WO2016090255A1 - Biological markers for predicting responsiveness to ibrutinib and r-chop combination therapy and methods of using the same - Google Patents

Biological markers for predicting responsiveness to ibrutinib and r-chop combination therapy and methods of using the same Download PDF

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WO2016090255A1
WO2016090255A1 PCT/US2015/064008 US2015064008W WO2016090255A1 WO 2016090255 A1 WO2016090255 A1 WO 2016090255A1 US 2015064008 W US2015064008 W US 2015064008W WO 2016090255 A1 WO2016090255 A1 WO 2016090255A1
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patient
secreted proteins
amount
biological sample
same
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PCT/US2015/064008
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French (fr)
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Sriram Balasubramanian
Michael Schaffer
Nele FOURNEAU
Shalini CHATURVEDI
Davis CUC
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Sriram Balasubramanian
Michael Schaffer
Fourneau Nele
Chaturvedi Shalini
Cuc Davis
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Publication of WO2016090255A1 publication Critical patent/WO2016090255A1/en

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    • 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
    • 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
    • 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
    • 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

  • the disclosure relates to biological markers useful in identifying and treating patients with diffuse large B-cell lymphoma.
  • the disclosed methods relate to biological markers for identifying a patient as having diffuse large B-cell lymphoma, treating a patient having diffuse large B-cell lymphoma, and methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma.
  • Non-Hodgkin lymphoma is the ninth and seventh most frequent cancer in males and females, respectively.
  • the incidence of NHL appears to be steadily increasing each year in Europe and the US.
  • DLBCL diffuse large B-cell lymphoma
  • R-CHOP the currently accepted standard regimen for frontline treatment of patients with DLBCL is R-CHOP with cure rates around 75%.
  • R-CHOP the currently accepted standard regimen for frontline treatment of patients with DLBCL
  • cure rates around 75% Despite the improvement in clinical outcomes following R-CHOP treatment, a substantial number of patients still have disease, which either fails to respond to initial therapy or relapses after initial remission.
  • the methods can comprise determining an amount of one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof, and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the
  • the methods can comprise determining an amount of one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof, and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
  • the determining can comprise assaying a biological sample from the patient to detect an amount of the one or more secreted proteins.
  • the methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma can comprise assaying a biological sample from the patient to detect an amount of one or more secreted proteins, wherein the one or more secreted proteins are CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFCl, FLRTl, IFN-g, JAM-C, prostatic binding protein, or any combination thereof and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and
  • the methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma can comprise assaying a biological sample from the patient to detect an amount of one or more secreted proteins, wherein the one or more secreted proteins are AURKB, HCK, TWEAKR, Nectin-like protein 1 , Angiogenin, CK-MB, or any combination thereof and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
  • the disclosed methods can comprise administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof.
  • the methods of treating a patient having diffuse large B-cell lymphoma can further comprise, prior to the administering, determining an amount of one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof and comparing the amount of the one or more secreted proteins in the biological sample to a mean amount of the same one or more secreted proteins from a control.
  • the one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM
  • the methods of treating a patient having diffuse large B-cell lymphoma can comprise administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof.
  • the methods of treating a patient having diffuse large B-cell lymphoma can further comprise, prior to the administering, determining the amount of the one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin- like protein 1 , Angiogenin, CK-MB, or any combination thereof, and comparing the amount of the one or more secreted proteins in the biological sample to the mean amount of the same one or more secreted proteins from the control.
  • Methods of identifying a patient as having diffuse large B-cell lymphoma comprise assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein the one or more genes comprise CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes identifies the patient as having diffuse large B-cell lymphoma.
  • Also disclosed are methods of treating a patient having diffuse large B-cell lymphoma comprising administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a mutation in one or more genes, wherein the one or more genes are CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof.
  • the one or more genes are CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSD1,
  • the method can further comprise, prior to the administering assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein a mutation in the one or more genes identifies the patient as having diffuse large B-cell lymphoma.
  • FIG. 1A to FIG. IT illustrate baseline SomaLogic Analyte Expression in DLBCL subtypes for: FIG. 1A) AURKB; FIG. IB) HCK; FIG. 1C) TWEAKR; FIG. ID) CPNE1; FIG. IE) kininogen, HMW; FIG. IF) nectin-like protein 1 ; FIG. 1G) PA2G4; FIG. 1H) peroxiredoxin-6; FIG. II) PSD7; FIG. 1J) UBE2N; FIG. IK) aflatoxin B l aldehyde reductase; FIG. 1L) angiogenin; FIG. 1M) AREG; FIG. IN) CD27; FIG.
  • FIG. IP germinal center B-cell
  • FIG. 1Q FLRT1
  • FIG. 1R IFN-g
  • FIG. IS JAM-C
  • FIG. IT prostatic binding protein.
  • the left-side of each panel represents germinal center B-cell (GCB) DLBCL; the right- side of each panel represents non-GCB DLBCL.
  • the y-axis in each is the log 2 RFU.
  • FIG. 2A to FIG. 2T illustrate baseline SomaLogic Analyte Expression between subjects with complete response (CR) versus those with partial response (PR) for: FIG. 2A) PSA-ACT; FIG. 2B) IL-17 RD; FIG. 2C) IL-2 sRa; FIG. 2D) C08A1 ; FIG. 2E) Coagulation Factor XI; FIG. 2F) p27Kipl; FIG. 2G) MICB; FIG. 2H) sICAM-5; FIG. 21) Spondin-1 ; FIG. 2J) b2-Microglobulin; FIG. 2K) Cl-Esterase Inhibitor; FIG. 2L) CD30 Ligand; FIG.
  • FIG. 2M HPLNl ; FIG. 2N) NPS-PLA2; FIG. 20) TNF sR-I; FIG. 2P) TNF sR-II; FIG. 2Q) TWEAKR; FIG. 2R) Antithrombin III; FIG. 2S) CTGF; and FIG. 2T) G-CSF-R.
  • the left-side of each panel represents complete response (CR); the right-side of each panel represents partial response (PR).
  • the y-axis in each is the log 2 RFU.
  • FIG. 3 illustrates an exemplary time-course of minimal residual disease (MRD) measurements.
  • any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed methods are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.
  • biological sample refers to any sample from a patient that contains, or can contain, the secreted proteins of interest, including, for example, tumor, blood, serum, or plasma.
  • the biological sample is blood, serum, or plasma.
  • treating and like terms refer to reducing the severity and/or frequency of DLBCL symptoms, eliminating DLBCL symptoms and/or the underlying cause of said symptoms, reducing the frequency or likelihood of DLBCL symptoms and/or their underlying cause, and improving or remediating damage caused, directly or indirectly, by DLBCL.
  • patient as used herein is intended to mean any animal, in particular, mammals. Thus, the methods are applicable to human and nonhuman animals, although preferably used with mice and humans, and most preferably with humans.
  • ibrutinib refers to l-[(3R)-3-[4- amino-3-(4-phenoxyphenyl)-lH-pyrazolo [3, 4 d] pyrimidin-l-yl]-l-piperidinyl]-2-propen-l- one, having a molecular weight of 440.50 g/mole (anhydrous basis), a single chiral center, and being an R-enantiomer.
  • Ibrutinib is a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK).
  • Ibrutinib has been designated as a breakthrough targeted therapy for certain types of B-cell malignancies and is now approved in the United States and European Union for patients with previously treated mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), as well as untreated del 17p or TP53 mutated CLL.
  • MCL mantle cell lymphoma
  • CLL chronic lymphocytic leukemia
  • R-CHOP refers to a drug combination containing rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone.
  • refractory DLBCL refers to DLBCL that is present after treatment.
  • relapsed DLBCL refers to DLBCL that has returned after treatment.
  • DLBCL diffuse large B-cell lymphoma
  • FFPE formalin fixed, paraffin-embedded
  • BTK Brunton's tyrosine kinase
  • MCL mantle cell lymphoma
  • CLL chronic lymphocytic leukemia
  • FL follicular lymphoma
  • R-CHOP rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone
  • NHL non-hodgkin's lymphoma
  • PBMCs peripheral blood mononuclear cells
  • GCB germinal center B-cell
  • ORR overall response rate
  • CR complete response
  • PR partial response
  • AURKB Aurora kinase B
  • HCK Tyrosine-protein kinase HCK
  • TWEAKR Tumor necrosis factor receptor super
  • DLBCL diffuse large B-cell lymphoma
  • DLBCL comprises at least two subgroups: germinal center B-cell like (GCB) BLBCL and non-GCB DLBCL.
  • GCB germinal center B-cell like
  • the disclosed methods can predict a likelihood of
  • the DLBCL can be GCB DLBCL.
  • the DLBCL can be non-GCB DLBCL.
  • the non-GCB DLBCL can be activated B-cell like (ABC) or unclassifiable.
  • the methods can comprise determining an amount of one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFCl, FLRTl, IFN-g, JAM-C, prostatic binding protein, or any combination thereof, and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
  • the one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7,
  • the determining can comprise assaying a biological sample from the patient to detect an amount of the one or more secreted proteins.
  • the methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma can comprise assaying a biological sample from the patient to detect an amount of one or more secreted proteins, wherein the one or more secreted proteins are CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFCl, FLRTl, IFN-g, JAM-C, prostatic binding protein, or any combination thereof, and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP
  • the methods can further comprise identifying the patient has having non-GCB diffuse large B-cell lymphoma if the amount of one or more of CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFCl, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof, from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
  • the one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFCl, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof.
  • the methods can comprise determining an amount of AREG in a biological sample from the patient.
  • the methods can comprise determining an amount of kininogen HMW in a biological sample from the patient.
  • the methods can comprise determining an amount of PA2G4 in a biological sample from the patient.
  • the methods can comprise determining an amount of peroxiredoxin-6 in a biological sample from the patient.
