WO2022125871A1 - Methods for tailoring analgesic regimen in cancer patient's based on tumor transcriptomics - Google Patents

Methods for tailoring analgesic regimen in cancer patient's based on tumor transcriptomics Download PDF

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WO2022125871A1
WO2022125871A1 PCT/US2021/062771 US2021062771W WO2022125871A1 WO 2022125871 A1 WO2022125871 A1 WO 2022125871A1 US 2021062771 W US2021062771 W US 2021062771W WO 2022125871 A1 WO2022125871 A1 WO 2022125871A1
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opioid
mmes
intraoperative
analgesic
effective amount
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PCT/US2021/062771
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French (fr)
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Joseph SCARPA
Gregory Fischer
Patrick Mccormick
Joshua MINCER
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Memorial Sloan Kettering Cancer Center
Cornell University
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Publication of WO2022125871A1 publication Critical patent/WO2022125871A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A61K31/24—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group having an amino or nitro group
    • A61K31/245—Amino benzoic acid types, e.g. procaine, novocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/46—8-Azabicyclo [3.2.1] octane; Derivatives thereof, e.g. atropine, cocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • 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
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present disclosure provides methods for determining whether a patient diagnosed with urological cancer and undergoing tumor resection surgery will benefit from treatment with intraoperative opioid analgesics or opioid-free intraoperative analgesics (e.g., intraoperative ester-type or amide-type local anesthetics) based on the expression levels of specific survival-associated gene expression networks in the cancer patient.
  • intraoperative opioid analgesics or opioid-free intraoperative analgesics e.g., intraoperative ester-type or amide-type local anesthetics
  • the present disclosure provides a method for selecting a cancer patient undergoing tumor resection surgery for renal cancer for treatment with an intraoperative opioid analgesic comprising (a) detecting expression levels of at least one survival-associated gene expression network in a biological sample obtained from the cancer patient that are either comparable or decreased relative to a control sample obtained from a healthy subject or a predetermined threshold, wherein the at least one survival-associated gene expression network is a NRF2-dependent macrophage network and/or a Th2 immune network; and (b) administering to the cancer patient an effective amount of an intraoperative opioid analgesic during the tumor resection surgery.
  • the expression levels of the at least one survival-associated gene expression network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • the biological sample comprises genomic DNA, cDNA, RNA, and/or mRNA.
  • the present disclosure provides a method for prolonging survival of a cancer patient undergoing tumor resection surgery for renal cancer comprising administering to the cancer patient an effective amount of an intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a NRF2-dependent macrophage network and/or a Th2 immune network in a biological sample obtained from the cancer patient are either comparable or decreased relative to a control sample obtained from a healthy subject or a predetermined threshold.
  • expression levels of the NRF2-dependent macrophage network and/or the Th2 immune network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • next-generation sequencing PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • the intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
  • the effective amount of the intraoperative opioid analgesic may range from about 1 MME to about 200 MMEs. In certain embodiments, the effective amount of the intraoperative opioid analgesic is about 1 MME to about 20 MMEs, about 20 MMEs to about 45 MMEs, or about 45 MMEs to about 200 MMEs.
  • the effective amount of the intraoperative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35 MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45
  • the effective amount of the intraoperative opioid analgesic is administered as a series of bolus doses or as a continuous infusion during the tumor resection surgery. In certain embodiments, the effective amount of the intraoperative opioid analgesic is administered to the cancer patient prior to incision. Additionally or alternatively, in some embodiments, the effective amount of the intraoperative opioid analgesic is administered intravenously.
  • the methods of the present technology further comprise administering to the cancer patient an effective amount of a local anesthetic solution via an epidural catheter before, during and/or after the tumor resection surgery.
  • the effective amount of the local anesthetic solution may range from about 0.05%- 4% local anesthetic solution in a volume of 1-10 ml per hour when administered via an epidural catheter.
  • the effective amount of the local anesthetic solution is administered as a series of bolus doses or as a continuous infusion during the tumor resection surgery.
  • the effective amount of the local anesthetic solution may be administered before, during and/or after the tumor resection surgery using any regional anesthesia technique directed at nerves innervating the thorax and chest wall (e.g., via serratus plane nerve block, intercostal nerve block, or paravertebral block).
  • the effective amount of the local anesthetic solution may range from about 0.05%-4% local anesthetic solution in a volume of 10-40 ml when administered using any regional anesthesia technique directed at nerves innervating the thorax and chest wall (e.g., via serratus plane nerve block, intercostal nerve block, or paravertebral block).
  • suitable local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine.
  • the local anesthetic solution may further comprise an opioid (e.g., fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil).
  • the local anesthetic solution may comprise 0.5 mcg/ml-50 mcg/ml opioid.
  • the methods of the present technology further comprise administering to the cancer patient an effective amount of a post-operative opioid analgesic after the tumor resection surgery.
  • post-operative opioid analgesics include, but are not limited to, fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
  • the post-operative opioid analgesic and the intraoperative opioid analgesic are the same opioid analgesic or different opioid analgesics.
  • the effective amount of the post-operative opioid analgesic and the effective amount of the intraoperative opioid analgesic are the same or different.
  • the effective amount of the post-operative opioid analgesic is administered to the cancer patient as a bolus of about 0.005 mg to about 100 mg. In some embodiments, the effective amount of the post-operative opioid analgesic is administered to the cancer patient as a bolus of about 0.005 mg, about 0.006 mg, about 0.007 mg, about 0.008 mg, about 0.009 mg, about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1-5 mg, about 5-10 mg, about 1-5 mg, about 5-10 mg, about 1-5 mg, about 5-10 mg, about 1- 5 mg, about 5-10 mg, about 10-15 mg, about 15-20 mg, about 20-25 mg, about 25-30 mg,
  • the effective amount of the post-operative opioid analgesic may be continuously delivered to the cancer patient at a per hour rate of about 0.01 mg/hr to about 10 mg/hr. In certain embodiments, the effective amount of the post-operative opioid analgesic is continuously delivered to the cancer patient at a per hour rate of about 0.01 mg/hr, about 0.02 mg/hr, about 0.03 mg/hr, about 0.04 mg/hr, about 0.05 mg/hr, about 0.06 mg/hr, about 0.07 mg/hr, about 0.08 mg/hr, about 0.09 mg/hr, about 0.1 mg/hr, about 0.2 mg/hr, about 0.3 mg/hr, about 0.4 mg/hr, about 0.5 mg/hr, about 0.6 mg/hr, about 0.7 mg/hr, about 0.8 mg/hr, about 0.9 mg/hr, about 1 mg/hr, about 1.5 mg/hr, about 2 mg/hr, about 2.5 mg/hr, about 3 mg
  • the present disclosure provides a method for selecting a cancer patient undergoing tumor resection surgery for renal cancer for treatment with an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic comprising (a) detecting expression levels of at least one survival-associated gene expression network in a biological sample obtained from the cancer patient that are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold, wherein the at least one survival-associated gene expression network is a NRF2-dependent macrophage network and/or a Th2 immune network; and (b) administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery.
  • the expression levels of the at least one survival-associated gene expression network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • the biological sample comprises genomic DNA, cDNA, RNA, and/or mRNA.
  • the present disclosure provides a method for prolonging survival of a cancer patient undergoing tumor resection surgery for renal cancer comprising administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low- dose intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a NRF2-dependent macrophage network and/or a Th2 immune network in a biological sample obtained from the cancer patient are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold.
  • expression levels of the NRF2-dependent macrophage network and/or the Th2 immune network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • next-generation sequencing PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • the low- dose intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. Additionally or alternatively, in some embodiments, the effective amount of the low-dose intraoperative opioid analgesic is about 1 MME to about 50 MMEs.
  • the effective amount of the low- dose intraoperative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25
  • MMEs about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30
  • MMEs about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35
  • MMEs about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45
  • MMEs or about 45-50 MMEs.
  • the opioid-free intraoperative analgesic is an amide-type local anesthetic or an ester- type local anesthetic.
  • amide-type local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, or levobupivacaine.
  • ester-type local anesthetics include, but are not limited to, cocaine, procaine, tetracaine, chloroprocaine, or benzocaine.
  • the opioid-free intraoperative analgesic may be administered via an epidural catheter.
  • the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 1-10 ml per hour when administered via an epidural catheter.
  • the opioid-free intraoperative analgesic may be administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
  • the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 10-40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
  • the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic may be administered as a series of bolus doses, or as a continuous infusion during the tumor resection surgery.
  • the methods of the present technology further comprise administering to the cancer patient an effective amount of an opioid-free post-operative analgesic or a low-dose post-operative opioid analgesic after the tumor resection surgery.
  • opioid-free post-operative analgesics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine.
  • suitable low-dose post-operative opioid analgesics include, but are not limited to, fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
  • the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same analgesic or different analgesics.
  • the effective amount of the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same or different.
  • the effective amount of the low-dose post-operative opioid analgesic is about 1 MME to about 50 MMEs. In some embodiments, the effective amount of the low-dose post-operative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs,
  • the effective amount of the opioid-free post-operative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about
  • amide-type or ester-type local anesthetic solution in a volume of about 10 ml, about 12.5 ml, about 15 ml, about 17.5 ml, about 20 ml, about 22.5 ml, about 25 ml, about 27.5 ml, about 30 ml, about 32.5 ml, about 35 ml, about 37.5 ml, or about 40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
  • the NRF2-dependent macrophage network comprises 5 or more genes selected from among A1CF, ABAT, ABCB1, ABCB9, ABCC2, ABCC6P1, ABCG1, ABHD6, ABLIM3, ABP1, ACAD11, ACADL, ACAT1, ACBD4, ACO2, ACOT4, ACOX2, ACSL1, ACSM5, ACY1, ADAMTS3, ADH6, AGPAT3, AGT, AIFM1, AKR1C1, AKR1C2, AKR7A2, AKR7A3, ALAD, ALDH1A1, ALDH1A2, ALDH1L1, ALDH2, ALDH3A2, ALDH4A1, ALDH7A1, ALDH8A1, ALDOB, ALPK2, ALPL, AMDHD1, ANK3, ANKRD56, ANPEP, ANXA13, ANXA2P2, ANXA2, AOX1, APITD1, AQP3, AQP9, ARHGAP1, ARHG
  • the Th2 immune network comprises 5 or more genes selected from among AIAP1, ANLN, ARHGAP11A, ASF1B, ASPM, ATAD2, AURKA, BRCA1, BUB1, C10orf2, C13orf34, C15orf23, C16orf75, CACNA2D4, CCDC99, CCNA2, CCNB1, CCNF, CDC6, CDCA7, CDK1, CDT1, CENPE, CENPF, CENPH, CENPL, CENPN, CENPO, CHAF1A, CHAF1B, CHEK1, CPOX, CTSF, DBF4, DERL1, DHFR, DIAPH3, DTL, E2F1, ECT2, EPR1, ESPL1, EZH2, FAM11 IB, FANCA, FANCD2, FANCI, FASN, FEN1, GGH, GINS1, GINS2, GINS3, GPRIN1, GPSM2, HELLS,
  • the tumor resection surgery comprises nephrectomy.
  • the renal cancer may have a histologic subtype selected from among clear cell renal cell carcinomas (ccRCC), papillary renal cell carcinomas (pRCC), chromophobe renal cell carcinomas (crRCC), multilocular cystic RCC, collecting duct carcinoma, medullary carcinoma, mucinous tubular and spindle cell carcinoma, neuroblastoma-associated RCC, Xpl l.2 translocation-TFE3 carcinoma, and unclassified lesions.
  • the cancer patient exhibits stage I, stage II, stage III, or stage IV renal cancer.
  • the present disclosure provides a method for selecting a cancer patient undergoing tumor resection surgery for bladder cancer for treatment with an opioid- free intraoperative analgesic or a low-dose intraoperative opioid analgesic comprising (a) detecting expression levels of a survival-associated gene expression network in a biological sample obtained from the cancer patient that are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold; and (b) administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery, wherein the survival- associated gene expression network comprises 5 or more genes selected from among ABCA6, ACTA2, ACTC1, ACTG2, ACTN1, ADAM12, ADAM19, ADAMTS2, ADH1B, ADRA2A, AEBP1, AG2, ALDH1L2, AMIG02, ANGPTL1, ATP10A, ATP8B2, AXIN2, BAG2, Cl lorf41,
  • the expression levels of the survival-associated gene expression network are detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • next-generation sequencing PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • qPCR real-time quantitative PCR
  • the present disclosure provides a method for prolonging survival of a cancer patient undergoing tumor resection surgery for bladder cancer comprising administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a survival-associated gene expression network in a biological sample obtained from the cancer patient are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold, wherein the survival-associated gene expression network comprises 5 or more genes selected from among ABCA6, ACTA2, ACTC1, ACTG2, ACTN1, ADAM12, ADAM I 9, ADAMTS2, ADH1B, ADRA2A, AEBP1, AG2, ALDH1L2, AMIG02, ANGPTL1, ATP10A, ATP8B2, AXIN2, BAG2, Cl lorf41, C14orfl32, Clorfl98, C2orf40, C7, C9orfl l0,
  • the expression levels of the survival-associated gene expression network are detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligationdependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • next-generation sequencing PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligationdependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • qPCR real-time quantitative PCR
  • the low- dose intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. Additionally or alternatively, in some embodiments, the effective amount of the low-dose intraoperative opioid analgesic is about 1 MME to about 50 MMEs.
  • the effective amount of the low- dose intraoperative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35 MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about
  • the opioid-free intraoperative analgesic is an amide-type local anesthetic or an ester- type local anesthetic.
  • amide-type local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, or levobupivacaine.
  • ester-type local anesthetics include, but are not limited to, cocaine, procaine, tetracaine, chloroprocaine, or benzocaine.
  • the opioid-free intraoperative analgesic may be administered via an epidural catheter.
  • the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 1-10 ml per hour when administered via an epidural catheter.
  • the opioid-free intraoperative analgesic may be administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
  • the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 10-40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
  • the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic may be administered as a series of bolus doses, or as a continuous infusion during the tumor resection surgery.
  • the methods of the present technology further comprise administering to the cancer patient an effective amount of an opioid-free post-operative analgesic or a low-dose post-operative opioid analgesic after the tumor resection surgery.
