcutaneously at about 75 mg/m2 on days 1 through 5, day 8, and day 9 of a 28-day cycle.
  • a hematological cancer patient with a confirmed mutation in IDH1 and/or IDH2 is treated with a combination of pevonedistat or a pharmaceutically acceptable salt thereof, venetoclax, and azacitidine, according to the following regimen: about 20 mg/m2 pevonedistat or a pharmaceutically acceptable salt thereof is administered intravenously on each of days 1, 3, and 5 of a 28 day cycle; about 100 mg venetoclax is administered orally on day 1, about 200 mg venetoclax is administered orally on day 2, and about 400 mg venetoclax is administered on days - 56 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 3 through 28 in a first 28-day cycle, and if tolerated in subsequent cycle(s), about 400 mg venetoclax is administered on days 1-28; and about 75 mg/m2 azacitidine is administered intravenously or subcutaneously on days 1 through 5, day 8, and day 9 of a
  • the concentration of a disclosed compound in a pharmaceutically acceptable mixture will vary depending on several factors, including the dosage of the compound to be administered, the pharmacokinetic characteristics of the compound(s) employed, and the route of administration.
  • the agent may be administered in a single dose or in repeat doses. Treatments may be administered daily or more frequently depending upon a number of factors, including the overall health of a patient, and the formulation and route of administration of the selected compound(s).
  • Electronic Apparatus Readable Arrays [00158] Electronic apparatus, including readable arrays comprising at least one predictive marker of the present invention is also contemplated for use in conjunction with the methods of the invention.
  • “electronic apparatus readable media” refers to any suitable medium for storing, holding or containing data or information that can be read and accessed directly by an electronic apparatus.
  • the term “electronic apparatus” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the present invention and monitoring of the recorded information include stand-alone computing apparatus; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.
  • LAN local area network
  • WAN wide area network
  • Extranet Intranet
  • electronic appliances such as personal digital assistants (PDAs), cellular phone, pager and the like
  • local and distributed processing systems local and distributed processing systems.
  • “recorded” refers to a process for storing or encoding information on the electronic apparatus readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the markers of the present invention.
  • microarray systems are well known and used in the art for assessment of samples, whether by assessment of gene expression (e.g., DNA detection, RNA detection, protein detection), or metabolite production, for example.
  • Microarrays for use according to the invention include one or more probes of predictive marker(s) of the invention characteristic of response and/or non-response to a therapeutic regimen as described herein.
  • the microarray comprises one or more probes corresponding to one or more of markers selected from the group consisting of markers which demonstrate increased expression - 57 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 in short term survivors, and genes which demonstrate increased expression in long term survivors in patients.
  • a number of different microarray configurations and methods for their production are known to those of skill in the art.
  • a microarray thus comprises one or more probes corresponding to one or more markers identified herein, e.g., those indicative of treatment outcome, e.g., to identify wild type marker genes, normal allelic variants and alterations of marker genes.
  • the microarray can comprise probes corresponding to, for example, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, or at least 100, markers and/or alterations thereof indicative of treatment outcome.
  • the microarray can comprise probes corresponding to one or more markers as set forth herein.
  • the microarray may comprise complete marker sets as set forth herein and which may be selected and compiled according to the methods set forth herein.
  • the microarray can be used to assay expression of one or more predictive markers or predictive marker sets in the array.
  • the array can be used to assay more than one predictive marker or marker set expression in a sample to ascertain an expression profile of markers in the array. In this manner, up to about 44,000 markers can be simultaneously assayed for expression. This allows an expression profile to be developed showing a battery of markers specifically expressed in one or more samples. Still further, this allows an expression profile to be developed to assess treatment outcome. [00161]
  • the array is also useful for ascertaining differential expression patterns of one or more markers in normal and abnormal (e.g., sample, e.g., tumor) cells. This provides a battery of markers that could serve as a tool for ease of identification of treatment outcome of patients. Further, the array is useful for ascertaining expression of reference markers for reference expression levels.
  • the array can be used to monitor the time course of expression of one or more markers in the array.
  • the invention allows the quantification of marker expression.
  • predictive markers can be grouped on the basis of marker sets or outcome indications by the amount of the marker in the sample. This is useful, for example, in ascertaining the outcome of the sample by virtue of scoring the amounts according to the methods provided herein.
  • kits for assaying a characteristic, e.g., size, sequence, composition or amount, of a marker, e.g., polypeptide or nucleic acid corresponding to a marker gene of the invention in a biological sample e.g., a bone marrow or blood sample, - 58 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 tumor biopsy or a reference sample).