  • the methods can comprise
  • the methods can comprise determining an amount of PSD7 in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of UBE2N in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of aflatoxin B l aldehyde reductase in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of AREG in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of CD27 in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of CFCl in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of FLRT1 in a biological sample from the patient.
  • the methods can comprise determining an amount of IFN-g in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of JAM-C in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of prostatic binding protein in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of any combination of the above listed proteins in a biological sample from the patient.
  • Suitable controls include, but are not limited to, a mean amount of the same one or more secreted proteins (i.e.
  • control can be a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL patient population. In other embodiments, the control can be a mean amount of the same one or more secreted proteins in a biological sample from a GCB patient population.
  • the methods further comprise: 1) determining an amount of a second one or more secreted proteins, wherein the second one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof in the biological sample from the patient; and 2) comparing the amount of the second one or more secreted proteins to a mean amount of the second one or more secreted proteins from a DLBCL patient population or a non-GCB patient population, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the second one or more secreted proteins are higher than the mean amount of the same second one or more secreted proteins in the DLBCL patient population or the non-GCB patients population.
  • the methods can comprise determining an amount of one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof, and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
  • the determining can comprise assaying a biological sample from the patient to detect an amount of the one or more secreted proteins.
  • the methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma can comprise assaying a biological sample from the patient to detect an amount of one or more secreted proteins, wherein the one or more secreted proteins are AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof, and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
  • the methods can further comprise identifying the patient has having GCB diffuse large B-cell lymphoma if the amount of one or more of AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof, from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
  • the one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin- like protein 1, Angiogenin, CK-MB, or any combination thereof.
  • the methods can comprise determining an amount of AURKB in a biological sample from the patient.
  • the methods can comprise determining an amount of HCK in a biological sample from the patient.
  • the methods can comprise determining an amount of TWEAKR in a biological sample from the patient.
  • the methods can comprise determining an amount of Nectin-like protein 1 in a biological sample from the patient.
  • the methods can comprise determining an amount of Angiogenin in a biological sample from the patient.
  • the methods can comprise determining an amount of CK-MB in a biological sample from the patient.
  • the methods can comprise determining an amount of any combination of the above secreted proteins in a biological sample from the patient.
  • Suitable controls include, but are not limited to, a mean amount of the same one or more secreted proteins (i.e. AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof) in a biological sample from a DLBCL patient population or a non-GCB patient population.
  • the control can be a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL patient population.
  • the control can be a mean amount of the same one or more secreted proteins in a biological sample from a non-GCB patient population.
  • the methods further comprise: 1) determining an amount of a second one or more secreted proteins, wherein the second one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof, in the biological sample from the patient; and 2) comparing the amount of the second one or more secreted proteins to a mean amount of the second one or more secreted proteins from a DLBCL patient population or a GCB patient population, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the second one or more secreted proteins are higher than the mean amount of the same second one or more secreted proteins in the DLBCL patient population or the GCB patient population.
  • the biological sample can be from any suitable stage in the treatment regimen.
  • the biological sample can be a pretreatment sample.
  • the biological sample can be a sample obtained after initiation of treatment. Accordingly, the disclosed methods can be used to determine a proper treatment regimen prior to the initiation of treatment or during treatment.
  • the disclosed methods can be performed and, if the amount of the one or more secreted proteins are higher than the mean amount of the same one or more secreted proteins in the control, the patient can be switched to an ibrutinib and R-CHOP combination therapy.
  • the amount of the one or more secreted proteins in a biological sample from the patient can be determined by known techniques for determining protein concentration and/or analyzing protein expression levels.
  • the methods comprise comparing the amount of the one or more secreted proteins to a mean amount of the same one or more secreted proteins from a control.
  • the comparison is performed based upon the same secreted protein, or combination thereof.
  • the method comprises determining an amount of AREG in a biological sample from the patient
  • the method comprises comparing the amount of AREG in the biological sample from the patient to a mean amount of AREG from a control.
  • the method comprises determining an amount of AREG and CD27 in a biological sample from the patient
  • the method comprises comparing the amount of AREG and CD27 in the biological sample from the patient to a mean amount of AREG and CD27 from a control.
  • the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 5% or greater.
  • the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%.
  • the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by greater than 500%.
  • the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 10 pg/ml or greater.
  • the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 10 pg/ml, 20 pg/ml, 30 pg/ml, 50 pg/ml, 75 pg/ml, 100 pg/ml, 200 pg/ml, 300 pg/ml, 400 pg/ml, 500 pg/ml, 750 pg/ml, 1000 pg/ml, 2000 pg/ml, 3000 pg/ml, 5000 pg/ml.
  • the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by greater than 5000 pg/ml.
  • the disclosed methods are equally applicable to patients having newly diagnosed DLBCL, relapsed DLBCL, or refractory DLBCL.
  • the methods can be used to predict a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having newly diagnosed DLBCL.
  • the methods can be used to predict a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having relapsed DLBCL.
  • the methods can be used to predict a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having refractory DLBCL.
  • the methods can be used to predict a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having both relapsed DLBCL and refractory DLBCL.
  • the disclosed methods can be used to treat a patient having GCB or non-GCB DLBCL.
  • the DLBCL can be GCB DLBCL.
  • the DLBCL can be non-GCB DLBCL.
  • the DLBCL can be ABC or nonclassifiable.
  • the methods can comprise administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof.
  • the methods of treating a patient having diffuse large B-cell lymphoma can further comprise, prior to the administering, determining an amount of one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFCl, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof and comparing the amount of the one or more secreted proteins in the biological sample to a mean amount of the same one or more secreted proteins from a control.
  • the one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFCl, FLRT1, IFN-g,
  • a suitable controls include, but are not limited to, a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL patient population or a GCB patient population.
  • the control can be a mean amount of the same one or more secreted proteins in a biological sample from DLBCL patient population.
  • the control can be a mean amount of the same one or more secreted proteins in a biological sample from a GCB population.
  • the one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFCl, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof.
  • the methods can comprise determining an amount of AREG in a biological sample from the patient.
  • the methods can comprise determining an amount of kininogen HMW in a biological sample from the patient.
  • the methods can comprise determining an amount of PA2G4 in a biological sample from the patient.
  • the methods can comprise determining an amount of peroxiredoxin-6 in a biological sample from the patient.
  • the methods can comprise
  • the methods can comprise determining an amount of PSD7 in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of UBE2N in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of aflatoxin B l aldehyde reductase in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of AREG in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of CD27 in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of CFCl in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of FLRT1 in a biological sample from the patient.
  • the methods can comprise determining an amount of IFN-g in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of JAM-C in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of prostatic binding protein in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of any combination of the above listed proteins in a biological sample from the patient.
  • the methods further comprise: 1) determining an amount of a second one or more secreted proteins, wherein the second one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof in the biological sample from the patient; 2) comparing the amount of the second one or more secreted proteins to a mean amount of the second one or more secreted proteins from a DLBCL patient population or a non-GCB patient population; and 3) treating the patient with ibrutinib and R-CHOP combination therapy if the amount of the second one or more secreted proteins are higher than the mean amount of the same second one or more secreted proteins in the DLBCL patient population or the non-GCB patient population.
  • the methods of treating a patient having diffuse large B-cell lymphoma can comprise administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof.
  • the methods of treating a patient having diffuse large B-cell lymphoma can further comprise, prior to the administering, determining the amount of the one or more secreted proteins in a biological sample from the patient, and comparing the amount of the one or more secreted proteins in the biological sample to the mean amount of the same one or more secreted proteins from the control.
  • a suitable controls include, but are not limited to, a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL patient population or a non- GCB patient population.
  • the control can be a mean amount of the same one or more secreted proteins in a biological sample from DLBCL patient population.
  • the control can be a mean amount of the same one or more secreted proteins in a biological sample from a non-GCB population.
  • the one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin- like protein 1, Angiogenin, CK-MB, or any combination thereof.
  • the methods can comprise determining an amount of AURKB in a biological sample from the patient.
  • the methods can comprise determining an amount of HCK in a biological sample from the patient.
  • the methods can comprise determining an amount of TWEAKR in a biological sample from the patient.
  • the methods can comprise determining an amount of Nectin-like protein lin a biological sample from the patient.
  • the methods can comprise determining an amount of Angiogenin in a biological sample from the patient.
  • the methods can comprise determining an amount of CK-MB in a biological sample from the patient.
  • the methods can comprise determining an amount of any one of the above secreted proteins in a biological sample from the patient.
  • the methods further comprise: 1) determining an amount of a second one or more secreted proteins, wherein the second one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof, in the biological sample from the patient; and 2) comparing the amount of the second one or more secreted proteins to a mean amount of the second one or more secreted proteins from a DLBCL patient population or a GCB patient population, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the second one or more secreted proteins are higher than the mean amount of the same second one or more secreted proteins in the DLBCL patient population or the GCB patient population.
  • Suitable biological samples, and techniques for determining the amount of proteins in the biological sample include those disclosed above.
  • the methods comprise comparing the amount of the one or more secreted proteins to a mean amount of the same one or more secreted proteins from a control.
  • the comparison is performed based upon the same secreted protein, or combination thereof.
  • the method comprises determining an amount of AREG in a biological sample from the patient
  • the method comprises comparing the amount of AREG in the biological sample from the patient to a mean amount of AREG from a control.
  • the method comprises determining an amount of AREG and CD27 in a biological sample from the patient
  • the method comprises comparing the amount of AREG and CD27 in the biological sample from the patient to a mean amount of AREG and CD27 from a control.
  • the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 5% or greater.
  • the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%.
  • the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by greater than 500%.
  • the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 10 pg/ml or greater.
  • the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 10 pg/ml, 20 pg/ml, 30 pg/ml, 50 pg/ml, 75 pg/ml, 100 pg/ml, 200 pg/ml, 300 pg/ml, 400 pg/ml, 500 pg/ml, 750 pg/ml, 1000 pg/ml, 2000 pg/ml, 3000 pg/ml, 5000 pg/ml.