  • opioid-free post-operative analgesics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine.
  • suitable low-dose post-operative opioid analgesics include, but are not limited to, fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
  • the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same analgesic or different analgesics.
  • the effective amount of the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same or different.
  • the effective amount of the low-dose post-operative opioid analgesic is about 1 MME to about 50 MMEs. In some embodiments, the effective amount of the low-dose post-operative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs
  • the effective amount of the opioid-free post-operative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about
  • the tumor resection surgery comprises cystectomy. Additionally or alternatively, in certain embodiments, the cancer patient exhibits stage I, stage II, stage III,
  • the patient is human.
  • the biological sample obtained from the cancer patient comprises biopsied tumor tissue, whole blood, plasma, or serum.
  • FIGs. 1A-1F show differential expression of opioid pathway genes in clear cell renal cell carcinoma.
  • FIG. 1A shows top fifty differentially expressed genes between cases and controls in clear cell renal cell carcinoma, clustered by gene expression. Shades of red represents lower expression, and blue expression represents greater expression.
  • FIG. IB shows P value and log2 fold change for a subset of five hundred differentially expressed genes. Horizontal dotted line represents P threshold of 0.001, and the vertical dotted line represents fold change threshold of 0.5. Genes with a log2 fold change > 0.5 are labeled in red and log2 fold change ⁇ 0.5 are labeled in blue. Genes in the opioid signaling pathway are represented by large labeled nodes.
  • FIGs. 1C-1F show comparison of gene expression distributions between cases and controls for OGFR, OGFRL1, TLR4, OPRL1, all of which have P ⁇ 0.05.
  • FIGs. 2A-2J show characterization of the 15 gene coexpression networks in clear cell renal cell carcinoma (ccRCC) and association with survival endpoints.
  • FIG. 2A shows that topological overlap matrix plot depicts gene coexpression. Darker yellow and red represents stronger correlations between genes.
  • FIG. 2B shows that Z statistic represents reproducibility of each module. Z > 10 (green dotted line) represents strong evidence of robustness and Z >1 (blue dotted line) represents weak evidence.
  • FIG. 2C shows that circos plot depicts module eigengene correlations with survival and pathology measurements. Modules are ranked by the strength of their association with cancer-specific survival, reflected by the height of the purple histogram in Row 1.
  • Rows 2-4 reflect the Cox model beta coefficient for overall survival, cancer-specific survival, and recurrence-free survival, respectively.
  • Rows 5-7 reflect rho values for T, N, and M stage, respectively. Bluer values reflect negative values, while brown values reflect strong positive values.
  • Rows 8-13 depict the -loglO(P) values for the same survival (8-10) and pathology variables (11-13).
  • FIG. 2D shows Cox model beta coefficient and 95% confidence interval for each module and its association with cancer-specific survival. Red bars depict P ⁇ 0.05.
  • FIGs. 2E-2J show survival curves comparing individuals with upregulated (blue) and downregulated (gold) network expression for green module (FIGs. 2E-2G) and tan module (FIGs. 2H-2J).
  • FIGs. 2E and 2H Cancer-specific survival (FIGs. 2E and 2H), recurrence-free survival (FIGs. 2F and 21), and overall survival (FIGs. 2G and 2J) are depicted, and their respective Cox P values are each less than 0.05.
  • FIGs. 3A-3E show prediction of drug effects on the survival-associated networks.
  • FIG. 3A shows a comparison of topological overlap matrices in cases (top right triangle) versus controls (bottom left triangle) for four modules. Greater coexpression is colored in dark yellow and red, while less coexpression is colored in light yellow and white.
  • Module differential connectivity (MDC) and FDR values are depicted for each module. Differential connectivity was considered significant by FDR ⁇ 0.01.
  • FIGs. 3B-3D show tau scores representing modulation of each survival network by leu-enkephalin (FIG. 3B), naloxone (FIG. 3C), and VEGF -inhibitor (FIG. 3D).
  • FIG. 3E shows the association between leu-enkephalin tau score and Cox model survival coefficients for overall survival, recurrence-free survival, and cancerspecific survival.
  • FIG. 4 shows reconstructing directed transcriptional networks and master regulators in ccRCC.
  • Directed networks representing relationships between modules (boxed), as well as gene-gene relationships within four separate modules.
  • Each node is outlined based on its module color.
  • Key drivers are represented by large nodes, and shaded key drivers are those with known associations to the opioid pathway.
  • FIG. 5 shows top gene ontology pathways enriched for differentially expressed genes in ccRCC.
  • the color of the circle reflects enrichment p value, and the size of the circle reflects the number of differentially expressed genes in the respective gene ontology categories.
  • FIG. 6 shows comparison of OPRM1, OPRK1, and OPRD1 gene expression between cases and controls. Each point represents gene expression for an individual sample in the cohort.
  • FIG. 7 shows that tumors with high and low opioid pathway gene expression have differential pathologic stage in ccRCC. Boxplots illustrate the opioid pathway gene expression distribution across pathologic stage for tumors with high and low gene expression of specific opioid signaling genes. For each opioid signaling gene, the respective top and bottom gene expression quartiles were determined and the sample subset were defined accordingly. Association between gene expression and stage for these high and low expressors were estimated by the Kruskal Wallis test. RSEM normalized gene expression is plotted on the y axis and pathologic stage on the x axis. Kruskal Wallis P values are listed for each gene.
  • FIGs. 8A-8C show individual sample expression across module eigengenes.
  • FIG. 8A shows that median module eigengene expression was calculated for each sample and the distribution across all samples was plotted.
  • FIG. 8B shows that the variance of module eigengene expression for each sample was calculated and distribution across all samples plotted.
  • FIG. 9 shows fisher’s exact test overrepresentation between TCGA and MSKCC ccRCC networks.
  • the blue boxes reflect significant Fisher’s exact test enrichment between specific MSK and KIRC networks (P ⁇ 0.05). The darker shades correspond to increased odds ratio.
  • the eight TCGA survival-associated networks are seen to strongly overlap with the set of networks independently derived in the ccRCC-MSKCC cohort.
  • FIGs. 10A-10H show correlation between TCGA and MSKCC gene expression for eight survival-associated networks. Gene expression is correlated between the TCGA KIRC and ccRCC-MSKCC for each of these eight networks. Spearman rho and p value listed.
  • FIG. 12A-12B show prediction of drug effects on a survival-associated network in bladder cancer (BLCA).
  • FIG. 12A shows that the “pink” network is associated with survival in BLCA.
  • FIG. 12B shows that Tau for pink network hub expression with various drugs is plotted along the x axis.
  • Leu-enkephalin modulates the “pink” network in an anti-survival direction, while naloxone is pro-survival.
  • Positive controls include various chemotherapeutic agents, which are seen to modulate this network in a pro-survival direction.
  • the dotted red (outside two) vertical lines represent
  • 90, which represents strong evidence of an effect.
  • the dotted blue (middle two) lines represent
  • FIGs. 13A-13D show gene expression correlation between immune signature genes (y axis) and four ccRCC master regulator genes (x axis): PLXNB1 (FIG. 13A), CREB5 (FIG. 13B), IL4R (FIG. 13C), CLEC2D (FIG. 13D).
  • PLXNB1 FIG. 13A
  • CREB5 FIG. 13B
  • IL4R FIG. 13C
  • CLEC2D FIG. 13D
  • One representative correlation plot is depicted here for each master regulator gene. Spearman Rho and P values are reported for each gene-pair comparison. All correlation statistics between master regulators and immune signature genes are listed in FIG. 21.
  • FIG. 14 shows mutations of the Reactome opioid signaling pathway in ccRCC.
  • FIGs. 15A-15B show module membership for each module.
  • FIG. 15A. hub status 0;
  • FIG. 15B. hub status 1.
  • FIG. 16 shows gene ontology coexpression network enrichment.
  • FIGs. 17A-17I show Cox survival analysis for association of the 15 networks and survival endpoints.
  • FIG. 18 shows clinical and demographic overrepresentation in sample subgroups with high module eigengene expression.
  • FIG. 19 shows list of hub genes for the eight cancer-specific survival-associated networks.
  • FIG. 20 shows master regulators of the transition from normal to disease state.
  • FIG. 21 shows gene expression correlation statistics between immune signature genes and four ccRCC master regulator genes involved in opioid immunomodulation (CREB5, IL4R, CLEC2D, PLXNB1).
  • Kidney cancer is the seventh most commonly diagnosed solid tumor in the United States, with clear cell being its most common subtype. Genetic and molecular changes are associated with survival, and gene network expression changes distinguish renal cell carcinoma subtypes (Ricketts CJ, et al., Cell Reports 23: 313-326 (2016)). Opioids play a critical role in perioperative analgesia in oncologic surgery, and recent in vitro evidence implicates opioids in proliferation, invasion and metastasis of ccRCC specifically (Ma et al., Renal Failure. 2016;39:258-64).
  • opioids constitute the major component of perioperative analgesic regimens for surgery in general, a variety of evidence points to an association between perioperative opioid exposure and longer-term oncological outcomes. The mechanistic details underlying these effects are not well understood. The relationship between opioids and cancer outcomes is controversial. Some retrospective studies have failed to replicate earlier findings and reported effect sizes are variable and often specific to cancer subtypes. In some cases, this may point to truly null effects or confounders. It may also indicate that opioids have cancer subtype-specific effects that are masked in larger heterogeneous cohorts. Without prospective experiments, quasi-experimental studies, or a molecular understanding of opioids in humans with cancer, it is difficult to adjudicate between these possibilities.
  • the present disclosure demonstrates that opioids modulate survival-associated coexpression networks in clear cell renal cell carcinoma (ccRCC).
  • ccRCC clear cell renal cell carcinoma
  • the present disclosure examines RNAseq and cancer-specific outcomes data in both the TCGA KIRC cohort and an independent ccRCC replication cohort who underwent nephrectomy. Undirected and directed gene networks were constructed and each were correlated with recurrence-free survival (RFS), cancer-specific survival (CSS), and overall survival (OS).
  • RFS recurrence-free survival
  • CSS cancer-specific survival
  • OS overall survival
  • the expression and network connectivity of opioid- and survival -related pathways between ccRCC and controls were compared and drug-induced transcriptional profiles from in vitro cancer cells were projected onto ccRCC gene networks to characterize pathways through which opioids may influence survival.
  • survival-associated networks associated with survival endpoints in ccRCC were identified, and master regulators of the transition from the normal to disease state were inferred, a number of which are linked to opioid pathways. These results are the first to suggest a mechanism for opioid effects on cancer outcomes through modulation of survival-associated coexpression networks. Accordingly, the survival-associated networks disclosed herein may function as potential pharmacogenomic biomarkers, helping to risk- stratify individual patients and predict individual response to a drug of interest. By better modeling underlying cancer biology and its response to pharmacologic perturbations, integrative systems-based models may use individual gene expression profiles to guide personalized anesthetic and analgesic plans and to optimize cancer-specific outcomes for individual cancer patients.
  • the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
  • adapter refers to a short, chemically synthesized, nucleic acid sequence which can be used to ligate to the end of a nucleic acid sequence in order to facilitate attachment to another molecule.
  • the adapter can be single-stranded or double-stranded.
  • An adapter can incorporate a short (typically less than 50 base pairs) sequence useful for PCR amplification or sequencing.
  • the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another.
  • an “alteration” of a gene or gene product refers to the presence of a mutation or mutations within the gene or gene product, e.g., a mutation, which affects the quantity or activity of the gene or gene product, as compared to the normal or wild-type gene.
  • the genetic alteration can result in changes in the quantity, structure, and/or activity of the gene or gene product in a cancer tissue or cancer cell, as compared to its quantity, structure, and/or activity, in a normal or healthy tissue or cell (e.g., a control).
  • an alteration which is associated with cancer, or predictive of responsiveness to an intraoperative analgesic can have an altered nucleotide sequence (e.g., a mutation), amino acid sequence, chromosomal translocation, intra-chromosomal inversion, copy number, expression level, protein level, protein activity, in a cancer tissue or cancer cell, as compared to a normal, healthy tissue or cell.
  • exemplary mutations include, but are not limited to, point mutations (e.g., silent, missense, or nonsense), deletions, insertions, inversions, linking mutations, duplications, translocations, inter- and intra- chromosomal rearrangements. Mutations can be present in the coding or non-coding region of the gene.
  • nucleic acid amplification methods are well known to the skilled artisan and include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Q-replicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two- step multiplexed amplifications, rolling circle amplification (RCA), recombinase- polymerase amplification (RPA)(TwistDx, Cambridge, UK), transcription mediated amplification, signal mediated amplification of RNA technology, loop-mediated isothermal amplification of DNA, helicase-dependent amplification, single primer isothermal amplification, and self
  • cancer or “tumor” are used interchangeably and refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell. As used herein, the term “cancer” includes premalignant, as well as malignant cancers.
  • cancer-specific survival refers to the time from the date of diagnosis of a cancer to the date of death from the cancer apart from other causes. Patients who die from causes unrelated to the cancer are not counted in this measurement.
  • complementary or “complementarity” as used herein with reference to polynucleotides (z.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) refer to the base-pairing rules.
  • nucleic acid sequence refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3’ end of the other, is in “antiparallel association.”
  • sequence “5'-A-G-T-3”’ is complementary to the sequence “3’-T-C-A-5.”
  • Certain bases not commonly found in naturally-occurring nucleic acids may be included in the nucleic acids described herein. These include, for example, inosine, 7- deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA).
  • Complementarity need not be perfect; stable duplexes may contain mismatched base pairs, degenerative, or unmatched bases.
  • Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.
  • a complement sequence can also be an RNA sequence complementary to the DNA sequence or its complement sequence, and can also be a cDNA.
  • control is an alternative sample used in an experiment for comparison purpose.
  • a control can be "positive” or “negative.”
  • a positive control a compound or composition known to exhibit the desired therapeutic effect
  • a negative control a subject or a sample that does not receive the therapy or receives a placebo
  • control nucleic acid sample refers to nucleic acid molecules from a control or reference sample.
  • the reference or control nucleic acid sample is a wild type or a non-mutated DNA or RNA sequence.
  • the reference nucleic acid sample is purified or isolated (e.g., it is removed from its natural state).