  • kits can be used, e.g., in the methods described herein, to determine mutational status of at least one marker gene for treating a patient who will experience a favorable outcome, e.g., after a treatment regimen comprising an NAE inhibitor, e.g., pevonedistat or a pharmaceutically acceptable salt thereof.
  • the kit can comprise a probe or reagent capable of detecting a genomic DNA segment, ctDNA, a polypeptide or a transcribed RNA corresponding to a marker of the invention or an alteration of a marker gene in a biological sample and means for determining the amount of the genomic DNA segment, the ctDNA, the polypeptide or the mRNA in the sample.
  • Suitable reagents for binding with a marker protein include antibodies, antibody derivatives, antibody fragments, and the like.
  • Suitable reagents for binding with a marker nucleic acid include complementary nucleic acids.
  • a label can be directly attached to the marker binding agent, e.g., probe, e.g., nucleic acid reagent such as a probe or primer or protein reagent, such as a specific binding agent or antibody, or a secondary reagent can comprise a label for indirect labeling.
  • the kit can also contain a control or reference sample or a series of control or reference samples which can be assayed and compared to the test sample.
  • the kit may have a positive control sample, e.g., including one or more markers or alterations described herein, or reference markers, e.g. housekeeping markers to standardize the assay among samples or timepoints or reference genomes, e.g., from subjects without tumor e.g., to establish diploid copy number baseline or reference expression level of a marker.
  • the kit may comprise fluids (e.g., buffer) suitable for annealing complementary nucleic acids or for binding an antibody with a protein with which it specifically binds and one or more sample compartments.
  • the kit of the invention may optionally comprise additional components useful for performing the methods of the invention, e.g., a sample collection vessel, e.g., a tube, and optionally, means for optimizing the amount of marker detected, for example if there may be time or adverse storage and handling conditions between the time of sampling and the time of analysis.
  • the kit can contain means for increasing the number of tumor cells in the sample, as described above, a buffering agent, a preservative, a stabilizing agent or additional reagents for preparation of cellular material or probes for use in the methods provided; and detectable label, alone or conjugated to or incorporated within the provided probe(s).
  • a kit comprising a sample collection vessel can comprise e.g., a tube comprising anti-coagulant and/or stabilizer, e.g., an RNA stabilizer, as described above, or known to those skilled in the art.
  • the kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate).
  • the kit can comprise a marker set array or chip for use in detecting - 59 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 the biomarkers. Kits also can include instructions for interpreting the results obtained using the kit.
  • the kit can contain reagents for detecting one or more markers, e.g., 2, 3, 4, 5, or more markers described herein.
  • the kit comprises a probe to detect at least one nucleic acid marker, e.g., a marker indicative of treatment outcome (e.g., upon NAE inhibitor treatment).
  • the kit comprises a nucleic acid probe to detect a marker nucleic acid corresponding to a marker gene described herein.
  • the marker nucleic acid can be selected from the group consisting of SEQ ID NO: 3 or 5 and SEQ ID NO: 4 or 6 listed in Table 1, a portion of a marker nucleic acid comprising a sequence or mutated codon listed in in the disclosure above, Table 2, and in the Examples, and a complement of any of the foregoing.
  • the kit comprises a probe to detect a nucleic acid corresponding to a marker gene selected from the group consisting of IDH1 and/or IDH2.
  • a kit comprises a probe to detect wild type IDH1 and a probe to detect a genetic alteration, e.g., a mutation or truncation, in IDH2.
  • a kit comprises a probe to detect wild type IDH2 and a probe to detect a genetic alteration, e.g., a mutation or truncation, in IDH1.
  • a kit comprises a probe to detect a genetic alteration, e.g., a mutation or truncation, in IDH1 and a probe to detect a genetic alteration, e.g., a mutation or truncation, in IDH2.
  • the kit can comprise, for example: one or more nucleic acid reagents such as an oligonucleotide (labeled or non-labeled) which hybridizes to a nucleic acid sequence corresponding to a marker of the invention, optionally fixed to a substrate; and can optionally further comprise labeled oligonucleotides not bound with a substrate, a primer, a pair of PCR primers, e.g., useful for amplifying a nucleic acid molecule corresponding to a marker of the invention, molecular beacon probes, a marker set comprising oligonucleotides which hybridize to at least two nucleic acid sequences corresponding to markers of the invention, and the like.