  • the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by greater than 5000 pg/ml.
  • the disclosed methods are equally applicable to patients having newly diagnosed DLBCL, relapsed DLBCL, or refractory DLBCL.
  • the methods can be used to treat a patient having newly diagnosed DLBCL.
  • the methods can be used to treat a patient having relapsed DLBCL.
  • the methods can be used to treat a patient having refractory DLBCL.
  • the methods can be used to treat a patient having both relapsed DLBCL and refractory DLBCL.
  • Also disclosed herein are methods of identifying a patient as having diffuse large B-cell lymphoma comprising assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein the one or more genes comprise CREBBP, LRPIB, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes indicates diffuse large B-cell lymphoma.
  • the methods comprise assaying a DNA sample from the patient to detect a mutation in two or more genes.
  • Methods of treating a patient having diffuse large B-cell lymphoma are also disclosed.
  • the methods can comprise administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a mutation in one or more genes, wherein the one or more genes are CREBBP, LRPIB, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH 1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof.
  • the patient has a mutation in two or more genes.
  • the methods can further comprise, prior to the administering: assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein the one or more genes are CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes identifies the patient as having diffuse large B-cell lymphoma.
  • the methods comprise assaying a DNA sample from the patient to detect a mutation in two or more genes.
  • the detecting step can comprise enriching the DNA for the one or more genes and sequencing the DNA.
  • Enriching the DNA for the one or more genes can be performed, for example, using the Ovation® Target Enrichment system (NuGEN).
  • Sequencing of the DNA can be performed, for example, by deep sequencing using an Illumina® HiSeq instrument.
  • the DNA sample from the patient can be obtained from any source that contains DLBCL cells, including, but not limited to, blood, bone marrow, lymph fluid, tumor, or any combination thereof.
  • the DNA sample can be from a tumor biopsy.
  • the DNA sample can be CD 19-enriched cells from peripheral blood mononucleated cells (PBMCs).
  • PBMCs peripheral blood mononucleated cells
  • the DNA sample can be from any suitable stage in the treatment regimen.
  • the DNA sample can be a pretreatment sample.
  • the pretreatment sample can be a pretreatment tumor sample.
  • the pretreatment sample can be a pretreatment blood sample.
  • the DNA sample can be a sample obtained after initiation of treatment.
  • the DNA sample can be a tumor sample obtained after initiation of treatment.
  • the DNA sample can be a blood sample obtained after initiation of treatment.
  • Also disclosed are methods of treating a patient having diffuse large B-cell lymphoma comprising administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a mutation in one or more genes, wherein the one or more genes are CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSDl, NTRKl, NTRK3, SPEN, ZNF703, or any combination thereof.
  • the one or more genes are CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, N
  • the method further comprises, prior to the administering assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein a mutation in the one or more genes identifies the patient as having diffuse large B-cell lymphoma.
  • the methods can further comprise, prior to the administering assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein the one or more genes are
  • CREBBP CREBBP
  • LRP1B LRP1B
  • MLL2 MLL2
  • GNAS GNAS
  • TNFRSF 14 AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSD l, NTRKl, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes identifies the patient as having diffuse large B-cell lymphoma.
  • the detecting step can comprise enriching the DNA for the one or more genes and sequencing the DNA.
  • Enriching the DNA for the one or more genes can be performed, for example, using the Ovation® Target Enrichment system (NuGEN).
  • Sequencing of the DNA can be performed, for example, by deep sequencing using an Illumina® HiSeq instrument.
  • the DNA sample from the patient can be obtained from any source that contains DLBCL cells, including, but not limited to, blood, bone marrow, lymph fluid, tumor, or any combination thereof.
  • the DNA sample can be from a tumor biopsy.
  • the DNA sample can be CD 19-enriched cells from peripheral blood mononucleated cells (PBMCs).
  • PBMCs peripheral blood mononucleated cells
  • the DNA sample can be from any suitable stage in the treatment regimen.
  • the DNA sample can be a pretreatment sample.
  • the pretreatment sample can be a pretreatment tumor sample.
  • the pretreatment sample can be a pretreatment blood sample.
  • the DNA sample can be a sample obtained after initiation of treatment.
  • the DNA sample can be a tumor sample obtained after initiation of treatment.
  • the DNA sample can be a blood sample obtained after initiation of treatment.
  • each patient received R-CHOP in combination with ibrutinib according to the following schedule: Intravenous administration of rituximab 375 mg/m 2 , cyclophosphamide 750 mg/m 2 , doxorubicin 50 mg/m 2 , vincristine 1.4 mg/m 2 (maximum total of 2 mg), and oral administration of prednisone 100 mg on Days 1 through 5 of each 21 -day (3 -week) cycle for up to 6 cycles; ibrutinib was administered orally every day from Day 3 onward).
  • PBMCs peripheral blood mononuclear cells
  • COO Cell-of-origin analysis was carried out on the DLBCL formalin fixed, paraffin-embedded (FFPE) samples by IHC using the Hans method for classification at the IHC testing laboratory, PhenoPath Laboratories (Seattle, WA). The Hans method classifies samples into the germinal center B-cell (GCB) and the non-GCB subtypes of DLBCL through analysis of CD 10, Bcl-6, and MUM1 staining.
  • FFPE paraffin-embedded
  • Tumor B-cells were isolated from tumor biopsy and bone marrow of patients who responded to ibrutinib and R-CHOP combination therapy by flow cytometry at Universidad de Salamanca, Spain, and DNA/RNA was extracted. For samples where tumor was not available or DNA yield was low by flow sorting, viable frozen PBMCs (derived from whole blood) were enriched for CD 19+ B-cells at Janssen and DNA/RNA was extracted. DNA from all samples was subjected to targeted deep sequencing using the T5 panel at Foundation Medicine.
  • SOMAmers convert a protein signal to a nucleotide signal in proportion to the amount of protein, which can then be quantified using relative florescence on microarrays as previously described (Gold L, Ayers D, Bertino J, et al. Aptamer-based multiplexed proteomic technology for biomarker discovery. Nature Proceedings. 2010.hdl: 10101/npre.2010.4538.1). The unadjusted p-values and top 20 differentially expressed markers at baseline are indicated in Table 6.
  • Interferon gamma receptor decreased post-treatment and is known to cause Thl differentiation and promotes NK cell activity.
  • Baseline SomaLogic Analyte expression for the DLBCL subtypes (GCB vs non-GCB) is shown in FIG. 1A-FIG. IT.
  • MRD analysis was performed using the ClonoSIGHTTM assay (Sequenta Biotechnologies), which is a high-throughput DNA sequencing method that can detect MRD of lymphoid malignancies in peripheral blood with a sensitivity of one lymphoma cell per million leukocytes.
  • Genomic DNA was isolated from formalin-fixed paraffin embedded samples and plasma samples, amplified, and disease-specific lymphoma-derived sequences identified at diagnosis were used to assess the presence of MRD in follow-up plasma samples.
  • MRD was measured at Cycle 1 ; Day 1 (C1D1), C2D1, C4D1, and end-of-treatment (EOT) or early withdrawal.
  • MCL subjects showed a slower but prolonged MRD response - of the four subjects (all of whom achieved a CR and were MRD positive at baseline and C2D1), three reached MRD negativity by C4D1 and maintained negativity through EOT, while the fourth showed a drop in MRD at C4D 1 but was not evaluated at EOT. Only two subjects had FL - one achieved PR while the other was not evaluable for response due to discontinuation within the first cycle. Neither subject achieved MRD negativity at these time points (Table 8 and FIG. 3).
  • the ORR was 100% for DLBCL regardless of subtype; the CR rate was 100% in non-GCB and 71.4% in GCB patients.
  • Median percentage occupancy in the ibrutinib 560 mg cohort was 97.3% by Cycle 1 Day 3, with a range from 88% to 100%, showing that BTK is fully occupied by ibrutinib at the recommended dose for lymphomas.
  • LRP1B was one of the most frequently mutated genes in this DLBCL cohort. No CARD 11 or MYD88 mutations (previously related to ibrutinib resistance) were identified in this cohort.
  • Embodiment 1 A method of predicting a likelihood of responsiveness to ibrutinib and R- CHOP combination therapy in a patient having diffuse large B-cell lymphoma, comprising:
  • the one or more secreted proteins are CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof; and
  • the patient has a likelihood of responsiveness to ibrutinib and R- CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
  • Embodiment 2 The method of embodiment 1, further comprising identifying the patient has having non-GCB diffuse large B-cell lymphoma if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
  • Embodiment 3 A method of predicting a likelihood of responsiveness to ibrutinib and R- CHOP combination therapy in a patient having diffuse large B-cell lymphoma, comprising:
  • the assaying a biological sample from the patient to detect an amount of one or more secreted proteins, wherein the one or more secreted proteins are AURKB, HCK, TWEAKR, Nectin-like protein 1 , Angiogenin, CK-MB, or any combination thereof; and
  • CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
  • Embodiment 4 The method of embodiment 3, further comprising identifying the patient has having GCB diffuse large B-cell lymphoma if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
  • Embodiment 5 The method of any one of the previous embodiments, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL population.
  • Embodiment 6 The method of embodiment 1 or 2, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a GCB patient population.
  • Embodiment 7 The method of embodiment 3 or 4, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a non-GCB population.
  • Embodiment 8 A method of treating a patient having diffuse large B-cell lymphoma, comprising:
  • a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from CPNEl, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof.
  • Embodiment 9 A method of treating a patient having diffuse large B-cell lymphoma, comprising:
  • a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from AURKB, HCK, TWEAKR, Nectin-like protein 1 , Angiogenin, CK-MB, or any combination thereof.