  • the reference nucleic acid sample is from a non-tumor sample, e.g., a blood control, a normal adjacent tumor (NAT), or any other non-cancerous sample from the same or a different subject.
  • NAT normal adjacent tumor
  • Detecting refers to determining the presence of a mutation or alteration in a nucleic acid of interest in a sample. Detection does not require the method to provide 100% sensitivity. Analysis of nucleic acid markers can be performed using techniques known in the art including, but not limited to, sequence analysis, and electrophoretic analysis. Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al. , Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al., Methods Mol.
  • sequencing with mass spectrometry such as matrix- assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al., Nat. Biotechnol, 16:381-384 (1998)), and sequencing by hybridization.
  • MALDI-TOF/MS matrix- assisted laser desorption/ionization time-of-flight mass spectrometry
  • Non-limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Additionally, next generation sequencing methods can be performed using commercially available kits and instruments from companies such as the Life Technologies/Ion Torrent PGM or Proton, the Illumina HiSEQ or MiSEQ, and the Roche/454 next generation sequencing system.
  • Detectable label refers to a molecule or a compound or a group of molecules or a group of compounds used to identify a nucleic acid or protein of interest.
  • the detectable label may be detected directly.
  • the detectable label may be a part of a binding pair, which can then be subsequently detected.
  • Signals from the detectable label may be detected by various means and will depend on the nature of the detectable label.
  • Detectable labels may be isotopes, fluorescent moieties, colored substances, and the like.
  • means to detect detectable labels include but are not limited to spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluorescence, or chemiluminescence, or any other appropriate means.
  • the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein.
  • the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions can also be administered in combination with one or more additional therapeutic compounds.
  • the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein.
  • a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated.
  • a therapeutically effective amount can be given in one or more administrations.
  • Gene refers to a DNA sequence that comprises regulatory and coding sequences necessary for the production of an RNA, which may have a non-coding function (e.g., a ribosomal or transfer RNA) or which may include a polypeptide or a polypeptide precursor.
  • the RNA or polypeptide may be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained.
  • a sequence of the nucleic acids may be shown in the form of DNA, a person of ordinary skill in the art recognizes that the corresponding RNA sequence will have a similar sequence with the thymine being replaced by uracil, i.e., "T" is replaced with "U.”
  • hybridize refers to a process where two substantially complementary nucleic acid strands (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex through formation of hydrogen bonds between complementary base pairs.
  • Hybridizations are typically and preferably conducted with probe-length nucleic acid molecules, preferably 15- 100 nucleotides in length, more preferably 18-50 nucleotides in length. Nucleic acid hybridization techniques are well known in the art.
  • Hybridization and the strength of hybridization is influenced by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, and the thermal melting point (Tm) of the formed hybrid.
  • Tm thermal melting point
  • hybridization conditions and parameters see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, N.J.
  • specific hybridization occurs under stringent hybridization conditions.
  • An oligonucleotide or polynucleotide e.g., a probe or a primer
  • a probe or a primer that is specific for a target nucleic acid will “hybridize” to the target nucleic acid under suitable conditions.
  • the term “library” refers to a collection of nucleic acid sequences, e.g., a collection of nucleic acids derived from whole genomic, subgenomic fragments, cDNA, cDNA fragments, RNA, RNA fragments, or a combination thereof.
  • a portion or all of the library nucleic acid sequences comprises an adapter sequence.
  • the adapter sequence can be located at one or both ends.
  • the adapter sequence can be useful, e.g., for a sequencing method (e.g., an NGS method), for amplification, for reverse transcription, or for cloning into a vector.
  • the library can comprise a collection of nucleic acid sequences, e.g., a target nucleic acid sequence (e.g., a tumor nucleic acid sequence), a reference nucleic acid sequence, or a combination thereof.
  • the nucleic acid sequences of the library can be derived from a single subject.
  • a library can comprise nucleic acid sequences from more than one subject (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more subjects).
  • two or more libraries from different subjects can be combined to form a library having nucleic acid sequences from more than one subject.
  • a “library nucleic acid sequence” refers to a nucleic acid molecule, e.g., a DNA, RNA, or a combination thereof, that is a member of a library.
  • a library nucleic acid sequence is a DNA molecule, e.g., genomic DNA or cDNA.
  • a library nucleic acid sequence is fragmented, e.g., sheared or enzymatically prepared, genomic DNA.
  • the library nucleic acid sequences comprise sequence from a subject and sequence not derived from the subject, e.g., adapter sequence, a primer sequence, or other sequences that allow for identification, e.g., “barcode” sequences.
  • multiplex PCR refers to amplification of two or more PCR products or amplicons which are each primed using a distinct primer pair.
  • next-generation sequencing or NGS refers to any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules (e.g., in single molecule sequencing) or clonally expanded proxies for individual nucleic acid molecules in a high throughput parallel fashion (e.g., greater than 10 3 , 10 4 , 10 5 or more molecules are sequenced simultaneously).
  • the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment.
  • Next generation sequencing methods are known in the art, and are described, e.g., in Metzker, M. Nature Biotechnology Reviews 11 :31-46 (2010).
  • oligonucleotide refers to a molecule that has a sequence of nucleic acid bases on a backbone comprised mainly of identical monomer units at defined intervals. The bases are arranged on the backbone in such a way that they can bind with a nucleic acid having a sequence of bases that are complementary to the bases of the oligonucleotide.
  • the most common oligonucleotides have a backbone of sugar phosphate units. A distinction may be made between oligodeoxyribonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group at the 2' position.
  • Oligonucleotides may also include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group.
  • Oligonucleotides of the method which function as primers or probes are generally at least about 10-15 nucleotides long and more preferably at least about 15 to 25 nucleotides long, although shorter or longer oligonucleotides may be used in the method. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide.
  • the oligonucleotide may be generated in any manner, including, for example, chemical synthesis, DNA replication, restriction endonuclease digestion of plasmids or phage DNA, reverse transcription, PCR, or a combination thereof.
  • the oligonucleotide may be modified e.g., by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides.
  • all survival means the observed length of life from the start of treatment to death or the date of last contact.
  • perioperative refers to the time period of a patient's surgical procedure. It commonly includes ward admission, anesthesia, surgery, and recovery.
  • the perioperative period is characterized by a sequence including the time preceding an operation when a patient is being prepared for surgery (“the preoperative period”), followed by the time spent in surgery (“the intraoperative period”), and by the time following an operation when the patient is closely monitored for complications while recovering from the effects of anesthesia (“the postoperative period”).
  • the term “primer” refers to an oligonucleotide, which is capable of acting as a point of initiation of nucleic acid sequence synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a target nucleic acid strand is induced, i.e., in the presence of different nucleotide triphosphates and a polymerase in an appropriate buffer (“buffer” includes pH, ionic strength, cofactors etc.) and at a suitable temperature.
  • buffer includes pH, ionic strength, cofactors etc.
  • One or more of the nucleotides of the primer can be modified for instance by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides.
  • a primer sequence need not reflect the exact sequence of the template.
  • a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the strand.
  • primer as used herein includes all forms of primers that may be synthesized including peptide nucleic acid primers, locked nucleic acid primers, phosphorothioate modified primers, labeled primers, and the like.
  • the term “forward primer” as used herein means a primer that anneals to the anti-sense strand of dsDNA.
  • a “reverse primer” anneals to the sense-strand of dsDNA.
  • primer pair refers to a forward and reverse primer pair (i.e., a left and right primer pair) that can be used together to amplify a given region of a nucleic acid of interest.
  • Probe refers to nucleic acid that interacts with a target nucleic acid via hybridization.
  • a probe may be fully complementary to a target nucleic acid sequence or partially complementary. The level of complementarity will depend on many factors based, in general, on the function of the probe.
  • a probe or probes can be used, for example to detect the presence or absence of a mutation in a nucleic acid sequence by virtue of the sequence characteristics of the target. Probes can be labeled or unlabeled, or modified in any of a number of ways well known in the art.
  • a probe may specifically hybridize to a target nucleic acid. Probes may be DNA, RNA or a RNA/DNA hybrid.
  • Probes may be oligonucleotides, artificial chromosomes, fragmented artificial chromosome, genomic nucleic acid, fragmented genomic nucleic acid, RNA, recombinant nucleic acid, fragmented recombinant nucleic acid, peptide nucleic acid (PNA), locked nucleic acid, oligomer of cyclic heterocycles, or conjugates of nucleic acid. Probes may comprise modified nucleobases, modified sugar moieties, and modified internucleotide linkages. A probe may be used to detect the presence or absence of a target nucleic acid. Probes are typically at least about 10, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100 nucleotides or more in length.
  • recurrence-specific survival means the observed length of life from the time of surgical resection to the time of first recurrence of the cancer, otherwise censored at the time of last follow-up. In RSS, deaths not involving recurrence of cancer are excluded.
  • a “sample” refers to a substance that is being assayed for the presence of a mutation in a nucleic acid of interest. Processing methods to release or otherwise make available a nucleic acid for detection are well known in the art and may include steps of nucleic acid manipulation.
  • a biological sample may be a body fluid or a tissue sample.
  • a biological sample may consist of or comprise blood, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and/or chorionic villi, cultured cells, and the like.
  • Fresh, fixed or frozen tissues may also be used.
  • the sample is preserved as a frozen sample or as formaldehyde- or paraformaldehyde-fixed paraffin- embedded (FFPE) tissue preparation.
  • FFPE paraffin- embedded
  • the sample can be embedded in a matrix, e.g., an FFPE block or a frozen sample.
  • Whole blood samples of about 0.5 to 5 ml collected with EDTA, ACD or heparin as anti-coagulant are suitable.
  • sensitivity is a measure of the ability of a method to detect a preselected sequence variant in a heterogeneous population of sequences.
  • a method has a sensitivity of S % for variants of F % if, given a sample in which the preselected sequence variant is present as at least F % of the sequences in the sample, the method can detect the preselected sequence at a preselected confidence of C %, S % of the time.
  • the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.
  • the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.
  • the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.
  • oligonucleotide primer means that the nucleotide sequence of the primer has at least 12 bases of sequence identity with a portion of the nucleic acid to be amplified when the oligonucleotide and the nucleic acid are aligned.
  • An oligonucleotide primer that is specific for a nucleic acid is one that, under the stringent hybridization or washing conditions, is capable of hybridizing to the target of interest and not substantially hybridizing to nucleic acids which are not of interest. Higher levels of sequence identity are preferred and include at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and more preferably at least 98% sequence identity.
  • “Specificity,” as used herein, is a measure of the ability of a method to distinguish a truly occurring preselected sequence variant from sequencing artifacts or other closely related sequences. It is the ability to avoid false positive detections. False positive detections can arise from errors introduced into the sequence of interest during sample preparation, sequencing error, or inadvertent sequencing of closely related sequences like pseudo-genes or members of a gene family.
  • a method has a specificity of X % if, when applied to a sample set of Nrotai sequences, in which sequences are truly variant and XNottme are not truly variant, the method selects at least X % of the not truly variant as not variant.
  • a method has a specificity of 90% if, when applied to a sample set of 1,000 sequences, in which 500 sequences are truly variant and 500 are not truly variant, the method selects 90% of the 500 not truly variant sequences as not variant.
  • Exemplary specificities include 90, 95, 98, and 99%.
  • stringent hybridization conditions refers to hybridization conditions at least as stringent as the following: hybridization in 50% formamide, 5xSSC, 50 mM NaHzPC , pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5x Denhart's solution at 42°C overnight; washing with 2x SSC, 0.1% SDS at 45° C; and washing with 0.2x SSC, 0.1% SDS at 45° C.
  • stringent hybridization conditions should not allow for hybridization of two nucleic acids which differ over a stretch of 20 contiguous nucleotides by more than two bases.
  • the terms “subject”, “patient”, or “individual” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the subject, patient or individual is a human.
  • target sequence and “target nucleic acid sequence” refer to a specific nucleic acid sequence to be detected and/or quantified in the sample to be analyzed.
  • Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder.
  • treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.
  • the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved.
  • the treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.
  • Polynucleotides associated with responsiveness to intraoperative opioid analgesics may be detected by a variety of methods known in the art. Non-limiting examples of detection methods are described below.
  • the detection assays in the methods of the present technology may include purified or isolated DNA (genomic or cDNA), RNA or protein or the detection step may be performed directly from a biological sample without the need for further DNA, RNA or protein purification/isolation.
  • Polynucleotides associated with responsiveness to intraoperative opioid analgesics can be detected by the use of nucleic acid amplification techniques that are well known in the art.
  • the starting material may be genomic DNA, cDNA, RNA or mRNA.
  • Nucleic acid amplification can be linear or exponential.
  • Specific variants or mutations may be detected by the use of amplification methods with the aid of oligonucleotide primers or probes designed to interact with or hybridize to a particular target sequence in a specific manner, thus amplifying only the target variant.
  • Non-limiting examples of nucleic acid amplification techniques include polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), digital PCR (dPCR), reverse transcriptase polymerase chain reaction (RT-PCR), nested PCR, ligase chain reaction (see Abravaya, K. et al., Nucleic Acids Res . (1995), 23:675-682), branched DNA signal amplification (see Urdea, M. S.
  • RNA reporters et al., AIDS (1993), 7(suppl 2): S 11- S14
  • amplifiable RNA reporters Q-beta replication
  • transcription-based amplification boomerang DNA amplification
  • strand displacement activation cycling probe technology
  • isothermal nucleic acid sequence based amplification NASBA
  • NASBA isothermal nucleic acid sequence based amplification
  • Oligonucleotide primers for use in amplification methods can be designed according to general guidance well known in the art as described herein, as well as with specific requirements as described herein for each step of the particular methods described.
  • oligonucleotide primers for cDNA synthesis and PCR are 10 to 100 nucleotides in length, preferably between about 15 and about 60 nucleotides in length, more preferably 25 and about 50 nucleotides in length, and most preferably between about 25 and about 40 nucleotides in length.
  • the oligonucleotide primer used in various steps selectively hybridizes to a target template or polynucleotides derived from the target template (i.e., first and second strand cDNAs and amplified products).
  • selective hybridization occurs when two polynucleotide sequences are substantially complementary (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary).
  • a certain degree of mismatch at the priming site is tolerated.