  • nucleic acid reagents such as an oligonucleotide (labeled or non-labeled) which hybridizes to a nucleic acid sequence corresponding to a marker of the invention, optionally fixed to a substrate
  • the kit can contain an RNA- stabilizing agent.
  • PCR-based kits to measure IDH1 and IDH2 mutations in blood and bone marrow samples from AML patients are available as companions to some AML treatments.
  • the Abbott REALTIME IDH1 assay measures five single nucleotide variants at R132 of IDH1 for TIBSOVO® (ivosidenib) or with REZLIDHIA TM (olutasidenib).
  • Abbott REALTIME IDH2 assay measures four single nucleotide variants at R140 and five single nucleotide variants at R172 of IDH2.
  • a kit to assay mutational status of a protein marker of the invention can comprise one or more reagents to measure the activity of the protein.
  • the kit may comprise a reporter gene for use in the methods described herein.
  • the kit may comprise a substrate of an enzyme, e.g., isocitrate.
  • the kit comprises a probe to assay for a characteristic, e.g., size, sequence, composition or amount, of a protein marker of the invention.
  • the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker of the invention, e.g., having a sequence selected from the group consisting of SEQ ID NO: 1 or SEQ ID NO:2 or to a genetically altered form(s) thereof, e.g., a mutation in the sequence or a truncation, e.g., comprising SEQ ID NO:17 or as described in the disclosure above, Table 2, and the Examples; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label.
  • a first antibody e.g., attached to a solid support
  • a polypeptide corresponding to a marker of the invention e.g., having a sequence selected from the group consisting of SEQ ID NO: 1 or SEQ ID NO:2 or to a genetically altered form(s) thereof, e
  • the kit can contain a protein stabilizing agent.
  • the kit can contain reagents to reduce the amount of non-specific binding of non-marker material from the sample to the probe.
  • reagents to reduce non-specific binding include nonioinic detergents, non-specific protein containing solutions, such as those containing albumin or casein, or other substances known to those skilled in the art.
  • an isolated polypeptide corresponding to a marker gene of the invention, or a fragment or mutant thereof can be used as an immunogen.
  • an immunogen typically is used to prepare antibodies by immunizing a suitable (i.e., immunocompetent) host such as a rabbit, sheep, goat, mouse, or other mammal, chicken or invertebrate.
  • a suitable (i.e., immunocompetent) host such as a rabbit, sheep, goat, mouse, or other mammal, chicken or invertebrate.
  • the invention provides monoclonal antibodies or antigen binding fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2, an amino acid sequence encoded by a cDNA of the present invention, a fragment of at least 8, 10, 12, 15, 20 or 25 consecutive amino acid residues of an amino acid sequence of the present invention, e.g., a fragment comprising the mutant amino acid sequence, e.g., SEQ ID NO:17 or as described in the disclosure above, Table 2, and the Examples, or comprising a region beyond a t
  • the monoclonal antibodies can be human, humanized, chimeric and/or non-human antibodies.
  • An appropriate immunogenic preparation can contain, for example, recombinantly-expressed or chemically-synthesized polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.
  • an adjuvant such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.
  • Methods for making human antibodies are known in the art.
  • One method for making human antibodies employs the use of transgenic animals, such as a transgenic mouse. These transgenic animals contain a substantial portion of the human antibody producing genome inserted into their own genome and the animal’s own endogenous antibody production is rendered deficient in the production of antibodies. Methods for making such transgenic animals are known in the art.
  • transgenic animals can be made using XENOMOUSE TM technology or by using a "minilocus” approach.
  • Methods for making XENOMICE TM are described in U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598 and 6,075,181, which are incorporated herein by reference.
  • Methods for making transgenic animals using the "minilocus” approach are described in U.S. Pat. Nos.5,545,807, 5,545,806 and 5,625,825; also see International Publication No. WO93/12227, which are each incorporated herein by reference.
  • Antibodies include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention or a genetically altered form thereof, e.g., a mutated amino acid or a truncation of the protein expressed by a marker gene of the invention.
  • a molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
  • Polyclonal and monoclonal antibodies can be produced by a variety of techniques, including conventional murine monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975) the human B cell hybridoma technique (see Kozbor et al., 1983, Immunol. Today 4:72), the EBV-hybridoma technique (see Cole et al., pp. 77-96 In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985) or trioma techniques. See generally, Harlow, E. and Lane, D.
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma - 62 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.