  • Embodiment 10 The method of embodiment 8 or 9, further comprising, prior to the
  • Embodiment 1 1. The method of any one of embodiments 8-10, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL population.
  • Embodiment 12 The method of embodiment 8 or 10, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a GCB patient population.
  • Embodiment 13 The method of embodiment 9 or 10, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a non-GCB population.
  • Embodiment 14 A method of identifying a patient as having diffuse large B-cell lymphoma comprising:
  • the one or more genes are CREBBP, LRPIB, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARDl, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes identifies the patient as having diffuse large B- cell lymphoma.
  • Embodiment 15 A method of treating a patient having diffuse large B-cell lymphoma comprising:
  • ibrutinib and R-CHOP administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a mutation in one or more genes, wherein the one or more genes are CREBBP, LRPIB, MLL2, BCL2, GNAS, TNFRSF 14, AR, ARID2, AXL, BARDl, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSDl, NTRKl, NTRK3, SPEN, ZNF703, or any combination thereof.
  • the one or more genes are CREBBP, LRPIB, MLL2, BCL2, GNAS, TNFRSF 14, AR, ARID2, AXL, BARDl, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSDl, NTRKl, N
  • Embodiment 16 The method of embodiment 15, further comprising, prior to the
  • the one or more genes are CREBBP, LRPIB, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSDl, NTRKl, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes identifies the patient as having diffuse large B- cell lymphoma.
  • Embodiment 17 The method of embodiment 14 or 16, the wherein the method comprises assaying a DNA sample from the patient to detect a mutation in two or more genes.
  • Embodiment 18 The method of embodiment 15, wherein the patient has a mutation in two or more genes.

Abstract

Disclosed herein are biological markers useful in identifying and treating patients with diffuse large B-cell lymphoma. Provided are methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma, methods of treating a patient having diffuse large B-cell lymphoma, and methods of identifying a patient as having diffuse large B-cell lymphoma.

Description

BIOLOGICAL MARKERS FOR PREDICTING RESPONSIVENESS TO IBRUTINIB AND R- CHOP COMBINATION THERAPY AND METHODS OF USING THE SAME
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 62/088,230, filed December 5, 2014, the disclosure of which is hereby incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The disclosure relates to biological markers useful in identifying and treating patients with diffuse large B-cell lymphoma. In particular, the disclosed methods relate to biological markers for identifying a patient as having diffuse large B-cell lymphoma, treating a patient having diffuse large B-cell lymphoma, and methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma.
BACKGROUND
[0003] Non-Hodgkin lymphoma (NHL) is the ninth and seventh most frequent cancer in males and females, respectively. The incidence of NHL appears to be steadily increasing each year in Europe and the US. In the Western World, diffuse large B-cell lymphoma (DLBCL) comprises one-third of all adult NHL cases. DLBCL is an aggressive, although potentially curable, disease. Worldwide, the currently accepted standard regimen for frontline treatment of patients with DLBCL is R-CHOP with cure rates around 75%. Despite the improvement in clinical outcomes following R-CHOP treatment, a substantial number of patients still have disease, which either fails to respond to initial therapy or relapses after initial remission.
SUMMARY
[0004] Disclosed herein are methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma. In some embodiments, the methods can comprise determining an amount of one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof, and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control. In some embodiments, the methods can comprise determining an amount of one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof, and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
[0005] The determining can comprise assaying a biological sample from the patient to detect an amount of the one or more secreted proteins. Thus, in some embodiments, the methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma can comprise assaying a biological sample from the patient to detect an amount of one or more secreted proteins, wherein the one or more secreted proteins are CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFCl, FLRTl, IFN-g, JAM-C, prostatic binding protein, or any combination thereof and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control. In some embodiments, the methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma can comprise assaying a biological sample from the patient to detect an amount of one or more secreted proteins, wherein the one or more secreted proteins are AURKB, HCK, TWEAKR, Nectin-like protein 1 , Angiogenin, CK-MB, or any combination thereof and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
[0006] Disclosed herein are methods of treating a patient having diffuse large B-cell lymphoma. The disclosed methods can comprise administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof. In some embodiments, the methods of treating a patient having diffuse large B-cell lymphoma can further comprise, prior to the administering, determining an amount of one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof and comparing the amount of the one or more secreted proteins in the biological sample to a mean amount of the same one or more secreted proteins from a control.
[0007] The methods of treating a patient having diffuse large B-cell lymphoma can comprise administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof. In some embodiments, the methods of treating a patient having diffuse large B-cell lymphoma can further comprise, prior to the administering, determining the amount of the one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin- like protein 1 , Angiogenin, CK-MB, or any combination thereof, and comparing the amount of the one or more secreted proteins in the biological sample to the mean amount of the same one or more secreted proteins from the control.
[0008] Methods of identifying a patient as having diffuse large B-cell lymphoma are also provided. The methods comprise assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein the one or more genes comprise CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes identifies the patient as having diffuse large B-cell lymphoma.
[0009] Also disclosed are methods of treating a patient having diffuse large B-cell lymphoma comprising administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a mutation in one or more genes, wherein the one or more genes are CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof. In some embodiments, the method can further comprise, prior to the administering assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein a mutation in the one or more genes identifies the patient as having diffuse large B-cell lymphoma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed methods, there are shown in the drawings exemplary embodiments of the methods; however, the methods are not limited to the specific embodiments disclosed. In the drawings:
[0011] FIG. 1A to FIG. IT illustrate baseline SomaLogic Analyte Expression in DLBCL subtypes for: FIG. 1A) AURKB; FIG. IB) HCK; FIG. 1C) TWEAKR; FIG. ID) CPNE1; FIG. IE) kininogen, HMW; FIG. IF) nectin-like protein 1 ; FIG. 1G) PA2G4; FIG. 1H) peroxiredoxin-6; FIG. II) PSD7; FIG. 1J) UBE2N; FIG. IK) aflatoxin B l aldehyde reductase; FIG. 1L) angiogenin; FIG. 1M) AREG; FIG. IN) CD27; FIG. lO) CFC1 ; FIG. IP) CK-MB; FIG. 1Q) FLRT1 ; FIG. 1R) IFN-g; FIG. IS) JAM-C; and FIG. IT) prostatic binding protein. The left-side of each panel represents germinal center B-cell (GCB) DLBCL; the right- side of each panel represents non-GCB DLBCL. The y-axis in each is the log2 RFU.
[0012] FIG. 2A to FIG. 2T illustrate baseline SomaLogic Analyte Expression between subjects with complete response (CR) versus those with partial response (PR) for: FIG. 2A) PSA-ACT; FIG. 2B) IL-17 RD; FIG. 2C) IL-2 sRa; FIG. 2D) C08A1 ; FIG. 2E) Coagulation Factor XI; FIG. 2F) p27Kipl; FIG. 2G) MICB; FIG. 2H) sICAM-5; FIG. 21) Spondin-1 ; FIG. 2J) b2-Microglobulin; FIG. 2K) Cl-Esterase Inhibitor; FIG. 2L) CD30 Ligand; FIG. 2M) HPLNl ; FIG. 2N) NPS-PLA2; FIG. 20) TNF sR-I; FIG. 2P) TNF sR-II; FIG. 2Q) TWEAKR; FIG. 2R) Antithrombin III; FIG. 2S) CTGF; and FIG. 2T) G-CSF-R. The left-side of each panel represents complete response (CR); the right-side of each panel represents partial response (PR). The y-axis in each is the log2 RFU.
[0013] FIG. 3 illustrates an exemplary time-course of minimal residual disease (MRD) measurements.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0014] The disclosed methods may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed methods are not limited to the specific methods described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods.
[0015] Similarly, unless specifically otherwise stated, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed methods are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.
[0016] It is to be appreciated that certain features of the disclosed methods which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.
[0017] As used herein, the singular forms "a," "an," and "the" include the plural.
[0018] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.
[0019] The term "about" when used in reference to numerical ranges, cutoffs, or specific values is used to indicate that the recited values may vary by up to as much as 10% from the listed value. Thus, the term "about" is used to encompass variations of ± 10% or less, variations of ± 5% or less, variations of ± 1% or less, variations of ± 0.5% or less, or variations of ± 0.1% or less from the specified value.
[0020] The term "biological sample" refers to any sample from a patient that contains, or can contain, the secreted proteins of interest, including, for example, tumor, blood, serum, or plasma.
Preferably, the biological sample is blood, serum, or plasma. [0021] As used herein, "treating" and like terms refer to reducing the severity and/or frequency of DLBCL symptoms, eliminating DLBCL symptoms and/or the underlying cause of said symptoms, reducing the frequency or likelihood of DLBCL symptoms and/or their underlying cause, and improving or remediating damage caused, directly or indirectly, by DLBCL.
[0022] The term "patient" as used herein is intended to mean any animal, in particular, mammals. Thus, the methods are applicable to human and nonhuman animals, although preferably used with mice and humans, and most preferably with humans.
[0023] As used herein, "ibrutinib" (also known as PCI-32765) refers to l-[(3R)-3-[4- amino-3-(4-phenoxyphenyl)-lH-pyrazolo [3, 4 d] pyrimidin-l-yl]-l-piperidinyl]-2-propen-l- one, having a molecular weight of 440.50 g/mole (anhydrous basis), a single chiral center, and being an R-enantiomer. Ibrutinib is a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK). Ibrutinib has been designated as a breakthrough targeted therapy for certain types of B-cell malignancies and is now approved in the United States and European Union for patients with previously treated mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), as well as untreated del 17p or TP53 mutated CLL.
[0024] As used herein, "R-CHOP" refers to a drug combination containing rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone.
[0025] As used herein, "refractory DLBCL" refers to DLBCL that is present after treatment.
[0026] As used herein, "relapsed DLBCL" refers to DLBCL that has returned after treatment.