  • Such mismatch may be small, such as a mono-, di- or tri -nucleotide. In certain embodiments, 100% complementarity exists.
  • Probes are capable of hybridizing to at least a portion of the nucleic acid of interest or a reference nucleic acid (i.e., wild-type sequence). Probes may be an oligonucleotide, artificial chromosome, fragmented artificial chromosome, genomic nucleic acid, fragmented genomic nucleic acid, RNA, recombinant nucleic acid, fragmented recombinant nucleic acid, peptide nucleic acid (PNA), locked nucleic acid, oligomer of cyclic heterocycles, or conjugates of nucleic acid. Probes may be used for detecting and/or capturing/purifying a nucleic acid of interest.
  • probes can be about 10 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 75 nucleotides, or about 100 nucleotides long. However, longer probes are possible.
  • Longer probes can be about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 750 nucleotides, about 1,000 nucleotides, about 1,500 nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about 3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides, about 5,000 nucleotides, about 7,500 nucleotides, or about 10,000 nucleotides long.
  • Probes may also include a detectable label or a plurality of detectable labels.
  • the detectable label associated with the probe can generate a detectable signal directly. Additionally, the detectable label associated with the probe can be detected indirectly using a reagent, wherein the reagent includes a detectable label, and binds to the label associated with the probe.
  • detectably labeled probes can be used in hybridization assays including, but not limited to Northern blots, Southern blots, microarray, dot or slot blots, and in situ hybridization assays such as fluorescent in situ hybridization (FISH) to detect a target nucleic acid sequence within a biological sample.
  • FISH fluorescent in situ hybridization
  • Certain embodiments may employ hybridization methods for measuring expression of a polynucleotide gene product, such as mRNA. Methods for conducting polynucleotide hybridization assays have been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al.
  • Detectably labeled probes can also be used to monitor the amplification of a target nucleic acid sequence.
  • detectably labeled probes present in an amplification reaction are suitable for monitoring the amount of amplicon(s) produced as a function of time.
  • probes include, but are not limited to, the 5'- exonuclease assay (TAQMAN® probes described herein (see also U.S. Pat. No. 5,538,848) various stemloop molecular beacons (see for example, U.S. Pat. Nos.
  • the detectable label is a fluorophore.
  • Suitable fluorescent moieties include but are not limited to the following fluorophores working individually or in combination: 4-acetamido-4'-isothiocyanatostilbene- 2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes); 5-(2- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino-l- naphthy
  • IR1446 lanthamide phosphors; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin, R- phycoerythrin; allophycocyanin; o-phthaldialdehyde; Oregon Green®; propidium iodide; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 -pyrene butyrate; QSY® 7; QSY® 9; QSY® 21; QSY® 35 (Molecular Probes); Reactive Red 4 (Cibacron®Brilliant Red 3B-A); rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, r
  • Detector probes can also comprise sulfonate derivatives of fluorescenin dyes with S03 instead of the carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of CY 5 (commercially available for example from Amersham).
  • Detectably labeled probes can also include quenchers, including without limitation black hole quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch).
  • quenchers including without limitation black hole quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch).
  • Detectably labeled probes can also include two probes, wherein for example a fluorophore is on one probe, and a quencher is on the other probe, wherein hybridization of the two probes together on a target quenches the signal, or wherein hybridization on the target alters the signal signature via a change in fluorescence.
  • interchelating labels such as ethidium bromide, SYBR® Green I (Molecular Probes), and PicoGreen® (Molecular Probes) are used, thereby allowing visualization in real-time, or at the end point, of an amplification product in the absence of a detector probe.
  • real-time visualization may involve the use of both an intercalating detector probe and a sequence-based detector probe.
  • the detector probe is at least partially quenched when not hybridized to a complementary sequence in the amplification reaction, and is at least partially unquenched when hybridized to a complementary sequence in the amplification reaction.
  • the amount of probe that gives a fluorescent signal in response to an excited light typically relates to the amount of nucleic acid produced in the amplification reaction.
  • the amount of fluorescent signal is related to the amount of product created in the amplification reaction. In such embodiments, one can therefore measure the amount of amplification product by measuring the intensity of the fluorescent signal from the fluorescent indicator.
  • Primers or probes may be designed to selectively hybridize to any portion of a nucleic acid sequence encoding a polypeptide selected from among:
  • A1CF ABAT, ABCB1, ABCB9, ABCC2, ABCC6P1, ABCG1, ABHD6, ABLIM3, ABP1, ACAD11, ACADL, ACAT1, ACBD4, ACO2, ACOT4, ACOX2, ACSL1, ACSM5, ACY1, ADAMTS3, ADH6, AGPAT3, AGT, AIFM1, AKR1C1, AKR1C2, AKR7A2, AKR7A3, ALAD, ALDH1A1, ALDH1A2, ALDH1L1, ALDH2, ALDH3A2, ALDH4A1, ALDH7A1, ALDH8A1, ALDOB, ALPK2, ALPL, AMDHD1, ANK3, ANKRD56, ANPEP, ANXA13, ANXA2P2, ANXA2, A0X1, APITD1, AQP3, AQP9, ARHGAP1, ARHGAP24, ARL4C, ASB13, ASRGL1, ASTN2, ATP6V0A1, AUH, AX
  • detection can occur through any of a variety of mobility dependent analytical techniques based on the differential rates of migration between different nucleic acid sequences.
  • mobility-dependent analysis techniques include electrophoresis, chromatography, mass spectroscopy, sedimentation, gradient centrifugation, field-flow fractionation, multi-stage extraction techniques, and the like.
  • mobility probes can be hybridized to amplification products, and the identity of the target nucleic acid sequence determined via a mobility dependent analysis technique of the eluted mobility probes, as described in Published PCT Applications WO04/46344 and WOO 1/92579.
  • detection can be achieved by various microarrays and related software such as the Applied Biosystems Array System with the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer and other commercially available array systems available from Affymetrix, Agilent, Illumina, and Amersham Biosciences, among others (see also Gerry et al., J. Mol. Biol. 292:251-62, 1999; De Bellis et al., Minerva Biotec 14:247-52, 2002; and Stears et al., Nat. Med. 9:14045, including supplements, 2003).
  • Applied Biosystems Array System with the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer and other commercially available array systems available from Affymetrix, Agilent, Illumina, and Amersham Biosciences, among others (see also Gerry et al., J. Mol. Biol. 292:251-62, 1999; De Bellis et al., Minerva Biotec 14:2
  • detection can comprise reporter groups that are incorporated into the reaction products, either as part of labeled primers or due to the incorporation of labeled dNTPs during an amplification, or attached to reaction products, for example but not limited to, via hybridization tag complements comprising reporter groups or via linker arms that are integral or attached to reaction products.
  • unlabeled reaction products may be detected using mass spectrometry.
  • high throughput, massively parallel sequencing employs sequencing-by-synthesis with reversible dye terminators.
  • sequencing is performed via sequencing-by-ligation.
  • sequencing is single molecule sequencing. Examples of Next Generation Sequencing techniques include, but are not limited to pyrosequencing, Reversible dye-terminator sequencing, SOLiD sequencing, Ion semiconductor sequencing, Helioscope single molecule sequencing etc.
  • the Ion TorrentTM (Life Technologies, Carlsbad, CA) amplicon sequencing system employs a flow-based approach that detects pH changes caused by the release of hydrogen ions during incorporation of unmodified nucleotides in DNA replication.
  • a sequencing library is initially produced by generating DNA fragments flanked by sequencing adapters. In some embodiments, these fragments can be clonally amplified on particles by emulsion PCR. The particles with the amplified template are then placed in a silicon semiconductor sequencing chip. During replication, the chip is flooded with one nucleotide after another, and if a nucleotide complements the DNA molecule in a particular microwell of the chip, then it will be incorporated.
  • a proton is naturally released when a nucleotide is incorporated by the polymerase in the DNA molecule, resulting in a detectable local change of pH.
  • the pH of the solution then changes in that well and is detected by the ion sensor. If homopolymer repeats are present in the template sequence, multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.
  • the 454TM GS FLX TM sequencing system employs a light-based detection methodology in a large-scale parallel pyrosequencing system. Pyrosequencing uses DNA polymerization, adding one nucleotide species at a time and detecting and quantifying the number of nucleotides added to a given location through the light emitted by the release of attached pyrophosphates.
  • adapter-ligated DNA fragments are fixed to small DNA-capture beads in a water-in-oil emulsion and amplified by PCR (emulsion PCR).
  • Each DNA-bound bead is placed into a well on a picotiter plate and sequencing reagents are delivered across the wells of the plate.
  • the four DNA nucleotides are added sequentially in a fixed order across the picotiter plate device during a sequencing run. During the nucleotide flow, millions of copies of DNA bound to each of the beads are sequenced in parallel.
  • the nucleotide complementary to the template strand is added to a well, the nucleotide is incorporated onto the existing DNA strand, generating a light signal that is recorded by a CCD camera in the instrument.
  • DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed.
  • Four types of reversible terminator bases (RT -bases) are added, and non-incorporated nucleotides are washed away.
  • RT -bases reversible terminator bases
  • the DNA can only be extended one nucleotide at a time.
  • a camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle.
  • Helicos's single-molecule sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface. At each cycle, DNA polymerase and a single species of fluorescently labeled nucleotide are added, resulting in template-dependent extension of the surface-immobilized primer-template duplexes. The reads are performed by the Helioscope sequencer. After acquisition of images tiling the full array, chemical cleavage and release of the fluorescent label permits the subsequent cycle of extension and imaging.
  • Sequencing by synthesis like the "old style" dye-termination electrophoretic sequencing, relies on incorporation of nucleotides by a DNA polymerase to determine the base sequence.
  • a DNA library with affixed adapters is denatured into single strands and grafted to a flow cell, followed by bridge amplification to form a high-density array of spots onto a glass chip.
  • Reversible terminator methods use reversible versions of dye-terminators, adding one nucleotide at a time, detecting fluorescence at each position by repeated removal of the blocking group to allow polymerization of another nucleotide.
  • the signal of nucleotide incorporation can vary with fluorescently labeled nucleotides, phosphate-driven light reactions and hydrogen ion sensing having all been used.
  • SBS platforms include Illumina GA and HiSeq 2000.
  • the MiSeq® personal sequencing system (Illumina, Inc.) also employs sequencing by synthesis with reversible terminator chemistry.
  • the sequencing by ligation method uses a DNA ligase to determine the target sequence.
  • This sequencing method relies on enzymatic ligation of oligonucleotides that are adjacent through local complementarity on a template DNA strand.
  • This technology employs a partition of all possible oligonucleotides of a fixed length, labeled according to the sequenced position.
  • Oligonucleotides are annealed and ligated and the preferential ligation by DNA ligase for matching sequences results in a dinucleotide encoded color space signal at that position (through the release of a fluorescently labeled probe that corresponds to a known nucleotide at a known position along the oligo).
  • This method is primarily used by Life Technologies’ SOLiDTM sequencers.
  • the DNA is amplified by emulsion PCR.
  • the resulting beads, each containing only copies of the same DNA molecule, are deposited on a solid planar substrate.
  • 10.1301 SMRTTM sequencing is based on the sequencing by synthesis approach.
  • the DNA is synthesized in zero-mode wave-guides (ZMWs)-small well-like containers with the capturing tools located at the bottom of the well.
  • ZMWs zero-mode wave-guides
  • the sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labeled nucleotides flowing freely in the solution.
  • the wells are constructed in a way that only the fluorescence occurring at the bottom of the well is detected.
  • the fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.
  • the present disclosure provides a method for selecting a cancer patient undergoing tumor resection surgery for renal cancer for treatment with an intraoperative opioid analgesic comprising (a) detecting expression levels of at least one survival-associated gene expression network in a biological sample obtained from the cancer patient that are either comparable or decreased relative to a control sample obtained from a healthy subject or a predetermined threshold, wherein the at least one survival-associated gene expression network is a NRF2-dependent macrophage network and/or a Th2 immune network; and (b) administering to the cancer patient an effective amount of an intraoperative opioid analgesic during the tumor resection surgery.
  • the expression levels of the at least one survival-associated gene expression network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • the biological sample comprises genomic DNA, cDNA, RNA, and/or mRNA.
  • the present disclosure provides a method for prolonging survival of a cancer patient undergoing tumor resection surgery for renal cancer comprising administering to the cancer patient an effective amount of an intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a NRF2-dependent macrophage network and/or a Th2 immune network in a biological sample obtained from the cancer patient are either comparable or decreased relative to a control sample obtained from a healthy subject or a predetermined threshold.
  • expression levels of the NRF2-dependent macrophage network and/or the Th2 immune network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • next-generation sequencing PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • the intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
  • the effective amount of the intraoperative opioid analgesic may range from about 1 MME to about 200 MMEs. In certain embodiments, the effective amount of the intraoperative opioid analgesic is about 1 MME to about 20 MMEs, about 20 MMEs to about 45 MMEs, or about 45 MMEs to about 200 MMEs.
  • the effective amount of the intraoperative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35 MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45
  • the effective amount of the intraoperative opioid analgesic is administered as a series of bolus doses or as a continuous infusion during the tumor resection surgery. In certain embodiments, the effective amount of the intraoperative opioid analgesic is administered to the cancer patient prior to incision. Additionally or alternatively, in some embodiments, the effective amount of the intraoperative opioid analgesic is administered intravenously.
  • the methods of the present technology further comprise administering to the cancer patient an effective amount of a local anesthetic solution via an epidural catheter before, during and/or after the tumor resection surgery.
  • the effective amount of the local anesthetic solution may range from about 0.05%- 4% local anesthetic solution in a volume of 1-10 ml per hour when administered via an epidural catheter.
  • the effective amount of the local anesthetic solution is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about 1.2 %, about
  • the effective amount of the local anesthetic solution is administered as a series of bolus doses or as a continuous infusion during the tumor resection surgery.
  • Suitable local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine.
  • the effective amount of the local anesthetic solution may be administered before, during and/or after the tumor resection surgery using any regional anesthesia technique directed at nerves innervating the thorax and chest wall (e.g., via serratus plane nerve block, intercostal nerve block, or paravertebral block).