  • the antibody molecules can be harvested or isolated from the host (e.g., from the blood or serum of the host) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibodies specific for a protein or polypeptide of the invention can be selected or (e.g., partially purified) or purified by, e.g., affinity chromatography to obtain substantially purified and purified antibody.
  • a substantially purified antibody composition is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those of the desired protein or polypeptide of the invention, at most 20%, at most 10%, or at most 5% (by dry weight) of the sample is contaminating antibodies.
  • a purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired protein or polypeptide of the invention.
  • the antibodies can also be used diagnostically to monitor protein levels in tissues or body fluids (e.g. in a blood sample or urine) as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • the invention provides substantially purified antibodies or fragments thereof, and non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence encoded by a marker identified herein.
  • the substantially purified antibodies of the invention, or fragments thereof can be human, non-human, chimeric and/or humanized antibodies. - 63 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 [00175]
  • the invention also provides a kit containing an antibody of the invention conjugated to a detectable substance, and instructions for use.
  • Still another aspect of the invention is a prognostic composition comprising a probe of the invention and a pharmaceutically acceptable carrier.
  • the prognostic composition contains an antibody of the invention, a detectable moiety, and a pharmaceutically acceptable carrier.
  • information e.g., about the mutational status of a patient’s cancer, e.g., the patient’s marker(s) characteristic, e.g., size, sequence, composition or amount (e.g., the result of evaluating a marker or marker set described herein), or about whether a patient is expected to have a favorable outcome, is provided (e.g., communicated, e.g., electronically communicated) to a third party, e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • a third party e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • choice of medical procedure whether to pay for a medical procedure, payment by a reimbursing party, or cost for a service or insurance can be function of the information.
  • the third party receives the information, makes a determination based at least in part on the information, and optionally communicates the information or makes a choice of procedure, payment, level of payment, coverage, etc., based on the information.
  • the characteristic e.g., size, sequence, composition or amount, of a marker or a marker set selected from or derived from Table 2and/or described herein above and in the Examples is determined.
  • an entity e.g., a hospital, care giver, government entity, or an insurance company or other entity which pays for, or reimburses medical expenses
  • a party e.g., a party other than the subject patient
  • the method further comprises paying for the procedure wherein the patient has a favorable outcome to therapy comprising an NAE inhibitor, e.g., pevonedistat or a pharmaceutically active salt thereof.
  • the method comprises paying for a procedure comprising treatment with pevonedistat, venetoclax and azacitidine.
  • a premium for insurance (e.g., life or medical) is evaluated as a function of information about one or more marker expression or activity levels, e.g., a marker or marker set, e.g., a level of expression or enzymatic activity associated with treatment outcome (e.g., the informative amount).
  • premiums can be increased (e.g., by a certain percentage) if the marker genes of a patient or a patient’s marker set described herein have different characteristic, e.g., size, sequence, composition or amount between an insured candidate (or a candidate seeking insurance coverage) and a reference value (e.g., a non-afflicted person) or a reference sample, e.g., matched control.
  • Premiums can also be scaled depending on - 64 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 the result of evaluating a marker or marker set described herein.
  • premiums can be assessed to distribute risk, e.g., as a function of marker, e.g., the result of evaluating a marker or marker set described herein.
  • premiums are assessed as a function of actuarial data that is obtained from patients that have known treatment outcomes.
  • Information about marker characteristic e.g., size, sequence, composition or amount, e.g., the result of evaluating a marker or marker set described herein, can be used, e.g., in an underwriting process for life insurance.
  • the information can be incorporated into a profile about a subject. Other information in the profile can include, for example, date of birth, gender, marital status, banking information, credit information, children, and so forth.
  • An insurance policy can be issued as a function of the information on marker characteristic, e.g., size, sequence, composition or amount, e.g., the result of evaluating a marker or marker set described herein, along with one or more other items of information in the profile.
  • a first entity e.g., an insurance company
  • the disclosure features a method of providing data.
  • the method includes providing data described herein, e.g., generated by a method described herein, to provide a record, e.g., a record described herein, to proceed a payment.
  • the data are provided by computer, compact disc, telephone, facsimile, email, or letter.
  • the data are provided by a first party to a second party.
  • the first party is selected from a healthcare provider, a treating physician, a health maintenance organization (HMO), a hospital, a governmental entity, or an entity which sells or supplies the drug.
  • the second party is a third party payor, an insurance company, employer, employer-sponsored health plan, HMO, or governmental entity.