[0027] The following abbreviations are used throughout the disclosure: DLBCL (diffuse large B-cell lymphoma); FFPE (formalin fixed, paraffin-embedded); BTK (Bruton's tyrosine kinase); MCL (mantle cell lymphoma); CLL (chronic lymphocytic leukemia); FL (follicular lymphoma); R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone); NHL (non-hodgkin's lymphoma); PBMCs (peripheral blood mononuclear cells); GCB (germinal center B-cell); ORR (overall response rate); CR (complete response); PR (partial response); AURKB (Aurora kinase B); HCK (Tyrosine-protein kinase HCK); TWEAKR (Tumor necrosis factor receptor superfamily member 12A); CPNE1 (Copine-1); Kininogen, HMW (Kininogen-1); Nectin-like protein 1 (Cell adhesion molecule 3); PA2G4 (Proliferation- associated protein 2G4); PSD7 (26S proteasome non-ATPase regulatory subunit 7); UBE2N (Ubiquitin-conjugating enzyme E2 N); Aflatoxin B l aldehyde reductase (Aflatoxin Bl aldehyde reductase member 2); AREG (Amphiregulin); CD27 (CD27 antigen); CFCl (Cryptic protein); CK-MB (Creatine kinase M-type: Creatine kinase B-type heterodimer); FLRTl (Leucine-rich repeat transmembrane protein FLRTl); IFN-g (Interferon gamma); JAM-C (Junctional adhesion molecule C); prostatic binding protein (Phosphatidylethanolamine-binding protein 1); MRD (minimal residual disease).
Secreted proteins as biomarkers for predicting a likelihood of responsiveness
[0028] Disclosed herein are methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma (DLBCL). DLBCL comprises at least two subgroups: germinal center B-cell like (GCB) BLBCL and non-GCB DLBCL. The disclosed methods can predict a likelihood of
responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma. In some embodiments, the DLBCL can be GCB DLBCL. In other embodiments, the DLBCL can be non-GCB DLBCL. In some aspects, the non-GCB DLBCL can be activated B-cell like (ABC) or unclassifiable.
[0029] The methods can comprise determining an amount of one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFCl, FLRTl, IFN-g, JAM-C, prostatic binding protein, or any combination thereof, and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
[0030] The determining can comprise assaying a biological sample from the patient to detect an amount of the one or more secreted proteins. Thus, the methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma can comprise assaying a biological sample from the patient to detect an amount of one or more secreted proteins, wherein the one or more secreted proteins are CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFCl, FLRTl, IFN-g, JAM-C, prostatic binding protein, or any combination thereof, and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
[0031] The methods can further comprise identifying the patient has having non-GCB diffuse large B-cell lymphoma if the amount of one or more of CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFCl, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof, from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
[0032] The one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFCl, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof. In some embodiments, the methods can comprise determining an amount of AREG in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of kininogen HMW in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of PA2G4 in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of peroxiredoxin-6 in a biological sample from the patient. In some embodiments, the methods can comprise
determining an amount of PSD7 in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of UBE2N in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of aflatoxin B l aldehyde reductase in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of AREG in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of CD27 in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of CFCl in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of FLRT1 in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of IFN-g in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of JAM-C in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of prostatic binding protein in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of any combination of the above listed proteins in a biological sample from the patient. [0033] Suitable controls include, but are not limited to, a mean amount of the same one or more secreted proteins (i.e. CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof) in a biological sample from a DLBCL patient population or a GCB patient population. In some aspects, the control can be a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL patient population. In other embodiments, the control can be a mean amount of the same one or more secreted proteins in a biological sample from a GCB patient population.
[0034] In some embodiments, the methods further comprise: 1) determining an amount of a second one or more secreted proteins, wherein the second one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof in the biological sample from the patient; and 2) comparing the amount of the second one or more secreted proteins to a mean amount of the second one or more secreted proteins from a DLBCL patient population or a non-GCB patient population, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the second one or more secreted proteins are higher than the mean amount of the same second one or more secreted proteins in the DLBCL patient population or the non-GCB patients population.
[0035] Conversely, the methods can comprise determining an amount of one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof, and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
[0036] The determining can comprise assaying a biological sample from the patient to detect an amount of the one or more secreted proteins. Thus, in some embodiments, the methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma can comprise assaying a biological sample from the patient to detect an amount of one or more secreted proteins, wherein the one or more secreted proteins are AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof, and comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
[0037] The methods can further comprise identifying the patient has having GCB diffuse large B-cell lymphoma if the amount of one or more of AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof, from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
[0038] The one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin- like protein 1, Angiogenin, CK-MB, or any combination thereof. In some embodiments, the methods can comprise determining an amount of AURKB in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of HCK in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of TWEAKR in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of Nectin-like protein 1 in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of Angiogenin in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of CK-MB in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of any combination of the above secreted proteins in a biological sample from the patient.
[0039] Suitable controls include, but are not limited to, a mean amount of the same one or more secreted proteins (i.e. AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof) in a biological sample from a DLBCL patient population or a non-GCB patient population. In some aspects, the control can be a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL patient population. In other embodiments, the control can be a mean amount of the same one or more secreted proteins in a biological sample from a non-GCB patient population.
[0040] In some embodiments, the methods further comprise: 1) determining an amount of a second one or more secreted proteins, wherein the second one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof, in the biological sample from the patient; and 2) comparing the amount of the second one or more secreted proteins to a mean amount of the second one or more secreted proteins from a DLBCL patient population or a GCB patient population, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the second one or more secreted proteins are higher than the mean amount of the same second one or more secreted proteins in the DLBCL patient population or the GCB patient population.
[0041] For any of the methods of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having (DLBCL), the biological sample can be from any suitable stage in the treatment regimen. In some embodiments, for example, the biological sample can be a pretreatment sample. In other embodiments, the biological sample can be a sample obtained after initiation of treatment. Accordingly, the disclosed methods can be used to determine a proper treatment regimen prior to the initiation of treatment or during treatment. For example, if a patient is being treated with another therapeutic, the disclosed methods can be performed and, if the amount of the one or more secreted proteins are higher than the mean amount of the same one or more secreted proteins in the control, the patient can be switched to an ibrutinib and R-CHOP combination therapy.
[0042] The amount of the one or more secreted proteins in a biological sample from the patient can be determined by known techniques for determining protein concentration and/or analyzing protein expression levels.
[0043] The methods comprise comparing the amount of the one or more secreted proteins to a mean amount of the same one or more secreted proteins from a control. The comparison is performed based upon the same secreted protein, or combination thereof. For example, and without intending to be limiting, in embodiments wherein the method comprises determining an amount of AREG in a biological sample from the patient, the method comprises comparing the amount of AREG in the biological sample from the patient to a mean amount of AREG from a control. Similarly, in embodiments wherein the method comprises determining an amount of AREG and CD27 in a biological sample from the patient, the method comprises comparing the amount of AREG and CD27 in the biological sample from the patient to a mean amount of AREG and CD27 from a control.
[0044] The amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 5% or greater. For example, the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%. In some aspects, the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by greater than 500%.
[0045] The amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 10 pg/ml or greater. For example, the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 10 pg/ml, 20 pg/ml, 30 pg/ml, 50 pg/ml, 75 pg/ml, 100 pg/ml, 200 pg/ml, 300 pg/ml, 400 pg/ml, 500 pg/ml, 750 pg/ml, 1000 pg/ml, 2000 pg/ml, 3000 pg/ml, 5000 pg/ml. In some aspects, the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by greater than 5000 pg/ml.
[0046] The disclosed methods are equally applicable to patients having newly diagnosed DLBCL, relapsed DLBCL, or refractory DLBCL. In some embodiments, the methods can be used to predict a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having newly diagnosed DLBCL. In some embodiments, the methods can be used to predict a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having relapsed DLBCL. In other embodiments, the methods can be used to predict a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having refractory DLBCL. In yet other embodiments, the methods can be used to predict a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having both relapsed DLBCL and refractory DLBCL.
Secreted proteins as biomarkers in treating a patient having DLBCL
[0047] Also disclosed herein are methods of treating a patient having diffuse large B- cell lymphoma. The disclosed methods can be used to treat a patient having GCB or non-GCB DLBCL. In some embodiments, the DLBCL can be GCB DLBCL. In other embodiments, the DLBCL can be non-GCB DLBCL. In aspects wherein the DLBCL is non-GCB, the DLBCL can be ABC or nonclassifiable.
[0048] The methods can comprise administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof. In some embodiments, the methods of treating a patient having diffuse large B-cell lymphoma can further comprise, prior to the administering, determining an amount of one or more secreted proteins in a biological sample from the patient, wherein the one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFCl, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof and comparing the amount of the one or more secreted proteins in the biological sample to a mean amount of the same one or more secreted proteins from a control.
[0049] A suitable controls include, but are not limited to, a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL patient population or a GCB patient population. In some embodiments, the control can be a mean amount of the same one or more secreted proteins in a biological sample from DLBCL patient population. In other embodiments, the control can be a mean amount of the same one or more secreted proteins in a biological sample from a GCB population.
[0050] The one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFCl, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof. In some embodiments, the methods can comprise determining an amount of AREG in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of kininogen HMW in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of PA2G4 in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of peroxiredoxin-6 in a biological sample from the patient. In some embodiments, the methods can comprise
determining an amount of PSD7 in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of UBE2N in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of aflatoxin B l aldehyde reductase in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of AREG in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of CD27 in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of CFCl in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of FLRT1 in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of IFN-g in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of JAM-C in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of prostatic binding protein in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of any combination of the above listed proteins in a biological sample from the patient.