  • the effective amount of the local anesthetic solution may range from about 0.05%-4% local anesthetic solution in a volume of 10-40 ml when administered using any regional anesthesia technique directed at nerves innervating the thorax and chest wall (e.g., via serratus plane nerve block, intercostal nerve block, or paravertebral block).
  • the effective amount of the local anesthetic solution is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about 1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6 %, about 1.7 %, about 1.8 %, about 1.9 %, about 2.0 %, about 2.1 %, about 2.2 %, about 2.3 %, about 2.4 %, about 2.5 %, about 2.6 %, about
  • Suitable local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine.
  • the local anesthetic solution may further comprise an opioid (e.g., fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil).
  • an opioid e.g., fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
  • the local anesthetic solution may comprise 0.5 mcg/ml-50 mcg/ml opioid.
  • the local anesthetic solution may comprise about 0.5 mcg/ml, about 0.6 mcg/ml, about 0.7 mcg/ml, about 0.8 mcg/ml, about 0.9 mcg/ml, about 1.0 mcg/ml, about 1.5 mcg/ml, about 2.0 mcg/ml, about 2.5 mcg/ml, about 3.0 mcg/ml, about 3.5 mcg/ml, about 4.0 mcg/ml, about 4.5 mcg/ml, about 5.0 mcg/ml, about 5.5 mcg/ml, about 6.0 mcg/ml, about 6.5 mcg/ml, about 7.0 mcg/ml, about 7.5 mcg/ml, about 8.0 mcg/ml, about 8.5 mcg/ml, about 9.0 mcg/ml, about 10
  • the methods of the present technology further comprise administering to the cancer patient an effective amount of a post-operative opioid analgesic after the tumor resection surgery.
  • postoperative opioid analgesics include, but are not limited to, fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
  • the post-operative opioid analgesic and the intraoperative opioid analgesic are the same opioid analgesic or different opioid analgesics.
  • the effective amount of the post-operative opioid analgesic and the effective amount of the intraoperative opioid analgesic are the same or different.
  • the effective amount of the post-operative opioid analgesic is administered to the cancer patient as a bolus of about 0.005 mg to about 100 mg. In some embodiments, the effective amount of the post-operative opioid analgesic is administered to the cancer patient as a bolus of about 0.005 mg, about 0.006 mg, about 0.007 mg, about 0.008 mg, about 0.009 mg, about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1-5 mg, about 5-10 mg, about 1-5 mg, about 5-10 mg, about 1-5 mg, about 5-10 mg, about 1-5 mg, about 5-10 mg, about 10-15 mg, about 15-20 mg, about 20-25 mg, about 25-30 mg,
  • the effective amount of the post-operative opioid analgesic may be continuously delivered to the cancer patient at a per hour rate of about 0.01 mg/hr to about 10 mg/hr. In certain embodiments, the effective amount of the post-operative opioid analgesic is continuously delivered to the cancer patient at a per hour rate of about 0.01 mg/hr, about 0.02 mg/hr, about 0.03 mg/hr, about 0.04 mg/hr, about 0.05 mg/hr, about 0.06 mg/hr, about 0.07 mg/hr, about 0.08 mg/hr, about 0.09 mg/hr, about 0.1 mg/hr, about 0.2 mg/hr, about 0.3 mg/hr, about 0.4 mg/hr, about 0.5 mg/hr, about 0.6 mg/hr, about 0.7 mg/hr, about 0.8 mg/hr, about 0.9 mg/hr, about 1 mg/hr, about 1.5 mg/hr, about 2 mg/hr, about 2.5 mg/hr, about 3 mg
  • the present disclosure provides a method for selecting a cancer patient undergoing tumor resection surgery for renal cancer for treatment with an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic comprising (a) detecting expression levels of at least one survival-associated gene expression network in a biological sample obtained from the cancer patient that are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold, wherein the at least one survival-associated gene expression network is a NRF2-dependent macrophage network and/or a Th2 immune network; and (b) administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery.
  • the expression levels of the at least one survival-associated gene expression network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • the biological sample comprises genomic DNA, cDNA, RNA, and/or mRNA.
  • the present disclosure provides a method for prolonging survival of a cancer patient undergoing tumor resection surgery for renal cancer comprising administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a NRF2-dependent macrophage network and/or a Th2 immune network in a biological sample obtained from the cancer patient are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold.
  • expression levels of the NRF2-dependent macrophage network and/or the Th2 immune network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • next-generation sequencing PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • the low- dose intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. Additionally or alternatively, in some embodiments, the effective amount of the low-dose intraoperative opioid analgesic is about 1 MME to about 50 MMEs.
  • the effective amount of the low- dose intraoperative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25
  • MMEs about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30
  • MMEs about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35
  • MMEs about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45
  • MMEs or about 45-50 MMEs.
  • the opioid-free intraoperative analgesic is an amide-type local anesthetic or an ester- type local anesthetic.
  • amide-type local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, or levobupivacaine.
  • ester-type local anesthetics include, but are not limited to, cocaine, procaine, tetracaine, chloroprocaine, or benzocaine.
  • the opioid-free intraoperative analgesic may be administered via an epidural catheter.
  • the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 1-10 ml per hour when administered via an epidural catheter.
  • the effective amount of the opioid-free intraoperative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about 1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6 %, about 1.7 %, about 1.8 %, about 1.9 %, about 2.0 %, about 2.1 %, about 2.2 %, about 2.3 %, about 2.4 %, about 2.5 %, about 2.6 %, about
  • the opioid-free intraoperative analgesic may be administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
  • the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 10-40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
  • the effective amount of the opioid-free intraoperative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about 1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6
  • amide-type or ester-type local anesthetic solution in a volume of about 10 ml, about 12.5 ml, about 15 ml, about 17.5 ml, about 20 ml, about 22.5 ml, about 25 ml, about 27.5 ml, about 30 ml, about 32.5 ml, about 35 ml, about 37.5 ml, or about 40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
  • the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic may be administered as a series of bolus doses, or as a continuous infusion during the tumor resection surgery.
  • the methods of the present technology further comprise administering to the cancer patient an effective amount of an opioid-free post-operative analgesic or a low-dose post-operative opioid analgesic after the tumor resection surgery.
  • opioid-free post-operative analgesics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine.
  • suitable low-dose post-operative opioid analgesics include, but are not limited to, fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
  • the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same analgesic or different analgesics.
  • the effective amount of the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same or different.
  • the effective amount of the low-dose post-operative opioid analgesic is about 1 MME to about 50 MMEs.
  • the effective amount of the low-dose post-operative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35 MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs,
  • the effective amount of the opioid-free post-operative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about
  • amide-type or ester-type local anesthetic solution in a volume of about 10 ml, about 12.5 ml, about 15 ml, about 17.5 ml, about 20 ml, about 22.5 ml, about 25 ml, about 27.5 ml, about 30 ml, about 32.5 ml, about 35 ml, about 37.5 ml, or about 40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
  • the NRF2-dependent macrophage network comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255,
  • the Th2 immune network comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 115, at least 120, at least 125, or at least 130 or more genes selected from among AJAP1, ANLN, ARHGAP11 A, ASF1B, ASPM, ATAD2, AURKA, BRCA1, BUB1, C10orf2, C13orf34, C15orf23, C16orf75, CACNA2D4, CCDC99, CCNA2, CCNB1, CCNF, CDC6, CDCA7, CDK1, CDT1, CENPE, CENPF, CENPH, CENPL
  • the tumor resection surgery comprises nephrectomy.
  • the renal cancer may have a histologic subtype selected from among clear cell renal cell carcinomas (ccRCC), papillary renal cell carcinomas (pRCC), chromophobe renal cell carcinomas (crRCC), multilocular cystic RCC, collecting duct carcinoma, medullary carcinoma, mucinous tubular and spindle cell carcinoma, neuroblastoma-associated RCC, Xpl l.2 translocation-TFE3 carcinoma, and unclassified lesions.
  • the cancer patient exhibits stage I, stage II, stage III, or stage IV renal cancer.
  • the present disclosure provides a method for selecting a cancer patient undergoing tumor resection surgery for bladder cancer for treatment with an opioid- free intraoperative analgesic or a low-dose intraoperative opioid analgesic comprising (a) detecting expression levels of a survival-associated gene expression network in a biological sample obtained from the cancer patient that are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold; and (b) administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery, wherein the survival- associated gene expression network comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100,
  • the expression levels of the survival-associated gene expression network are detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligationdependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • next-generation sequencing PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligationdependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • qPCR real-time quantitative PCR
  • the present disclosure provides a method for prolonging survival of a cancer patient undergoing tumor resection surgery for bladder cancer comprising administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a survival-associated gene expression network in a biological sample obtained from the cancer patient are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold, wherein the survival-associated gene expression network comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least
  • the expression levels of the survival-associated gene expression network are detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligationdependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • next-generation sequencing PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligationdependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
  • qPCR real-time quantitative PCR
  • the low- dose intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. Additionally or alternatively, in some embodiments, the effective amount of the low-dose intraoperative opioid analgesic is about 1 MME to about 50 MMEs.
  • the effective amount of the low- dose intraoperative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35 MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about
  • the opioid-free intraoperative analgesic is an amide-type local anesthetic or an ester- type local anesthetic.
  • amide-type local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, or levobupivacaine.
  • ester-type local anesthetics include, but are not limited to, cocaine, procaine, tetracaine, chloroprocaine, or benzocaine.
  • the opioid-free intraoperative analgesic may be administered via an epidural catheter.
  • the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 1-10 ml per hour when administered via an epidural catheter.
  • the effective amount of the opioid-free intraoperative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about 1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6 %, about 1.7 %, about 1.8 %, about 1.9 %, about 2.0 %, about 2.1 %, about 2.2 %, about 2.3 %, about 2.4 %, about 2.5 %, about 2.6 %, about
  • the opioid-free intraoperative analgesic may be administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
  • the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 10-40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
  • the effective amount of the opioid-free intraoperative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about 1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6
  • amide-type or ester-type local anesthetic solution in a volume of about 10 ml, about 12.5 ml, about 15 ml, about 17.5 ml, about 20 ml, about 22.5 ml, about 25 ml, about 27.5 ml, about 30 ml, about 32.5 ml, about 35 ml, about 37.5 ml, or about 40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
  • the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic may be administered as a series of bolus doses, or as a continuous infusion during the tumor resection surgery.
  • the methods of the present technology further comprise administering to the cancer patient an effective amount of an opioid-free post-operative analgesic or a low-dose post-operative opioid analgesic after the tumor resection surgery.
  • opioid-free post-operative analgesics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine.
  • suitable low-dose post-operative opioid analgesics include, but are not limited to, fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
  • the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same analgesic or different analgesics.
  • the effective amount of the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same or different.
  • the effective amount of the low-dose post-operative opioid analgesic is about 1 MME to about 50 MMEs. In some embodiments, the effective amount of the low-dose post-operative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs,
  • the effective amount of the opioid-free post-operative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about
  • amide-type or ester-type local anesthetic solution in a volume of about 10 ml, about 12.5 ml, about 15 ml, about 17.5 ml, about 20 ml, about 22.5 ml, about 25 ml, about 27.5 ml, about 30 ml, about 32.5 ml, about 35 ml, about 37.5 ml, or about 40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
  • the tumor resection surgery comprises cystectomy. Additionally or alternatively, in certain embodiments, the cancer patient exhibits stage I, stage II, stage III, or stage IV bladder cancer.
  • the patient is human.
  • the biological sample obtained from the cancer patient comprises biopsied tumor tissue, whole blood, plasma, or serum.
  • Administration of any of the intraoperative or postoperative analgesics disclosed herein can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically.
  • the present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.
  • the examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the methods of the present technology.
  • the examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims.
  • the examples can include or incorporate any of the variations, aspects, or embodiments of the present technology described above.
  • the variations, aspects, or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present technology.
  • RNAseq raw read sequences were aligned against human genome assembly hgl9 by STAR 2-pass alignment (Dobin A, et al., Bioinform OxfEngl. 29: 15-21 (2012)).
  • RNAseq gene level count values were computed by using the R package GenomicAlignments (Lawrence M, et al., Pios Comput Biol. 9: el 003118 (2013)) over aligned reads with UCSC KnownGene (Rosenbloom KR, et al., Nucleic Acids Res. 43: 670-81 (2014)) in hgl9 as the base gene model.
  • gene expression was measured in an independent cohort and corrected for batch, sex, and age using linear regression. Gene coexpression was calculated independently in this cohort as previously described, without explicit reference or parameterization from the TCGA population. Module membership was directly compared between TCGA-KIRC and MSKCC-KIRC networks, and Fisher’s exact test was used to calculate enrichment. Modules were considered preserved if enrichment odds ratio > 1 and Bonferroni p-value ⁇ 0.05.
  • Differential network connectivity was calculated by comparing the mean intramodular connectivity for each disease network with those same network genes in the control cohort (Zhang B, et al., Cell 153: 707-720 (2013)). The ratio of average network connectivity between cases and controls was used as an estimate of differential connectivity. For example, a measure of 2 signifies that the average correlation strength for a group of genes in a network is two times greater in disease than in controls.
  • Two separate false discovery rates (FDR) were estimated by randomly shuffling samples and genes of disease and control networks. Shuffling samples creates networks with random edges and shuffling genes creates networks with random nodes. The final FDR was quantified by selecting the larger estimate and a conservative FDR threshold was used to assess significance (FDR ⁇ 0.001).
  • Connectivity scores were calculated between network hubs and drug-induced transcriptional profiles for leu-enkephalin, naloxone, and the VEGF-inhibitor class.
  • the drug profiles were catalogued by Connectivity Map and the CLUE Research platform was used to calculate connectivity scores (Lamb J, et al., Science 313: 1929-35 (2006)).
  • Connectivity Map has catalogued gene expression profiles for thousands of chemical and genetic perturbations across nine cell lines, and connectivity scores between all reference perturbations were calculated based on a weighted Kolmogorov Smirnov statistic, normalized for cell line and perturbation type (Subramanian A, et al., Proc National Acad Sci. 102: 15545-50 (2005)).