  • the first party is selected from a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the drug and the second party is a governmental entity.
  • the first party is selected from a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the drug and the second party is an insurance company.
  • a record e.g., computer readable record
  • the record includes more than one value for each marker.
  • - 65 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01
  • EXAMPLES Example 1 Phase 2 clinical study of Pevonedistat and Venetoclax Combined With Azacitidine.
  • the clinical trial (NCT04266795) centered on a study administering pevonedistat (PEV) with venetoclax (VEN) combined with azacitidine (AZA) in adults unable to receive intensive chemotherapy.
  • PEV pevonedistat
  • VEN venetoclax
  • AZA azacitidine
  • the main aim was to see how the combination of PEV+ VEN+ AZA compares to VEN + AZA in adults recently diagnosed with AML who are unable to be treated with intensive chemotherapy.
  • the Primary objectives included disease response, measured as event free survival (EFS) up to 36 months.
  • EFS event free survival
  • PEV single-agent pevonedistat
  • NAE NEDD8 activating enzyme
  • NEDD8 Neural Cell Developmentally-Downregulated 8
  • CTLs Cullin-RING E3 ubiquitin ligases
  • IV intravenous
  • SC subcutaneous
  • Patients Eligible patients were ⁇ 18 years old with untreated AML, considered unlikely to benefit from standard induction defined by ⁇ 1 of the following: Has morphologically confirmed diagnosis of AML (World Health Organization [WHO] criteria 2008). Participants may have newly diagnosed primary de novo AML or secondary AML (sAML), defined as AML after myelodysplastic syndromes (MDS) or myeloproliferative neoplasm (MPN), or therapy- related AML (t-AML) following cytotoxic therapy, and/or radiotherapy for a malignant or nonmalignant disease.
  • sAML primary de novo AML or secondary AML
  • MDS myelodysplastic syndromes
  • MPN myeloproliferative neoplasm
  • t-AML therapy- related AML
  • Patients may also be unfit for treatment with a standard arabinosylcytosine (Ara-C) and anthracycline induction regimen due to age or co-morbidities defined by 1 of the following: ⁇ 75 years of age. OR ⁇ 18 to ⁇ 75 years of age with at least one of the following: [00189]
  • the patient has a Eastern Cooperative Oncology Group (ECOG) performance status of 2 or 3.
  • ECG Eastern Cooperative Oncology Group
  • Severe cardiac disorder e.g., congestive heart failure requiring treatment, ejection fraction ⁇ 50%, or chronic stable angina.
  • Severe pulmonary disorder e.g., carbon monoxide lung diffusion capacity ⁇ 65% or forced expiratory volume in 1 second ⁇ 65%).
  • Participants who are cytoreduced with leukapheresis or with hydroxyurea may be enrolled if they meet the eligibility criteria before starting therapy.
  • Exclusion Criteria [00196] Patients were excluded if one or more of the following applied: [00197] Has history of MPN with BCR-ABL1 translocation or AML with BCR-ABL1 translocation. [00198] Has genetic diagnosis of acute promyelocytic leukemia. [00199] Is eligible for intensive chemotherapy and/or allogeneic stem cell transplantation. [00200] Has extramedullary AML without evidence of bone marrow involvement. [00201] Had prior treatment with hypomethylating agents for AML (hypomethylating agent treatment for prior MDS is not exclusionary).
  • [00202] Has clinical evidence of or history of central nervous system involvement by AML. [00203] Had diagnosed or treated for another malignancy (except for adequately treated carcinoma in situ of any organ or nonmelanoma skin cancer) within 1 year before randomization or previously diagnosed with another malignancy and have any evidence of residual disease that may compromise the administration of pevonedistat, venetoclax or azacitidine. Prior MDS is also allowed, but the participant cannot have received treatment for MDS within 14 days before first dose of any study drug. [00204] Has a WBC count ⁇ 25 ⁇ 109/L [00205] Has uncontrolled human immunodeficiency virus (HIV) infection. Note: Known HIV positive participants who meet the following criteria will be considered eligible: [00206] A.
  • HIV human immunodeficiency virus
  • Participant is known to be positive for hepatitis B or C infection, with the exception of those with an undetectable viral load within 3 months (hepatitis B or C testing is not required for eligibility assessment).
  • the primary objectives were to see how the combination of pevonedistat + venetoclax + azacitidine compares to venetoclax + azacitidine in adults recently diagnosed with AML who are unable to be treated with intensive chemotherapy.