[0051] In some embodiments, the methods further comprise: 1) determining an amount of a second one or more secreted proteins, wherein the second one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof in the biological sample from the patient; 2) comparing the amount of the second one or more secreted proteins to a mean amount of the second one or more secreted proteins from a DLBCL patient population or a non-GCB patient population; and 3) treating the patient with ibrutinib and R-CHOP combination therapy if the amount of the second one or more secreted proteins are higher than the mean amount of the same second one or more secreted proteins in the DLBCL patient population or the non-GCB patient population.
[0052] The methods of treating a patient having diffuse large B-cell lymphoma can comprise administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from AURKB, HCK, TWEAKR, Nectin-like protein 1, Angiogenin, CK-MB, or any combination thereof. In some embodiments, the methods of treating a patient having diffuse large B-cell lymphoma can further comprise, prior to the administering, determining the amount of the one or more secreted proteins in a biological sample from the patient, and comparing the amount of the one or more secreted proteins in the biological sample to the mean amount of the same one or more secreted proteins from the control.
[0053] A suitable controls include, but are not limited to, a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL patient population or a non- GCB patient population. In some embodiments, the control can be a mean amount of the same one or more secreted proteins in a biological sample from DLBCL patient population. In other embodiments, the control can be a mean amount of the same one or more secreted proteins in a biological sample from a non-GCB population.
[0054] The one or more secreted proteins comprise AURKB, HCK, TWEAKR, Nectin- like protein 1, Angiogenin, CK-MB, or any combination thereof. In some embodiments, the methods can comprise determining an amount of AURKB in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of HCK in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of TWEAKR in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of Nectin-like protein lin a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of Angiogenin in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of CK-MB in a biological sample from the patient. In some embodiments, the methods can comprise determining an amount of any one of the above secreted proteins in a biological sample from the patient.
[0055] In some embodiments, the methods further comprise: 1) determining an amount of a second one or more secreted proteins, wherein the second one or more secreted proteins comprise CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof, in the biological sample from the patient; and 2) comparing the amount of the second one or more secreted proteins to a mean amount of the second one or more secreted proteins from a DLBCL patient population or a GCB patient population, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the second one or more secreted proteins are higher than the mean amount of the same second one or more secreted proteins in the DLBCL patient population or the GCB patient population.
[0056] Suitable biological samples, and techniques for determining the amount of proteins in the biological sample, include those disclosed above.
[0057] For any of the above disclosed methods of treatment, the methods comprise comparing the amount of the one or more secreted proteins to a mean amount of the same one or more secreted proteins from a control. The comparison is performed based upon the same secreted protein, or combination thereof. For example, and without intending to be limiting, in embodiments wherein the method comprises determining an amount of AREG in a biological sample from the patient, the method comprises comparing the amount of AREG in the biological sample from the patient to a mean amount of AREG from a control. Similarly, in embodiments wherein the method comprises determining an amount of AREG and CD27 in a biological sample from the patient, the method comprises comparing the amount of AREG and CD27 in the biological sample from the patient to a mean amount of AREG and CD27 from a control.
[0058] The amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 5% or greater. For example, the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%. In some aspects, the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by greater than 500%.
[0059] The amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 10 pg/ml or greater. For example, the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by about 10 pg/ml, 20 pg/ml, 30 pg/ml, 50 pg/ml, 75 pg/ml, 100 pg/ml, 200 pg/ml, 300 pg/ml, 400 pg/ml, 500 pg/ml, 750 pg/ml, 1000 pg/ml, 2000 pg/ml, 3000 pg/ml, 5000 pg/ml. In some aspects, the amount of the one or more secreted proteins can be higher than the mean amount of the same one or more secreted proteins in the control by greater than 5000 pg/ml.
[0060] The disclosed methods are equally applicable to patients having newly diagnosed DLBCL, relapsed DLBCL, or refractory DLBCL. In some embodiments, the methods can be used to treat a patient having newly diagnosed DLBCL. In some embodiments, the methods can be used to treat a patient having relapsed DLBCL. In other embodiments, the methods can be used to treat a patient having refractory DLBCL. In yet other embodiments, the methods can be used to treat a patient having both relapsed DLBCL and refractory DLBCL.
Genetic mutations as biomarkers for identifying a patient as having, and treating, diffuse large B- cell lymphoma
[0061] Also disclosed herein are methods of identifying a patient as having diffuse large B-cell lymphoma comprising assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein the one or more genes comprise CREBBP, LRPIB, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes indicates diffuse large B-cell lymphoma. In some embodiments, the methods comprise assaying a DNA sample from the patient to detect a mutation in two or more genes.
[0062] Methods of treating a patient having diffuse large B-cell lymphoma are also disclosed. The methods can comprise administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a mutation in one or more genes, wherein the one or more genes are CREBBP, LRPIB, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH 1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof. In some aspects, the patient has a mutation in two or more genes. In some
embodiments, the methods can further comprise, prior to the administering: assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein the one or more genes are CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes identifies the patient as having diffuse large B-cell lymphoma. In some embodiments, the methods comprise assaying a DNA sample from the patient to detect a mutation in two or more genes.
[0063] Numerous techniques are known in the art for detecting mutations in genes. In some embodiments, for example, the detecting step can comprise enriching the DNA for the one or more genes and sequencing the DNA. Enriching the DNA for the one or more genes can be performed, for example, using the Ovation® Target Enrichment system (NuGEN). Sequencing of the DNA can be performed, for example, by deep sequencing using an Illumina® HiSeq instrument.
[0064] The DNA sample from the patient can be obtained from any source that contains DLBCL cells, including, but not limited to, blood, bone marrow, lymph fluid, tumor, or any combination thereof. In some embodiments, the DNA sample can be from a tumor biopsy. In other embodiments, the DNA sample can be CD 19-enriched cells from peripheral blood mononucleated cells (PBMCs).
[0065] The DNA sample can be from any suitable stage in the treatment regimen. In some embodiments, for example, the DNA sample can be a pretreatment sample. In some aspects, the pretreatment sample can be a pretreatment tumor sample. In other aspects, the pretreatment sample can be a pretreatment blood sample. In other embodiments, the DNA sample can be a sample obtained after initiation of treatment. In some aspects, for example, the DNA sample can be a tumor sample obtained after initiation of treatment. In other aspects, the DNA sample can be a blood sample obtained after initiation of treatment.
[0066] Also disclosed are methods of treating a patient having diffuse large B-cell lymphoma comprising administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a mutation in one or more genes, wherein the one or more genes are CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSDl, NTRKl, NTRK3, SPEN, ZNF703, or any combination thereof.
[0067] In some embodiments, the method further comprises, prior to the administering assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein a mutation in the one or more genes identifies the patient as having diffuse large B-cell lymphoma. Thus, the methods can further comprise, prior to the administering assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein the one or more genes are
CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF 14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSD l, NTRKl, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes identifies the patient as having diffuse large B-cell lymphoma.
[0068] Numerous techniques are known in the art for detecting mutations in genes. In some embodiments, for example, the detecting step can comprise enriching the DNA for the one or more genes and sequencing the DNA. Enriching the DNA for the one or more genes can be performed, for example, using the Ovation® Target Enrichment system (NuGEN). Sequencing of the DNA can be performed, for example, by deep sequencing using an Illumina® HiSeq instrument.
[0069] The DNA sample from the patient can be obtained from any source that contains DLBCL cells, including, but not limited to, blood, bone marrow, lymph fluid, tumor, or any combination thereof. In some embodiments, the DNA sample can be from a tumor biopsy. In other embodiments, the DNA sample can be CD 19-enriched cells from peripheral blood mononucleated cells (PBMCs).
[0070] The DNA sample can be from any suitable stage in the treatment regimen. In some embodiments, for example, the DNA sample can be a pretreatment sample. In some aspects, the pretreatment sample can be a pretreatment tumor sample. In other aspects, the pretreatment sample can be a pretreatment blood sample. In other embodiments, the DNA sample can be a sample obtained after initiation of treatment. In some aspects, for example, the DNA sample can be a tumor sample obtained after initiation of treatment. In other aspects, the DNA sample can be a blood sample obtained after initiation of treatment.
EXAMPLES [0071] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.
Primary Study Results
[0072] The DBL1002 study is described in Younes A, et ah, Combination of ibrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for treatment-naive patients with CD20-positive B-cell non-Hodgkin lymphoma: a non-randomised, phase lb study. Lancet Oncol. 2014; 15: 1019-1026, which is incorporated herein in its entirety. The herein disclosed analysis focused on the secondary study objectives, including the assessment of pharmacodynamic markers of ibrutinib in peripheral blood mononuclear cells (PBMCs), as well as identification of biomarkers predictive of response to the combination regimen. Briefly, each patient received R-CHOP in combination with ibrutinib according to the following schedule: Intravenous administration of rituximab 375 mg/m2, cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2, vincristine 1.4 mg/m2 (maximum total of 2 mg), and oral administration of prednisone 100 mg on Days 1 through 5 of each 21 -day (3 -week) cycle for up to 6 cycles; ibrutinib was administered orally every day from Day 3 onward). The maximum tolerated dose for ibrutinib in combination with R-CHOP was not reached and the recommended dose of ibrutinib was 560 mg per day. 33 patients were enrolled in the study, of whom 32 received at least 1 dose of therapy; the distribution of patients in the various doe groups and histologies is shown in Table 1.
Table 1. Study enrollment by histology
Figure imgf000020_0001
[0073] 29 of the 32 dosed patients completed 6 cycles. The ibrutinib + R-CHOP combination demonstrated clinical activity across all dose cohorts and all treated histologies, with an overall response rate (ORR) of 94% (30/32 patients who received 1 or more doses of combination therapy); of these, 22/23 patients (96%) with DLBCL who received 1 or more doses of ibrutinib plus R-CHOP had an overall response, including 18/18 (100%) patients who received the recommended phase 2 dose of ibrutinib (560 mg).