  • T A non-parametric weighted connectivity score and an enrichment score, T, was then calculated for each module-drug pair of interest, ranging from -100 to +100 (Subramanian A, et al., Cell 171 : 1437-1452 (2017)). T measures the fraction of reference connectivity scores greater than the tested module-drug pair. A positive score shows that hub expression and drug-induced expression are in the same direction, while negative scores reflect expression in the opposite direction. A score of 90 indicates that only 10% of the reference set had a stronger score. Unlike a null distribution generated by random permutation, this empirical test avoids strong assumptions about the distribution of gene expression data under perturbed conditions.
  • master regulators were inferred using MARINa, leveraging a phenotype transition signature derived from t-test analysis comparing gene expression between cases and controls (Lefebvre C, et al., Mol Syst Biol. 6: 377(2010)).
  • Opioid signaling is not nearly as overrepresented as large pathways more proximal to pathogenesis, like immune regulation and cell migration, but these analyses provide evidence for its association with ccRCC.
  • Canonical opioid receptor genes 0PRM1, OPRD1, OPRK1 are all poorly expressed in renal tissue and there is no evidence in this analysis that they are differentially expressed in ccRCC, though the expression of each shows greater variability in the disease state (FIG. 6).
  • OPRL1 nociception receptor
  • OGFR OGFR
  • TLR4 receptor genes are upregulated in ccRCC (FIGs. 1C-1F).
  • Example 4 Tumor microenvironment and onco enesis pathways associated with survival- associated sene networks
  • Survival-related networks also include approximately 40% (9 out of 22) of the intOGen RCC mutational driver genes (Gonzalez-Perez A, et al., Nat Methods 10: 1081-2 (2013)).
  • leu-enkephalin has significant anti-survival effects on seven survival -related networks, upregulating modules negatively correlated with survival and downregulating modules positively correlated with survival (FIG. 3B).
  • bladder cancer is a relevant comparison to ccRCC given that it is also a urological cancer where opioid excretion in urine could enable direct effects on tumorigenesis.
  • ccRCC a relevant comparison to ccRCC given that it is also a urological cancer where opioid excretion in urine could enable direct effects on tumorigenesis.
  • pink one network
  • Several of their regulators have been experimentally validated drivers of oncogenesis, tumor progression, and metastasis in ccRCC, suggesting possible transcriptional mechanisms through which opioids may affect ccRCC development and metastasis.
  • CDH2 the MR with the strongest enrichment score, is known to be involved in the opioid pathway, and CDH2 variants influence methadone response.
  • MRs associated with other survival -related networks also are tightly linked with opioid pathway (FIG.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

Abstract

The present disclosure provides methods for determining whether a patient diagnosed with urological cancer and undergoing tumor resection surgery will benefit from treatment with intraoperative opioid analgesics or opioid-free intraoperative analgesics (e.g., intraoperative ester-type or amide-type local anesthetics) based on the expression levels of specific survival-associated gene expression networks in the cancer patient.

Description

METHODS FOR TAILORING ANALGESIC REGIMEN IN CANCER PATIENT'S BASED ON TUMOR TRANSCRIPTOMICS
CROSS-REFERENCE TO RELATED APPLICATIONS
[(HMD ] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/124,239, filed December 11, 2020, the entire contents of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present disclosure provides methods for determining whether a patient diagnosed with urological cancer and undergoing tumor resection surgery will benefit from treatment with intraoperative opioid analgesics or opioid-free intraoperative analgesics (e.g., intraoperative ester-type or amide-type local anesthetics) based on the expression levels of specific survival-associated gene expression networks in the cancer patient.
STATEMENT OF GOVERNMENT SUPPORT
[0003] This invention was made with government support under CA008748 awarded by the National Cancer Institute. The government has certain rights in the invention.
BACKGROUND
[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.
[0005] Retrospective clinical evidence suggests that perioperative opioid exposure may be associated with cancer recurrence and survival (Wigmore T, Farquhar- Smith P. Curr Opin Support Pa. 2016; 10: 109-18; Kim R. J Transl Med. 2018; 16:8; Wall T et al. Brit J Anae sth. 2019;123: 135-50; Sekandarzad MW et al., Anesthesia Analgesia. 2017;124: 1697-708), and its specific effects are likely cancer-specific. For instance, while opioids are thought to have a negative effect on immune-mediated cancers (Topalian SL, et al. Jama Oncol.
2019;5: 1411-20) like lung adenocarcinoma (Cata JP, et al. Cancer Med-us. 2014;3:900-8) and renal cell carcinoma (Silagy AW, et al. Brit J Anaesth. 2020; 125 :e402-4), they may play a protective role in esophageal cancer (Du KN, et al. Anesthesia Analgesia. 2018;127:210-6). Despite numerous studies indicating an association, causal evidence and biological rationale are both lacking in human populations.
[0006] Accordingly, there is an urgent need for developing reliable and accurate methods for predicting whether a cancer patient undergoing tumor resection surgery will benefit from treatment with intraoperative opioid analgesics.
SUMMARY OF THE PRESENT TECHNOLOGY
[0007] In one aspect, the present disclosure provides a method for selecting a cancer patient undergoing tumor resection surgery for renal cancer for treatment with an intraoperative opioid analgesic comprising (a) detecting expression levels of at least one survival-associated gene expression network in a biological sample obtained from the cancer patient that are either comparable or decreased relative to a control sample obtained from a healthy subject or a predetermined threshold, wherein the at least one survival-associated gene expression network is a NRF2-dependent macrophage network and/or a Th2 immune network; and (b) administering to the cancer patient an effective amount of an intraoperative opioid analgesic during the tumor resection surgery. The expression levels of the at least one survival-associated gene expression network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH). In some embodiments, the biological sample comprises genomic DNA, cDNA, RNA, and/or mRNA.
[0008] In one aspect, the present disclosure provides a method for prolonging survival of a cancer patient undergoing tumor resection surgery for renal cancer comprising administering to the cancer patient an effective amount of an intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a NRF2-dependent macrophage network and/or a Th2 immune network in a biological sample obtained from the cancer patient are either comparable or decreased relative to a control sample obtained from a healthy subject or a predetermined threshold. Additionally or alternatively, in some embodiments, expression levels of the NRF2-dependent macrophage network and/or the Th2 immune network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
[0009] In any of the preceding embodiments of methods disclosed herein, the intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. The effective amount of the intraoperative opioid analgesic may range from about 1 MME to about 200 MMEs. In certain embodiments, the effective amount of the intraoperative opioid analgesic is about 1 MME to about 20 MMEs, about 20 MMEs to about 45 MMEs, or about 45 MMEs to about 200 MMEs. In some embodiments, the effective amount of the intraoperative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35 MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45 MMEs, about 45-50 MMEs, about 50-55 MMEs, about 55-60 MMEs, about 60-65 MMEs, about 65-70 MMEs, about 70-75 MMEs, about 75-80 MMEs, about 80-85 MMEs, about 85-90 MMEs, about 90-95 MMEs, about 95-100 MMEs, about 100-110 MMEs, about 110-120 MMEs, about 120-130 MMEs, about 130-140 MMEs, about 140-150 MMEs, about 150-160 MMEs, about 160-170 MMEs, about 170-180 MMEs, about 180-190 MMEs, or about 190-200 MMEs. Additionally or alternatively, in some embodiments, the effective amount of the intraoperative opioid analgesic is administered as a series of bolus doses or as a continuous infusion during the tumor resection surgery. In certain embodiments, the effective amount of the intraoperative opioid analgesic is administered to the cancer patient prior to incision. Additionally or alternatively, in some embodiments, the effective amount of the intraoperative opioid analgesic is administered intravenously.
[0010] Additionally or alternatively, in some embodiments, the methods of the present technology further comprise administering to the cancer patient an effective amount of a local anesthetic solution via an epidural catheter before, during and/or after the tumor resection surgery. The effective amount of the local anesthetic solution may range from about 0.05%- 4% local anesthetic solution in a volume of 1-10 ml per hour when administered via an epidural catheter. Additionally or alternatively, in some embodiments, the effective amount of the local anesthetic solution is administered as a series of bolus doses or as a continuous infusion during the tumor resection surgery. In other embodiments, the effective amount of the local anesthetic solution may be administered before, during and/or after the tumor resection surgery using any regional anesthesia technique directed at nerves innervating the thorax and chest wall (e.g., via serratus plane nerve block, intercostal nerve block, or paravertebral block). The effective amount of the local anesthetic solution may range from about 0.05%-4% local anesthetic solution in a volume of 10-40 ml when administered using any regional anesthesia technique directed at nerves innervating the thorax and chest wall (e.g., via serratus plane nerve block, intercostal nerve block, or paravertebral block). Examples of suitable local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine. Additionally or alternatively, in certain embodiments, the local anesthetic solution may further comprise an opioid (e.g., fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil). In some embodiments, the local anesthetic solution may comprise 0.5 mcg/ml-50 mcg/ml opioid.
[00111 Additionally or alternatively, in certain embodiments, the methods of the present technology further comprise administering to the cancer patient an effective amount of a post-operative opioid analgesic after the tumor resection surgery. Examples of post-operative opioid analgesics include, but are not limited to, fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. In some embodiments, the post-operative opioid analgesic and the intraoperative opioid analgesic are the same opioid analgesic or different opioid analgesics. In other embodiments, the effective amount of the post-operative opioid analgesic and the effective amount of the intraoperative opioid analgesic are the same or different. Additionally or alternatively, in some embodiments, the effective amount of the post-operative opioid analgesic is administered to the cancer patient as a bolus of about 0.005 mg to about 100 mg. In some embodiments, the effective amount of the post-operative opioid analgesic is administered to the cancer patient as a bolus of about 0.005 mg, about 0.006 mg, about 0.007 mg, about 0.008 mg, about 0.009 mg, about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1-5 mg, about 5-10 mg, about 1-5 mg, about 5-10 mg, about 1-5 mg, about 5-10 mg, about 1- 5 mg, about 5-10 mg, about 10-15 mg, about 15-20 mg, about 20-25 mg, about 25-30 mg, about 30-35 mg, about 35-40 mg, about 40-45 mg, about 45-50 mg, about 50-55 mg, about 55-60 mg, about 60-65 mg, about 65-70 mg, about 70-75 mg, about 75-80 mg, about 80-85 mg, about 85-90 mg, about 90-95 mg, or about 95-100 mg. In other embodiments, the effective amount of the post-operative opioid analgesic may be continuously delivered to the cancer patient at a per hour rate of about 0.01 mg/hr to about 10 mg/hr. In certain embodiments, the effective amount of the post-operative opioid analgesic is continuously delivered to the cancer patient at a per hour rate of about 0.01 mg/hr, about 0.02 mg/hr, about 0.03 mg/hr, about 0.04 mg/hr, about 0.05 mg/hr, about 0.06 mg/hr, about 0.07 mg/hr, about 0.08 mg/hr, about 0.09 mg/hr, about 0.1 mg/hr, about 0.2 mg/hr, about 0.3 mg/hr, about 0.4 mg/hr, about 0.5 mg/hr, about 0.6 mg/hr, about 0.7 mg/hr, about 0.8 mg/hr, about 0.9 mg/hr, about 1 mg/hr, about 1.5 mg/hr, about 2 mg/hr, about 2.5 mg/hr, about 3 mg/hr, about 3.5 mg/hr, about 4 mg/hr, about 4.5 mg/hr, about 5 mg/hr, about 5.5 mg/hr, about 6 mg/hr, about 6.5 mg/hr, about 7 mg/hr, about 7.5 mg/hr, about 8 mg/hr, about 8.5 mg/hr, about 9 mg/hr, about 9.5 mg/hr, or about 10 mg/hr. Additionally or alternatively, in some embodiments, the effective amount of the post-operative opioid analgesic is administered intravenously, orally, or transdermally.
[0012] In one aspect, the present disclosure provides a method for selecting a cancer patient undergoing tumor resection surgery for renal cancer for treatment with an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic comprising (a) detecting expression levels of at least one survival-associated gene expression network in a biological sample obtained from the cancer patient that are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold, wherein the at least one survival-associated gene expression network is a NRF2-dependent macrophage network and/or a Th2 immune network; and (b) administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery. The expression levels of the at least one survival-associated gene expression network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH). In some embodiments, the biological sample comprises genomic DNA, cDNA, RNA, and/or mRNA.
[0013] In one aspect, the present disclosure provides a method for prolonging survival of a cancer patient undergoing tumor resection surgery for renal cancer comprising administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low- dose intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a NRF2-dependent macrophage network and/or a Th2 immune network in a biological sample obtained from the cancer patient are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold. Additionally or alternatively, in some embodiments, expression levels of the NRF2-dependent macrophage network and/or the Th2 immune network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
100141 In any of the preceding embodiments of methods disclosed herein, the low- dose intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. Additionally or alternatively, in some embodiments, the effective amount of the low-dose intraoperative opioid analgesic is about 1 MME to about 50 MMEs. In some embodiments, the effective amount of the low- dose intraoperative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25
MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30
MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35
MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45
MMEs, or about 45-50 MMEs.
[0015] Additionally or alternatively, in some embodiments of the methods disclosed herein, the opioid-free intraoperative analgesic is an amide-type local anesthetic or an ester- type local anesthetic. Examples of amide-type local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, or levobupivacaine. Examples of ester-type local anesthetics include, but are not limited to, cocaine, procaine, tetracaine, chloroprocaine, or benzocaine. The opioid-free intraoperative analgesic may be administered via an epidural catheter. Additionally or alternatively, in some embodiments of the methods disclosed herein, the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 1-10 ml per hour when administered via an epidural catheter.
[0016] In certain embodiments, the opioid-free intraoperative analgesic may be administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block. In some embodiments, the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 10-40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block. [0017] Additionally or alternatively, in certain embodiments of the methods disclosed herein, the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic may be administered as a series of bolus doses, or as a continuous infusion during the tumor resection surgery.
10018 [ Additionally or alternatively, in some embodiments, the methods of the present technology further comprise administering to the cancer patient an effective amount of an opioid-free post-operative analgesic or a low-dose post-operative opioid analgesic after the tumor resection surgery. Examples of suitable opioid-free post-operative analgesics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine. Examples of suitable low-dose post-operative opioid analgesics include, but are not limited to, fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. In some embodiments, the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same analgesic or different analgesics. In other embodiments, the effective amount of the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same or different.