  • Secondary objectives included descriptions of PEV pharmacokinetics (PK) in whole blood and a preliminary assessment of anti-tumor activity. The study enrolled approximately 164 patients.
  • Pevonedistat will be given as an intravenous (IV) infusion and Azacitidine will be given through IV or subcutaneous (under the skin). VEN will be administered as an oral tablet. Study treatments may continue as long as the participant is receiving benefit from it. Participants may choose to stop treatment at any time. The study will enroll approximately 164 patients.
  • VEN 400 mg can be administered on Day 1 through 21 or 28 as per Investigator's discretion, plus AZA at 75 mg/m 2 intravenous (IV) or subcutaneous (SC) dosing on Day 1 through 7 or Days 1 through 5, Days 8, and 9 in each cycle.
  • IV intravenous
  • SC subcutaneous
  • VEN dosed at 400 mg can be administered on Day 1 through 21 or 28 as per Investigator's discretion, plus AZA 75 mg/m 2 IV or SC dosing on Days 1 through 7 or Days 1 through 5, Days 8, and 9 in each cycle.
  • Safety and efficacy assessments [00230] Patient demographics and medical history were recorded at baseline. Adverse event (AE) assessments, physical examination, vital signs, and ECOG PS were documented at baseline and on day 1 of subsequent cycles for the duration of the study. Safety was assessed from informed consent to 30 days after final doses of study therapy.
  • AE Adverse event
  • EFS Event-Free Survival
  • CR European Leukemia Net 2017 guidelines.
  • CR is defined as bone marrow blasts ⁇ 5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; absolute neutrophil count (ANC) ⁇ 1.0 ⁇ 10 9 /L (1000/ ⁇ L); platelet count ⁇ 100 ⁇ 10 9 /L (100,000/ ⁇ L).
  • ANC absolute neutrophil count
  • CRi is defined as all CR criteria except for residual neutropenia ( ⁇ 1.0 ⁇ 10 9 /L [1000/ ⁇ L]) or thrombocytopenia ( ⁇ 100 ⁇ 10 9 /L [100,000/ ⁇ L]).
  • OS Overall Survival
  • Mortality rate is defined as percentage of participants who survive at most 60 days from the first dose of study drug.
  • Percentage of Participants with Complete Remission (CR) [ Time Frame: Up to 36 months ].
  • CR rate is defined as the percentage of participants who achieve the CR as evaluated by the investigator. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines.
  • CCR is defined as bone marrow blasts ⁇ 5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC ⁇ 1.0 ⁇ 10 9 /L (1000/ ⁇ L); platelet count ⁇ 100 ⁇ 10 9 /L (100,000/ ⁇ L).
  • CCR rate is defined as the percentage of participants who achieve the CR + CRi as evaluated by the investigator. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines.
  • CR is defined as bone marrow blasts ⁇ 5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC ⁇ 1.0 ⁇ 10 9 /L - 72 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 (1000/ ⁇ L); platelet count ⁇ 100 ⁇ 10 9 /L (100,000/ ⁇ L).
  • CRi is defined as all CR criteria except for residual neutropenia ( ⁇ 1.0 ⁇ 10 9 /L [1000/ ⁇ L]) or thrombocytopenia ( ⁇ 100 ⁇ 10 9 /L [100,000/ ⁇ L]).
  • ORR Overall Response Rate
  • ORR is defined as the percentage of participants who achieve the CR + CRi + Partial Remission (PR) as evaluated by the investigator. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines.
  • CR is defined as bone marrow blasts ⁇ 5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC ⁇ 1.0 ⁇ 10 9 /L (1000/ ⁇ L); platelet count ⁇ 100 ⁇ 10 9 /L (100,000/ ⁇ L).
  • CRi is defined as all CR criteria except for residual neutropenia ( ⁇ 1.0 ⁇ 10 9 /L [1000/ ⁇ L]) or thrombocytopenia ( ⁇ 100 ⁇ 10 9 /L [100,000/ ⁇ L]).
  • PR is defined as all hematologic criteria of CR; decrease of bone marrow blast percentage to 5% to 25%; and decrease of pretreatment bone marrow blast percentage by at least 50%.
  • Percentage of Participants with CR + CRh [ Time Frame: Up to 36 months ].
  • CR + CRh rate is defined as the percentage of participants who achieve the CR + CRh as evaluated by the investigator. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines.