Immunohistochemistry (IHC) Analysis
[0074] Cell-of-origin (COO) analysis was carried out on the DLBCL formalin fixed, paraffin-embedded (FFPE) samples by IHC using the Hans method for classification at the IHC testing laboratory, PhenoPath Laboratories (Seattle, WA). The Hans method classifies samples into the germinal center B-cell (GCB) and the non-GCB subtypes of DLBCL through analysis of CD 10, Bcl-6, and MUM1 staining.
[0075] 13/24 patients had IHC subtyping data available by this method; 9/13 patients were classified as GCB and 4/13 as non-GCB subtype (Table 2). Of these 13 patients, 1 patient was nonevaluable (in 280 mg cohort) and 1 was discontinued (in 560 mg cohort). For the remaining patients, ORR was 100%, with 9/1 1 complete responses (CR) and 2/11 partial responses (PR). Both subjects with a PR were of the GCB subtype.
Table 2. Distribution of DLBCL subtypes based on Hans classification
Figure imgf000021_0001
HTG Analysis for Molecular Subtyping
[0076] COO classification of the DLBCL samples was also performed using the HTG Edge DLBCL COO Assay (HTG Molecular, Tucson, AZ). This assay was designed to provide DLBCL COO classification using as little as a single section of FFPE tissue. The 12
classification genes (http://www.htgmolecular.com/products/htg-edge-dlbcl-coo) provide a 92% concordance of classification result when compared with gene expression-based classifications. Results were available on 7 of 14 samples assessed in this study and are shown in Table 3.
Table 3. Results from the HTG classification Ι ΓΚ , Resu lt 1 I K -H ans M i l hod
Patient 1 GCB GCB
Patient 2 U C GCB
Patient 3 GCB GCB
Patient 4 ABC GCB
Patient 5 GCB GCB
Patient 6 GCB NA
Patient 7 ABC Non-GCB
UNC, unclassified; ABC, activated B-cell; NA, not available.
DNA Sequencing
[0077] Tumor B-cells were isolated from tumor biopsy and bone marrow of patients who responded to ibrutinib and R-CHOP combination therapy by flow cytometry at Universidad de Salamanca, Spain, and DNA/RNA was extracted. For samples where tumor was not available or DNA yield was low by flow sorting, viable frozen PBMCs (derived from whole blood) were enriched for CD 19+ B-cells at Janssen and DNA/RNA was extracted. DNA from all samples was subjected to targeted deep sequencing using the T5 panel at Foundation Medicine.
[0078] 30 samples from 28 patients were sequenced, of which 19 produced reportable short variant mutation data. These data are summarized in Table 4 for genes with variants identified in at least 2 of the 19 sequenced samples, sorted by mutation frequency. LRP1B was one of the most frequently mutated genes in this DLBCL cohort. Other variants were found in the apoptosis regulator BCL2 as well as TNFRSF14, a TNF superfamily member known to bind TRAF proteins, which are involved in NF-kB signaling.
Table 4. The number of DLBCL patients with at least 2 mutations (Freq) as well as the specific amino acid (AA) changes
Figure imgf000022_0001
Figure imgf000023_0001
Gene Expression Profiling
[0079] RNA extracted from tumor B-cells (as described above) was also analyzed by gene expression profiling (Labcorp). Of the 8 samples for which gene expression profiling was performed, only 5 passed quality control and were considered acceptable; these were a mix of DLBCL (N = 3; 2/3 GCB and 1/3 non-GCB) and MCL (N = 2).
FACS Analysis
[0080] Immunophenotyping and phosphoprotein analysis of several key phosphorylated proteins active in the BCR signaling pathway were evaluated by flow cytometry. Cryopreserved PBMCs isolated from patient whole blood were thawed and stained with a 9-fluorochrome conjugated antibody cocktail specific for the detection of human CD38, CD40, CD43, CD79b, CD123, CXCR4 (CD184), and CD279 (PD-1) on CD19+ B cells. Aliquots of the thawed PBMCs were stimulated with either: 1) H202 + anti-IgG and anti-IgM for pBTK, pPLCy2, pZAP70/pSYK, and pERKl/2; or 2) PMA + anti-IgG and anti-IgM for pAKT and pNFKB expression. Samples were fixed, permeabilized, and stained with an 8-fluorochrome conjugated antibody cocktail specific for the detection of phosphorylated forms of BTK, PLCy2,
ZAP70/SYK, NFKB, AKT, and ERKl/2 on CD19+ B-cells. Preliminary data analysis suggested that, post ibrutinib treatment, there was a trend toward decreased stimulation as measured by phosphoflow marker proteins (data not shown). BTK Occupancy
[0081] To assess BTK occupancy, a novel, sensitive, and robust assay was developed to study target engagement on the Meso Scale Discovery platform. Viably frozen PBMCs were used for this analysis, and percentage occupancy determined with respect to the baseline samples (assuming 0 % occupancy at baseline). Samples were collected at baseline and 4 hours post- treatment, and assessed for BTK occupancy (Table 5). Percent occupancy ranged from 88.78 % to 99.76% in the 560 mg cohort indicating acceptable occupancy of the target 4 hours post treatment.
Table 5. Percentage occupancy for 560 mg cohort
Figure imgf000024_0001
Secreted Protein Analysis
[0082] Differentially expressed proteins in the SomaLogic panel were identified from plasma protein samples with a Wilcoxon rank sum test in the DLBCL subtypes (GCB vs non- GCB) and adjusted for multiple testing using the Benjamini-Hochberg false discovery rate correction. In particular, proteins were measured using a Slow Off-rate Modified Aptamer (SOMAmer)-based capture array called "SOMAscan" (SomaLogic, Inc, Boulder, CO). The pre- validated SOMAScan assay measures 1 129 protein analytes using SOMAmer affinity-based molecules. This approach uses chemically modified nucleotides, called SOMAmers, which are single stranded DNA-based protein affinity reagents. SOMAmers convert a protein signal to a nucleotide signal in proportion to the amount of protein, which can then be quantified using relative florescence on microarrays as previously described (Gold L, Ayers D, Bertino J, et al. Aptamer-based multiplexed proteomic technology for biomarker discovery. Nature Proceedings. 2010.hdl: 10101/npre.2010.4538.1). The unadjusted p-values and top 20 differentially expressed markers at baseline are indicated in Table 6.
[0083] In order to identify pharmacodynamics markers, differentially expressed analytes in the SomaLogic panel were identified with a Wilcoxon Signed Rank test between Cycle 4 Day 1 and Cycle 1 Day 1 (baseline) as well as Cycle 2 Day 1 and Cycle 1 Day 1 (baseline) for all indications as well as for DLBCL only. P-values from this test were subsequently adjusted for multiple testing using the Benjamini-Hochberg False Discovery Rate correction. Top 20 differentially expressed proteins with p-value < 0.05 were identified. These 20 analytes were compared between both the sets; only Interferon gamma receptor protein was common to both the sets. Interferon gamma receptor decreased post-treatment and is known to cause Thl differentiation and promotes NK cell activity. Baseline SomaLogic Analyte expression for the DLBCL subtypes (GCB vs non-GCB) is shown in FIG. 1A-FIG. IT.
[0084] The secreted protein analysis demonstrated distinct differences in the expression profiles of GCB DLBCL compared to non-GCB DLBCL. For example, non-GCB DLBCL, which is more aggressive, had a higher expression of proteins previously implicated in the NF- KB pathway (peroxiredoxin-6, interferon gamma, CD27 antigen).
Table 6. Top 20 differentially expressed proteins in GCB versus non-GCB
Figure imgf000025_0001
26S proteasome non-ATPase 256.5 413.2
PSD7 0.024 regulatory subunit 7 (26.5) (148.3)
Ubiquitin-conjugating enzyme E2 3752 9049.3
UBE2N 0.024
N (1851.4) (5341.8)
Aflatoxin B 1
Aflatoxin B 1 aldehyde reductase 1321 3765.1
aldehyde 0.042 member 2
reductase (1036.6) (3087)
4186.9 3082.8
Angiogenin Angiogenin 0.042
(909.5) (491.9)
1508.6 2886.1
AREG Amphiregulin 0.042
(756.5) (1882)
2865.7 5699.9
CD27 CD27 antigen 0.042
(549.6) (4359.8)
1342.1 1683.7
CFC1 Cryptic protein 0.042
(197.6) (356.1)
Creatine kinase M-type:Creatine 1814.2 639
CK-MB 0.042 kinase B-type heterodimer (1010.6) (270.1)
Leucine-rich repeat 612.7 872.2
FLRT1 0.042 transmembrane protein FLRT1 (143.6) (218.9)
528.4 939.4
IFN-g Interferon gamma 0.042
(212.6) (427.5)
2053.9 3136.5
JAM-C Junctional adhesion molecule C 0.042
(787) (1728.3)
Prostatic
Phosphatidylethanolamine- 5550.6 10558.8 binding 0.042 binding protein 1
protein (1880.6) (5693.3)
[0085] In order to find characteristic proteins predictive of response to ibrutinib, baseline SomaLogic Analyte expression for the top 20 differentially expressed proteins in all DLBCL was also performed to compare levels between subjects with CR versus those with PR. The results are shown in Table 7 and FIG. 2A-FIG. 2T. Several members of the soluble TNF receptor family (CD30 ligand, TNF sR-I, TNF sR-II, TWEAKR) had differential expression and were observed at lower levels in subjects with CR versus subjects with PR. The maximum difference in secreted protein level was demonstrated by C08A1 expression, which was much higher in subjects with PR.