100191 In some embodiments, the effective amount of the low-dose post-operative opioid analgesic is about 1 MME to about 50 MMEs. In some embodiments, the effective amount of the low-dose post-operative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35 MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45 MMEs, or about 45-50 MMEs. [0020] In some embodiments, the effective amount of the opioid-free post-operative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about
1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6 %, about 1.7 %, about 1.8 %, about
1.9 %, about 2.0 %, about 2.1 %, about 2.2 %, about 2.3 %, about 2.4 %, about 2.5 %, about
2.6 %, about 2.7 %, about 2.8 %, about 2.9 %, about 3.0 %, about 3.1 %, about 3.2 %, about
3.3 %, about 3.4 %, about 3.5 %, about 3.6 %, about 3.7 %, about 3.8 %, about 3.9 %, or about 4.0 % amide-type or ester-type local anesthetic solution in a volume of about 10 ml, about 12.5 ml, about 15 ml, about 17.5 ml, about 20 ml, about 22.5 ml, about 25 ml, about 27.5 ml, about 30 ml, about 32.5 ml, about 35 ml, about 37.5 ml, or about 40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
[00211 In any and all of the preceding embodiments of the methods disclosed herein, the NRF2-dependent macrophage network comprises 5 or more genes selected from among A1CF, ABAT, ABCB1, ABCB9, ABCC2, ABCC6P1, ABCG1, ABHD6, ABLIM3, ABP1, ACAD11, ACADL, ACAT1, ACBD4, ACO2, ACOT4, ACOX2, ACSL1, ACSM5, ACY1, ADAMTS3, ADH6, AGPAT3, AGT, AIFM1, AKR1C1, AKR1C2, AKR7A2, AKR7A3, ALAD, ALDH1A1, ALDH1A2, ALDH1L1, ALDH2, ALDH3A2, ALDH4A1, ALDH7A1, ALDH8A1, ALDOB, ALPK2, ALPL, AMDHD1, ANK3, ANKRD56, ANPEP, ANXA13, ANXA2P2, ANXA2, AOX1, APITD1, AQP3, AQP9, ARHGAP1, ARHGAP24, ARL4C, ASB13, ASRGL1, ASTN2, ATP6V0A1, AUH, AXL, AZGP1, B3GNT7, B4GALNT1, B4GALT1, BACE2, BAIAP2L1, BAIAP2L2, BAMBI, BARX2, BCAT1, BCMO1, BDH2, BDKRB2, BEND3, BHMT2, BHMT, BNIP3, BPHL, BTG3, C10orfl08, Cl lorf45, Cl lorf52, C14orf64, C14orf73, C17orf51, C18orfl8, C19orf77, C1RL, C1R, CIS, Clorfl l5, Clorf201, Clorf203, Clorf210, Clorf21, Clorf89, Clorf96, C21orf7, C22orf45, C2orf24, C2orf67, C3, C4orfl9, C5orf23, C6, C7orfl0, C8orf47, C9orfl25, CABCI, CABLES1, CADM3, CALML4, CBR4, CBWD1, CBX7, CCDC146, CCDC64, CCDC68, CD276, CD44, CD47, CD55, CD82, CDADC1, CDC42SE2, CDCA2, CDCP1, CDH16, CDH2, CDHR2, CDHR5, CDK18, CDK20, CDON, CEACAM1, CES2, CFB, CGREF1, CHDH, CHI3L1, CHPF2, CHST13, CIDEB, CISH, CIT, CKAP4, CLDN10, CLDN2, CLEC18A, CLEC18B, CLEC18C, CLIC6, CLPTM1L, CMBL, CNDP2, CNNM1, COBL, COL22A1, COL23A1, COL4A5, COL8A2, COLEC12, COPG2, CPT2, CRAT, CRB3, CREB3L3, CRY2, CRYM, CRYZ, CTHRC1, CYB5A, CYB5D1, CYP1B1, CYP27A1, CYP2J2, CYP4V2, CYS1, DAB2, DAPK2, DCBLD2, DDAH1, DEPDC7, DGKG, DHDH, DHTKD1, DIRAS2, DLG5, DMGDH, DMRTA1, DPF3, DPYS, DSEL, ECHDC3, EFNA5, EGOT, ELFN2, EMX1, EMX2OS, EMX2, ENAM, ENPEP, ENPP3, EPB49, EPHA7, EPHX2, ERBB3, ETFA, ETFDH, ETNK2, ETV1, ETV6, EZR, FAAH, FABP3, FAHD1, FAM149A, FAM164C, FAM60A, FAM69A, FANCC, FBP1, FBXL16, FBXO32, FCAMR, FGFR1OP, FGFR3, FGGY, FHL2, FLJ23867, FLJ36031, FLNC, FMO4, FNDC4, FREM2, FTCD, GAL3ST1, GALM, GALNT2, GALNT7, GATM, GATS, GBAS, GDA, GFPT2, GGT3P, GJB1, GK, GLB1L, GLIS1, GLRB, GLT25D1, GOT1, GPD1, GPER, GPT, GPX3, GPX8, GRAMD1C, GRTP1, GSTA1, GSTA2, GXYLT2, GYLTL1B, HAAO, HABP2, HABP4, HGD, HHLA2, HIBCH, HIGD1A, HLF, HMGCL, HMOX1, HNF4A, HOXCIO, HSDL2, HSP90B1, HSPA2, HSPB8, HYAL1, HYOU1, IDH1, IGDCC4, IGF2BP2, IL17RB, IL1R2, IL22RA1, IMPA2, IMPDH1, IRS2, ITGB6, JPH2, KCNIP3, KCNJ15, KCNJ16, KCNS3, KCTD17, KCTD1, KIAA1543, KLC4, KLF15, KLHDC7A, KL, KRT19, KRT80, KSR1, LAD1, LAMA3, LAMB1, LAMB3, LAMC2, LDHD, LEF1, LIMK2, LNP1, LOC100126784, LOC100131551, LOC151534, LOC388387, LOC389332, LOC723809, LRG1, LRRC19, LRRC8E, LYG1, MAF, MAGED1, MAN1C1, MAOB, MAP7, MAPK8IP1, MAPT, MARVELD3, METTL7A, METTL7B, MFI2, MINA, MLYCD, MMD, MME, MMP14, MMP7, MMP9, MOBKL2B, MOSC2, MPI, MPV17L, MPZL1, MSRA, MTHFD1L, MTHFD2, MUC1, MXRA8, MYO1E, MYO3A, MYO7B, MYOM3, NAMPT, NAP1L1, NAPSA, NEFL, NGEF, NHEJ1, NIPSNAP1, NOMO1, NOMO3, NPR3, NRXN2, NTN4, NUDT6, OPN3, 0SCP1, OSTalpha, PANK1, PAPP A, PAQR7, PARD6B, PBLD, PBX3, PCBD2, PCCA, PCK1, PCOLCE2, PC, PDE10A, PDIA3P, PDIA4, PDIA5, PDK2, PDXP, PDZD3, PDZK1P1, PECI, PECR, PEPD, PER3, PGPEP1, PHYH, PIGT, PIPOX, PKHD1, PLA2G4C, PL AU, PLIN2, PLIN3, PLOD2, PLTP, PMAIP1, PNMA6A, PON2, PPFIBP2, PPL, PPP1R14C, PRODH2, PRODH, PSD3, PTGER2, PTGFRN, PTGR2, PTH1R, PTH2R, PVR, PXMP2, QDPR, QRFPR, QSOX1, RAB17, RAB3IP, RAB7L1, RAI2, RARRES1, RCN1, RGN, RGS14, RHOBTB1, RIT1, RND3, RNF5P1, RORC, RPN2, RUNDC3B, RUNX1, RUNX2, SAMD5, SATB2, SCARB1, SCD, SCGN, SCLY, SCNN1A, SEMA3C, SEMA3D, SEMA4B, SEMA4F, SEMA6A, SEPHS2, SEPSECS, SERPINA3, SERPINA6, SERPINF1, SERPINF2, SGSM1, SH3BGRL2, SH3PXD2B, SH3YL1, SHISA4, SHMT1, SLC10A2, SLC12A7, SLC16A12, SLC16A13, SLC16A4, SLC16A5, SLC17A4, SLC1A1, SLC22A2, SLC22A4, SLC22A5, SLC23A1, SLC25A23, SLC25A34, SLC25A42, SLC25A44, SLC26A1, SLC28A1, SLC2A2, SLC2A5, SLC2A9, SLC38A5, SLC3A1, SLC46A1, SLC5A12, SLC5A1, SLC5A8, SLC5A9, SLC6A12, SLC6A19, SLC6A3, SLC7A5, SLC9A1, SLC9A3R1, SLCO4C1, SLITRK2, SLITRK4, SLPI, SMPDL3A, SMTNL2, SPATA18, SPATS2L, SPNS2, SPOCK1, SPON2, SQLE, STAMBPL1, STEAP3, STK17A, STK32B, STK39, STON2, STX3, SULF2, SYBU, SYT12, SYT9, SYTL2, TBC1D2, TCEA3, TCFL5, TCN2, TCTA, TEF, TFEC, TFPI2, TGFBI, THAP9, THSD4, TLN2, TMCC1, TMCO4, TMEM125, TMEM130, TMEM139, TMEM140, TMEM164, TMEM171, TMEM176A, TMEM176B, TMEM195, TMEM26, TMEM37, TMEM45A, TMPRSS3, TNFAIP6, TNFRSF10C, TNFRSF21, TPBG, TPMT, TPST2, TRAP1, TRIM55, TRPV4, TSGA14, TSPAN1, TTC39C, TUBB3, UBA5, UGT1A6, UGT1A9, UGT2A3, UGT2B7, UGT3A1, USP2, VCAM1, VIL1, WDR72, WDR81, WFDC2, WFS1, WNT5A, WNT5B, ZBTB7C, ZFAT, ZFHX4, ZNF385B, ZNF711, ZSCAN2, ABCC6P2, ABCC6, ACAA2, ACE2, ACMSD, ACSM2A, ACSM2B, ACY3, ADM2, AGMAT, AGXT2, AMN, ANKS4B, APOM, ASP A, BBOX1, Cllorf54, C9orf66, CLCN5, CLRN3, CRYL1, CUBN, CYP4A11, DDC, EHHADH, FMO1, FUT6, GBA3, GIPC2, GLYATL1, GLYAT, HAO2, HNF1A, HRSP12, KHK, LGALS2, LRP2, MIOX, NAT8, PDZK1, PHYHIPL, PKLR, RBP5, SLC13A1, SLC16A9, SLC17A1, SLC17A3, SLC22A11, SLC22A12, SLC23A3, SLC27A2, SLC37A4, SLC39A5, SLC47A1, SLC5A10, SLC6A13, SLC7A9, TINAG, TM4SF5, TMEM27, TRIM10, TRIM15, TRPM3, TTC38, UPB1, and USH1C.
[0022] In any and all of the preceding embodiments of the methods disclosed herein, the Th2 immune network comprises 5 or more genes selected from among AIAP1, ANLN, ARHGAP11A, ASF1B, ASPM, ATAD2, AURKA, BRCA1, BUB1, C10orf2, C13orf34, C15orf23, C16orf75, CACNA2D4, CCDC99, CCNA2, CCNB1, CCNF, CDC6, CDCA7, CDK1, CDT1, CENPE, CENPF, CENPH, CENPL, CENPN, CENPO, CHAF1A, CHAF1B, CHEK1, CPOX, CTSF, DBF4, DERL1, DHFR, DIAPH3, DTL, E2F1, ECT2, EPR1, ESPL1, EZH2, FAM11 IB, FANCA, FANCD2, FANCI, FASN, FEN1, GGH, GINS1, GINS2, GINS3, GPRIN1, GPSM2, HELLS, HMMR, HPS3, IQGAP3, KIAA0101, KIF11, KIF18B, KIF20A, KIF23, KIF24, KIF2C, KIF4A, KIFC1, KPNA2, LMNB1, LMNB2, LRP8, MAD2L1, MCM2, MCM5, MCM6, MCM7, MELK, MKI67, MLF1IP, MTMR4, MYBL2, NCAPD2, NCAPH, NDC80, NEB, NUSAP1, PAQR4, PBXIP1, PLK4, PPARG, PPRC1, PRC1, PTTG1, RACGAP1, RAD51AP1, RFC4, RIPK2, RRP12, SG0L2, SHCBP1, SMC4, SPATA7, STIL, STMN1, TACC3, TCF19, TIMELESS, TK1, TMEM25, TRIM59, TRIP13, TUBB, UBE2C, UHRF1, WDR4, WDR67, WSCD1, ZWILCH, ZWINT, BUB IB, CCNB2, CDC20, CDCA5, CDCA8, CEP55, FOXM1, GTSE1, HJURP, NCAPG, PLK1, RRM2, T0P2A, and TPX2.
[00231 Additionally or alternatively, in some embodiments, the tumor resection surgery comprises nephrectomy. The renal cancer may have a histologic subtype selected from among clear cell renal cell carcinomas (ccRCC), papillary renal cell carcinomas (pRCC), chromophobe renal cell carcinomas (crRCC), multilocular cystic RCC, collecting duct carcinoma, medullary carcinoma, mucinous tubular and spindle cell carcinoma, neuroblastoma-associated RCC, Xpl l.2 translocation-TFE3 carcinoma, and unclassified lesions. Additionally or alternatively, in certain embodiments, the cancer patient exhibits stage I, stage II, stage III, or stage IV renal cancer.