  • CR is defined as bone marrow blasts ⁇ 5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC ⁇ 1.0 ⁇ 10 9 /L (1000/ ⁇ L); platelet count ⁇ 100 ⁇ 10 9 /L (100,000/ ⁇ L).
  • CRh is defined as bone marrow with ⁇ 5% blasts, peripheral blood neutrophil count >0.5 ⁇ 10 3 / ⁇ L and peripheral blood platelet count >0.5 ⁇ 10 5 / ⁇ L.
  • Leukemia response rate is defined as the percentage of participants who achieve the CR + CRi + PR + morphological leukemia-free state [MLFS, marrow CR (mCR)]) as evaluated by the investigator. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines.
  • CR is defined as bone marrow blasts ⁇ 5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC ⁇ 1.0 ⁇ 10 9 /L (1000/ ⁇ L); platelet count ⁇ 100 ⁇ 10 9 /L (100,000/ ⁇ L).
  • CRi is defined as all CR criteria except for residual neutropenia ( ⁇ 1.0 ⁇ 10 9 /L [1000/ ⁇ L]) or thrombocytopenia ( ⁇ 100 ⁇ 10 9 /L [100,000/ ⁇ L]).
  • PR is defined as all hematologic criteria of CR; decrease of bone marrow blast percentage to 5% to 25%; and decrease of pretreatment bone marrow blast percentage by at least 50%.
  • MLFS is defined as bone marrow blasts ⁇ 5%; absence of blasts with Auer rods; absence of extramedullary disease; no hematologic recovery required. [00249] Duration of CR and CRi [ Time Frame: Up to 36 months ].
  • Duration of CR and CRi is defined as the time from first documentation of CR or CRi to the date of first documentation of PD or relapse from CR or CRi, and will be summarized descriptively using the K-M method based on the responders. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines.
  • CR is defined as bone marrow blasts ⁇ 5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC ⁇ 1.0 ⁇ 10 9 /L (1000/ ⁇ L); platelet count ⁇ 100 ⁇ 10 9 /L (100,000/ ⁇ L).
  • CRi is defined as all CR criteria except for residual neutropenia ( ⁇ 1.0 ⁇ 10 9 /L [1000/ ⁇ L]) or thrombocytopenia ( ⁇ 100 ⁇ 10 9 /L [100,000/ ⁇ L]).
  • Time to First CR, CRi and PR [ Time Frame: Up to 36 months ].
  • Time to first CR, CRi, and PR is defined as the time from randomization until the first documented CR, CRi or PR, whichever occurs first, and will be analyzed using the K-M method. Assessments of disease response are based on the criteria outlined in the ELN 2017 guidelines.
  • CR is defined as bone marrow blasts ⁇ 5%; absence of circulating blasts and blasts with Auer rods; absence of extramedullary disease; ANC ⁇ 1.0 ⁇ 10 9 /L (1000/ ⁇ L); platelet count ⁇ 100 ⁇ 10 9 /L (100,000/ ⁇ L).
  • CRi is defined as all CR criteria except for residual neutropenia ( ⁇ 1.0 ⁇ 10 9 /L [1000/ ⁇ L]) or thrombocytopenia ( ⁇ 100 ⁇ 10 9 /L [100,000/ ⁇ L]).
  • PR is defined as all hematologic criteria of CR; decrease of bone marrow blast percentage to 5% to 25%; and decrease of pretreatment bone marrow blast percentage by at least 50%.
  • Bone Marrow Mononuclear Cell Processing Cell pellets were prepared at Q 2 Solutions from bone marrow aspirates collected in K2EDTA. After mononuclear cell separation using standard Ficoll-Paque gradient centrifugation, the cell interface layer was harvested carefully, followed by cell lysis with RBC lysis buffer.
  • DNA and RNA isolation Bone marrow cell pellets were shipped from Q 2 Solutions to the Broad institute (Cambridge, MA) for DNA/RNA isolation. The All-Prep DNA/RNA/miRNA Universal Kit (Qiagen Inc.) was used following manufacturer’s protocol. DNA samples were used for further mutational analyses.
  • NGS Analyses Molecular mutational analysis by NGS was performed at Center for Advanced Molecular Diagnostics at Brigham and Women’s Hospital (Boston, MA) using their Rapid Heme Panel (RHP) assay.
  • the Rapid heme panel identifies gene alterations associated with various neoplasms that are frequently driven by recurrent, somatically acquired genetic variants that affect DNA sequence or copy number.