Table 7. Baseline secreted protein analysis in all DLBCL CR vs PR
Figure imgf000026_0001
Figure imgf000027_0001
stimulating factor receptor 16(293.7) 3 (59.0) (-388.3,-11.4) Minimal Residual Disease (MRD) Analysis
[0086] MRD analysis was performed using the ClonoSIGHT™ assay (Sequenta Biotechnologies), which is a high-throughput DNA sequencing method that can detect MRD of lymphoid malignancies in peripheral blood with a sensitivity of one lymphoma cell per million leukocytes. Genomic DNA was isolated from formalin-fixed paraffin embedded samples and plasma samples, amplified, and disease-specific lymphoma-derived sequences identified at diagnosis were used to assess the presence of MRD in follow-up plasma samples. MRD was measured at Cycle 1 ; Day 1 (C1D1), C2D1, C4D1, and end-of-treatment (EOT) or early withdrawal.
[0087] All samples tested (12 DLBCL) were positive for MRD at baseline. Most DLBCL subjects achieved MRD negativity by C2D1 (75%); by C4D1, only one subject remained MRD positive. This subject, who achieved a CR, finally became MRD negative by EOT (FIG. 3). Two subjects in this DLBCL cohort, who both achieved a CR, reached MRD negativity only at C4D 1 - one was non-GCB and still MRD-negative at EOT, while the other (who did not have subtype data) became MRD-positive again at EOT. MCL subjects showed a slower but prolonged MRD response - of the four subjects (all of whom achieved a CR and were MRD positive at baseline and C2D1), three reached MRD negativity by C4D1 and maintained negativity through EOT, while the fourth showed a drop in MRD at C4D 1 but was not evaluated at EOT. Only two subjects had FL - one achieved PR while the other was not evaluable for response due to discontinuation within the first cycle. Neither subject achieved MRD negativity at these time points (Table 8 and FIG. 3).
Table 8. MRD positivity of available samples
Figure imgf000028_0001
Conclusions
[0088] Once-daily ibrutinib in combination with R-CHOP is active in the treatment of naive patients with NHL, with a high ORR (96%) in DLBCL patients, with 100% ORR at the recommended phase 2 dose of ibrutinib. In patients with available GCB/non-GCB subtyping by
IHC, the ORR was 100% for DLBCL regardless of subtype; the CR rate was 100% in non-GCB and 71.4% in GCB patients. Median percentage occupancy in the ibrutinib 560 mg cohort was 97.3% by Cycle 1 Day 3, with a range from 88% to 100%, showing that BTK is fully occupied by ibrutinib at the recommended dose for lymphomas. LRP1B was one of the most frequently mutated genes in this DLBCL cohort. No CARD 11 or MYD88 mutations (previously related to ibrutinib resistance) were identified in this cohort. Secreted protein analyses highlighted certain key differences between the GCB and non-GCB samples in this study: IFN-gamma and peroxiredoxin-6, which have been implicated in aggressive tumors including DLBCL, were higher in the non-GCB cohort; and CD27, a TNFR superfamily member, which was higher in non-GCB subjects, has been implicated in NF-kB signaling.
[0089] Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.
[0090] The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference, in its entirety.
EMBODIMENTS
The following list of embodiments is intended to complement, rather than displace or supersede, the previous descriptions.
Embodiment 1. A method of predicting a likelihood of responsiveness to ibrutinib and R- CHOP combination therapy in a patient having diffuse large B-cell lymphoma, comprising:
assaying a biological sample from the patient to detect an amount of one or more secreted proteins, wherein the one or more secreted proteins are CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof; and
comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control,
wherein the patient has a likelihood of responsiveness to ibrutinib and R- CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
Embodiment 2. The method of embodiment 1, further comprising identifying the patient has having non-GCB diffuse large B-cell lymphoma if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
Embodiment 3. A method of predicting a likelihood of responsiveness to ibrutinib and R- CHOP combination therapy in a patient having diffuse large B-cell lymphoma, comprising:
assaying a biological sample from the patient to detect an amount of one or more secreted proteins, wherein the one or more secreted proteins are AURKB, HCK, TWEAKR, Nectin-like protein 1 , Angiogenin, CK-MB, or any combination thereof; and
comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-
CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
Embodiment 4. The method of embodiment 3, further comprising identifying the patient has having GCB diffuse large B-cell lymphoma if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
Embodiment 5. The method of any one of the previous embodiments, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL population.
Embodiment 6. The method of embodiment 1 or 2, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a GCB patient population.
Embodiment 7. The method of embodiment 3 or 4, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a non-GCB population.
Embodiment 8. A method of treating a patient having diffuse large B-cell lymphoma, comprising:
administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from CPNEl, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin Bl aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof.
Embodiment 9. A method of treating a patient having diffuse large B-cell lymphoma, comprising:
administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from AURKB, HCK, TWEAKR, Nectin-like protein 1 , Angiogenin, CK-MB, or any combination thereof.
Embodiment 10. The method of embodiment 8 or 9, further comprising, prior to the
administering:
determining the amount of the one or more secreted proteins in a biological sample from the patient; and
comparing the amount of the one or more secreted proteins in the biological sample to the mean amount of the same one or more secreted proteins from the control.
Embodiment 1 1. The method of any one of embodiments 8-10, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL population.
Embodiment 12. The method of embodiment 8 or 10, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a GCB patient population.
Embodiment 13. The method of embodiment 9 or 10, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a non-GCB population.
Embodiment 14. A method of identifying a patient as having diffuse large B-cell lymphoma comprising:
assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein the one or more genes are CREBBP, LRPIB, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARDl, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes identifies the patient as having diffuse large B- cell lymphoma.
Embodiment 15. A method of treating a patient having diffuse large B-cell lymphoma comprising:
administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a mutation in one or more genes, wherein the one or more genes are CREBBP, LRPIB, MLL2, BCL2, GNAS, TNFRSF 14, AR, ARID2, AXL, BARDl, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSDl, NTRKl, NTRK3, SPEN, ZNF703, or any combination thereof.
Embodiment 16. The method of embodiment 15, further comprising, prior to the
administering:
assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein the one or more genes are CREBBP, LRPIB, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH1, NOTCH2, NOTCH3, NSDl, NTRKl, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes identifies the patient as having diffuse large B- cell lymphoma.
Embodiment 17. The method of embodiment 14 or 16, the wherein the method comprises assaying a DNA sample from the patient to detect a mutation in two or more genes.
Embodiment 18. The method of embodiment 15, wherein the patient has a mutation in two or more genes.

Claims

What is Claimed:
1. A method of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma, comprising: assaying a biological sample from the patient to detect an amount of one or more secreted proteins, wherein the one or more secreted proteins are CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof; and
comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
2. The method of claim 1, further comprising identifying the patient has having non- GCB diffuse large B-cell lymphoma if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
3. A method of predicting a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy in a patient having diffuse large B-cell lymphoma, comprising: assaying a biological sample from the patient to detect an amount of one or more secreted proteins, wherein the one or more secreted proteins are AURKB, HCK, TWEAKR, Nectin-like protein 1 , Angiogenin, CK-MB, or any combination thereof; and
comparing the amount of the one or more secreted proteins from the biological sample to a mean amount of the same one or more secreted proteins from a control, wherein the patient has a likelihood of responsiveness to ibrutinib and R-CHOP combination therapy if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control.
4. The method of claim 3, further comprising identifying the patient has having GCB diffuse large B-cell lymphoma if the amount of the one or more secreted proteins from the biological sample is higher than the mean amount of the same one or more secreted proteins in the control
5. The method of claim 1 or 3, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL population.
6. The method of claim 1 or 2, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a GCB patient population.
7. The method of claim 3 or 4, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a non-GCB population.
8. A method of treating a patient having diffuse large B-cell lymphoma, comprising: administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from CPNE1, kininogen HMW, PA2G4, peroxiredoxin-6, PSD7, UBE2N, aflatoxin B l aldehyde reductase, AREG, CD27, CFC1, FLRT1, IFN-g, JAM-C, prostatic binding protein, or any combination thereof.
9. A method of treating a patient having diffuse large B-cell lymphoma, comprising: administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a higher amount of one or more secreted proteins as compared to a mean amount of the same one or more secreted proteins from a control, the one or more secreted proteins selected from AURKB, HCK, TWEAKR, Nectin- like protein 1, Angiogenin, CK-MB, or any combination thereof.
10. The method of claim 8 or 9, further comprising, prior to the administering:
determining the amount of the one or more secreted proteins in a biological sample from the patient; and comparing the amount of the one or more secreted proteins in the biological sample to the mean amount of the same one or more secreted proteins from the control.
11. The method of claim 8 or 9, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a DLBCL population.
12. The method of claim 8, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a GCB patient population.
13. The method of claim 9, wherein the control is a mean amount of the same one or more secreted proteins in a biological sample from a non-GCB population.
14. A method of identifying a patient as having diffuse large B-cell lymphoma
comprising:
assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein the one or more genes are CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF 14, AR, ARID2, AXL, BARDl, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCORl, NOTCH 1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes identifies the patient as having diffuse large B-cell lymphoma.
15. A method of treating a patient having diffuse large B-cell lymphoma comprising: administering a pharmaceutically effective dose of ibrutinib and R-CHOP to the patient, wherein the patient has a mutation in one or more genes, wherein the one or more genes are CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF14, AR, ARID2, AXL, BARDl, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCORl, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof.
16. The method of claim 15, further comprising, prior to the administering:
assaying a DNA sample from the patient to detect a mutation in one or more genes, wherein the one or more genes are CREBBP, LRP1B, MLL2, BCL2, GNAS, TNFRSF 14, AR, ARID2, AXL, BARD1, BRAF, ERBB2, FANCC, FAT3, FLT4, HGF, MET, MYC, NCOR1, NOTCH 1, NOTCH2, NOTCH3, NSD1, NTRK1, NTRK3, SPEN, ZNF703, or any combination thereof, wherein a mutation in the one or more genes identifies the patient as having diffuse large B-cell lymphoma.
17. The method of claim 14 or 16, the wherein the method comprises assaying a DNA sample from the patient to detect a mutation in two or more genes.
18. The method of claim 15, wherein the patient has a mutation in two or more genes.
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