  • the RHPv3 assay identifies somatic single nucleotide variants (SNVs), small insertion/deletions (indels), and copy number variants (CNV) implicated in hematologic neoplasms in a targeted panel consisting of 88 genes (Table 3), using NEBNext Direct technology. Parallel sequencing was performed on an Illumina NextSeq 550Dx.
  • Table 3 List of genes in the Rapid Heme Panel (RHP) for NGS BTK EZH2 NFE2 SMC1A - 75 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 CALR FBXW7 NOTCH1 SMC3 [00261] DNA samples were processed in duplicate using the NEBNext Direct hybridization-based target enrichment method (New England Biolabs, Ipswich, MA, USA). As used herein, nucleic acids in a sample can be subject to target enrichment, in which molecules having target sequences are captured for subsequent analysis.
  • RHP Rapid Heme Panel
  • Target enrichment can involve use of a bait set comprising oligonucleotide baits labeled with a capture moiety, such as biotin.
  • the probes can have sequences selected to tile across a panel of regions, such as the genes in the RHP v3. Genomic DNA is enzymatically nicked and then hybridized to biotinylated oligonucleotide baits that capture both strands of the target DNA and define the 3' ends of the regions of interest. After hybridization, the bait-target hybrids are bound to streptavidin beads and any 3' off-target sequences are removed enzymatically.
  • the trimmed targets are then converted into Illumina-compatible libraries that include a 12 bp unique molecular identifier in the i5 index location and an 8bp sample barcode in the Illumina i7 index location.
  • the Flt3-ITD allelic ratio was determined by a PCR-based assay using DNA as input. PCR products were amplified from the juxtamembrane domain (exons 14 and 15) of the FLT3 gene using fluorescently labeled primers. If present, an ITD results in a larger PCR product than normal.
  • PCR products were then visualized by capillary electrophoresis (CE) using ABL 3500xL Dx Genetic Analyzer with a Genescan LIZ size standard. Traces were analyzed on the GeneMapper 6.0 software. Allelic ratio was calculated using the area under the curve (AUC) calculations for the mutant(s) over wild type peaks.
  • CE capillary electrophoresis
  • AML risk assessment was performed by a single pathologist for consistency at BWH by combining cytogenetic abnormalities, mutation data from the RHP panel, and FLT3- - 76 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 ITD results to stratify patients into three outcome groups, i.e., Adverse, Intermediate, and Favorable, using the European Leukemia Net (ELN) 2017 risk stratification system. For nine of 139 samples where cytogenetic results were not available from internal assessments at BWH (e.g., poor sample quality), cytogenetic results from pathology reports from the local sites were used instead.
  • ESN European Leukemia Net
  • ENN-2017 European LeukemiaNet
  • AML acute myeloid leukemia
  • This stratification analysis is broadly accepted by physicians as a gold standard and provide guidelines to stratify patients into three outcome groups: favorable, intermediate, and adverse based on cytogenetics and mutation status of commonly mutated and oncogenic genes in AML, such as ASXL1, CEBPA, FLT3, NPM1, RUNX1, DNMT3A, IDH1, IDH2, SRSF2, TET2 and TP53.
  • This stratification scheme provides a simple, yet powerful means to triage patients for appropriate therapies.
  • Table 6 lists the mutations found in sam les from the atient o ulation: , R R R R R172K Substitution* 9 - 78 - 56340669.1 Attorney Docket No.223266-549182 / PAT27156PCT01 L143-T146 In-frame deletion *m nt-to- treat (ITT) patients that were not RE.
  • ITT In-frame deletion *m nt-to- treat
  • FLT3 FLT3 internal tandem duplication mutations
  • FLT3-ITD FLT3 internal tandem duplication mutations
  • AML with a FLT3 internal tandem duplication (FLT3/ITD) mutation has been previously thought to be an aggressive hematologic malignancy with a generally poor prognosis. It can be successfully treated into remission with intensive chemotherapy, but it routinely relapses.

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Abstract

L'invention concerne des marqueurs dont l'état mutationnel est associé à la sensibilité au traitement par des inhibiteurs de NAE. L'état mutationnel est déterminé par la mesure de caractéristiques de marqueurs correspondant aux gènes marqueurs. L'invention concerne des compositions et des procédés pour évaluer des marqueurs de gènes marqueurs pour prédire une réponse à un traitement d'inhibition de NAE.
PCT/IB2024/055802 2023-06-16 2024-06-13 Biomarqueurs et méthodes de traitement avec des inhibiteurs de nae Pending WO2024257013A1 (fr)

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