[0024] In another aspect, the present disclosure provides a method for selecting a cancer patient undergoing tumor resection surgery for bladder cancer for treatment with an opioid- free intraoperative analgesic or a low-dose intraoperative opioid analgesic comprising (a) detecting expression levels of a survival-associated gene expression network in a biological sample obtained from the cancer patient that are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold; and (b) administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery, wherein the survival- associated gene expression network comprises 5 or more genes selected from among ABCA6, ACTA2, ACTC1, ACTG2, ACTN1, ADAM12, ADAM19, ADAMTS2, ADH1B, ADRA2A, AEBP1, AG2, ALDH1L2, AMIG02, ANGPTL1, ATP10A, ATP8B2, AXIN2, BAG2, Cl lorf41, C14orfl32, Clorfl98, C2orf40, C7, C9orfl l0, CACNA1H, CAMK1G, CAND2, CCDC3, CCL19, CCL21, CDH11, CES1, CFH, CLEC11A, CNN1, COL11A1, COL12A1, COL16A1, COL6A1, COL6A2, COL6A3, CORO2B, CPXM1, CPXM2, CPZ, CREB3L1, CRYBG3, CSRP1, CTGF, CTSK, CYBRD1, CYTSB, DBN1, DCN, DES, DIO2, ECM1, EFEMP1, ELN, ENAH, F3, FAM109B, FAM126A, FAM129A, FAM129B, FAM83D, FBLN1, FBLN5, FBLN7, FBN1, FEZ1, FGD1, FGF14, FGF1, FGF7, FHL3, FLRT2, FMO3, FMOD, FN1, FNDC1, FOXP1, FOXS1, FZD7, GALNTL1, GAS1, GATA6, GEFT, GEM, GLT8D2, GNAO1, GPC3, GREM1, GRID1, H19, HDGFRP3, HOPX, HTR2B, IGF2, IGFBP2, IGFBP7, IL18R1, IL1R1, INHBA, INMT, ITGBL1, ITPRIPL2, KCNMB1, KIAA1199, KIAA1211, KIAA1755, KRT7, LAMA2, LARGE, LDB3, LEPREL2, LGI2, LIPC, LMCD1, LOC145820, LOC399959, LOC401093, LOXL1, LPHN1, LPHN3, LRFN3, LTBP1, LTBP4, LUM, LYNX1, MAGED4B, MAP1A, MAP6, MARK1, MARVELD1, MDGA1, MEIS3, MEST, MFAP2, MFAP4, MFGE8, MLPH, MMP11, MOXD1, MRC2, MRGPRF, MRVI1, MSRB3, MUSTN1, MYH10, MYH11, MYL9, MYO10, MYOID, NACAD, NAP1L3, NAV3, NDRG4, NEXN, NFASC, NOV, NPTXR, NR2F2, NT5DC2, NXN, OXTR, PCDH7, PCOLCE, PDE1 A, PDE5A, PDLIM3, PDLIM7, PGR, PHYHD1, PID1, PLEKHA4, PLN, POSTN, PPP1R12B, PPP1R14A, PRR15L, PRRX1, PTGIR, PTGIS, PTK7, PVRL1, RAB23, RAB31, RAMP1, RBP1, RGMA, RGS16, RGS4, RICH2, ROR2, SCARF2, SCG5, SETMAR, SFRP1, SFRP2, SFRP4, SGCD, SHISA3, SLC20A2, SLC24A3, SLC2A10, SLIT2, SLIT3, SMOC2, SNAI1, SOBP, SOD3, S0RCS3, SPAG1, SPEG, SRPX2, SRPX, SSC5D, ST5, ST6GALNAC6, SULF1, SVEP1, SYT17, TACSTD2, TCF21, TGFB3, THBS2, TIMP2, TMEM117, TMEM119, TMEM30B, TMEM90B, TNC, TNFAIP8L3, TNXB, TPM2, TPM4, TRAM2, TSPAN2, VANGL2, VOL, VGLL3, WFDC1, ZAK, ZBTB47, ZCCHC24, ZNF469, ZNF503, ZNF703, ZNF853, ANTXR1, ASPN, CCDC80, COL14A1, COL1A1, COL1A2, COL3A1, COL5A1, DACT1, DACT3, EMILIN1, FAP, FBLIM1, GGT5, HSPB6, ISLR, ITGA11, LHFP, LMOD1, LTBP2, MGP, MICAL2, PALLD, PDGFRA, PODN, PRELP, SYNPO2, and TAGLN. In some embodiments, the expression levels of the survival-associated gene expression network are detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
[0025] In another aspect, the present disclosure provides a method for prolonging survival of a cancer patient undergoing tumor resection surgery for bladder cancer comprising administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a survival-associated gene expression network in a biological sample obtained from the cancer patient are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold, wherein the survival-associated gene expression network comprises 5 or more genes selected from among ABCA6, ACTA2, ACTC1, ACTG2, ACTN1, ADAM12, ADAM I 9, ADAMTS2, ADH1B, ADRA2A, AEBP1, AG2, ALDH1L2, AMIG02, ANGPTL1, ATP10A, ATP8B2, AXIN2, BAG2, Cl lorf41, C14orfl32, Clorfl98, C2orf40, C7, C9orfl l0, CACNA1H, CAMK1G, CAND2, CCDC3, CCL19, CCL21, CDH11, CES1, CFH, CLEC11A, CNN1, COL11A1, COL12A1, COL16A1, COL6A1, COL6A2, COL6A3, CORO2B, CPXM1, CPXM2, CPZ, CREB3L1, CRYBG3, CSRP1, CTGF, CTSK, CYBRD1, CYTSB, DBN1, DCN, DES, DIO2, ECM1, EFEMP1, ELN, ENAH, F3, FAM109B, FAM126A, FAM129A, FAM129B, FAM83D, FBLN1, FBLN5, FBLN7, FBN1, FEZ1, FGD1, FGF14, FGF1, FGF7, FHL3, FLRT2, FM03, FMOD, FN1, FNDC1, FOXP1, FOXS1, FZD7, GALNTL1, GAS1, GATA6, GEFT, GEM, GLT8D2, GNAO1, GPC3, GREM1, GRID1, H19, HDGFRP3, HOPX, HTR2B, IGF2, IGFBP2, IGFBP7, IL18R1, IL1R1, INHBA, INMT, ITGBL1, ITPRIPL2, KCNMB1, KIAA1199, KIAA1211, KIAA1755, KRT7, LAMA2, LARGE, LDB3, LEPREL2, LGI2, LIPC, LMCD1, LOC145820, LOC399959, LOC401093, LOXL1, LPHN1, LPHN3, LRFN3, LTBP1, LTBP4, LUM, LYNX1, MAGED4B, MAP1A, MAP6, MARK1, MARVELD1, MDGA1, MEIS3, MEST, MFAP2, MFAP4, MFGE8, MLPH, MMP11, M0XD1, MRC2, MRGPRF, MRVI1, MSRB3, MUSTN1, MYH10, MYH11, MYL9, MYO10, MYOID, NACAD, NAP1L3, NAV3, NDRG4, NEXN, NFASC, NOV, NPTXR, NR2F2, NT5DC2, NXN, OXTR, PCDH7, PCOLCE, PDE1A, PDE5A, PDLIM3, PDLIM7, PGR, PHYHD1, PID1, PLEKHA4, PLN, POSTN, PPP1R12B, PPP1R14A, PRR15L, PRRX1, PTGIR, PTGIS, PTK7, PVRL1, RAB23, RAB31, RAMP1, RBP1, RGMA, RGS16, RGS4, RICH2, ROR2, SCARF2, SCG5, SETMAR, SFRP1, SFRP2, SFRP4, SGCD, SHISA3, SLC20A2, SLC24A3, SLC2A10, SLIT2, SLIT3, SMOC2, SNAI1, SOBP, SOD3, SORCS3, SPAG1, SPEG, SRPX2, SRPX, SSC5D, ST5, ST6GALNAC6, SULF1, SVEP1, SYT17, TACSTD2, TCF21, TGFB3, THBS2, TIMP2, TMEM117, TMEM119, TMEM30B, TMEM90B, TNC, TNFAIP8L3, TNXB, TPM2, TPM4, TRAM2, TSPAN2, VANGL2, VCL, VGLL3, WFDC1, ZAK, ZBTB47, ZCCHC24, ZNF469, ZNF503, ZNF703, ZNF853, ANTXR1, ASPN, CCDC80, COL14A1, COL1A1, COL1A2, COL3A1, COL5A1, DACT1, DACT3, EMILIN1, FAP, FBLIM1, GGT5, HSPB6, ISLR, ITGA11, LHFP, LMOD1, LTBP2, MGP, MICAL2, PALLD, PDGFRA, PODN, PRELP, SYNPO2, and TAGLN. In certain embodiments, the expression levels of the survival-associated gene expression network are detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligationdependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
[0026] In any of the preceding embodiments of methods disclosed herein, the low- dose intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. Additionally or alternatively, in some embodiments, the effective amount of the low-dose intraoperative opioid analgesic is about 1 MME to about 50 MMEs. In some embodiments, the effective amount of the low- dose intraoperative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35 MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45 MMEs, or about 45-50 MMEs.
[0027] Additionally or alternatively, in some embodiments of the methods disclosed herein, the opioid-free intraoperative analgesic is an amide-type local anesthetic or an ester- type local anesthetic. Examples of amide-type local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, or levobupivacaine. Examples of ester-type local anesthetics include, but are not limited to, cocaine, procaine, tetracaine, chloroprocaine, or benzocaine. The opioid-free intraoperative analgesic may be administered via an epidural catheter. Additionally or alternatively, in some embodiments of the methods disclosed herein, the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 1-10 ml per hour when administered via an epidural catheter.
[0028] In certain embodiments, the opioid-free intraoperative analgesic may be administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block. In some embodiments, the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 10-40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
100291 Additionally or alternatively, in certain embodiments of the methods disclosed herein, the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic may be administered as a series of bolus doses, or as a continuous infusion during the tumor resection surgery.
100301 Additionally or alternatively, in some embodiments, the methods of the present technology further comprise administering to the cancer patient an effective amount of an opioid-free post-operative analgesic or a low-dose post-operative opioid analgesic after the tumor resection surgery. Examples of suitable opioid-free post-operative analgesics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine. Examples of suitable low-dose post-operative opioid analgesics include, but are not limited to, fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. In some embodiments, the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same analgesic or different analgesics. In other embodiments, the effective amount of the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same or different.
10031 ] In some embodiments, the effective amount of the low-dose post-operative opioid analgesic is about 1 MME to about 50 MMEs. In some embodiments, the effective amount of the low-dose post-operative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35 MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45 MMEs, or about 45-50 MMEs.
100321 In some embodiments, the effective amount of the opioid-free post-operative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about
1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6 %, about 1.7 %, about 1.8 %, about
1.9 %, about 2.0 %, about 2.1 %, about 2.2 %, about 2.3 %, about 2.4 %, about 2.5 %, about
2.6 %, about 2.7 %, about 2.8 %, about 2.9 %, about 3.0 %, about 3.1 %, about 3.2 %, about
3.3 %, about 3.4 %, about 3.5 %, about 3.6 %, about 3.7 %, about 3.8 %, about 3.9 %, or about 4.0 % amide-type or ester-type local anesthetic solution in a volume of about 10 ml, about 12.5 ml, about 15 ml, about 17.5 ml, about 20 ml, about 22.5 ml, about 25 ml, about 27.5 ml, about 30 ml, about 32.5 ml, about 35 ml, about 37.5 ml, or about 40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block. [0033] Additionally or alternatively, in some embodiments, the tumor resection surgery comprises cystectomy. Additionally or alternatively, in certain embodiments, the cancer patient exhibits stage I, stage II, stage III, or stage IV bladder cancer.
[0034] In any and all embodiments of the methods disclosed herein, the patient is human.
(0035] In any and all embodiments of the methods disclosed herein, the biological sample obtained from the cancer patient comprises biopsied tumor tissue, whole blood, plasma, or serum.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIGs. 1A-1F show differential expression of opioid pathway genes in clear cell renal cell carcinoma. FIG. 1A shows top fifty differentially expressed genes between cases and controls in clear cell renal cell carcinoma, clustered by gene expression. Shades of red represents lower expression, and blue expression represents greater expression. FIG. IB shows P value and log2 fold change for a subset of five hundred differentially expressed genes. Horizontal dotted line represents P threshold of 0.001, and the vertical dotted line represents fold change threshold of 0.5. Genes with a log2 fold change > 0.5 are labeled in red and log2 fold change < 0.5 are labeled in blue. Genes in the opioid signaling pathway are represented by large labeled nodes. FIGs. 1C-1F show comparison of gene expression distributions between cases and controls for OGFR, OGFRL1, TLR4, OPRL1, all of which have P < 0.05.
[0037] FIGs. 2A-2J show characterization of the 15 gene coexpression networks in clear cell renal cell carcinoma (ccRCC) and association with survival endpoints. FIG. 2A shows that topological overlap matrix plot depicts gene coexpression. Darker yellow and red represents stronger correlations between genes. FIG. 2B shows that Z statistic represents reproducibility of each module. Z > 10 (green dotted line) represents strong evidence of robustness and Z >1 (blue dotted line) represents weak evidence. FIG. 2C shows that circos plot depicts module eigengene correlations with survival and pathology measurements. Modules are ranked by the strength of their association with cancer-specific survival, reflected by the height of the purple histogram in Row 1. Rows 2-4 reflect the Cox model beta coefficient for overall survival, cancer-specific survival, and recurrence-free survival, respectively. Rows 5-7 reflect rho values for T, N, and M stage, respectively. Bluer values reflect negative values, while brown values reflect strong positive values. Rows 8-13 depict the -loglO(P) values for the same survival (8-10) and pathology variables (11-13). FIG. 2D shows Cox model beta coefficient and 95% confidence interval for each module and its association with cancer-specific survival. Red bars depict P < 0.05. FIGs. 2E-2J show survival curves comparing individuals with upregulated (blue) and downregulated (gold) network expression for green module (FIGs. 2E-2G) and tan module (FIGs. 2H-2J). Cancer-specific survival (FIGs. 2E and 2H), recurrence-free survival (FIGs. 2F and 21), and overall survival (FIGs. 2G and 2J) are depicted, and their respective Cox P values are each less than 0.05.
[0038] FIGs. 3A-3E show prediction of drug effects on the survival-associated networks. FIG. 3A shows a comparison of topological overlap matrices in cases (top right triangle) versus controls (bottom left triangle) for four modules. Greater coexpression is colored in dark yellow and red, while less coexpression is colored in light yellow and white. Module differential connectivity (MDC) and FDR values are depicted for each module. Differential connectivity was considered significant by FDR < 0.01. FIGs. 3B-3D show tau scores representing modulation of each survival network by leu-enkephalin (FIG. 3B), naloxone (FIG. 3C), and VEGF -inhibitor (FIG. 3D). Red dotted (outside two) lines represent strong evidence of drug modulation, |tau| > 90. Blue dotted (middle two) lines represent suggestive evidence, |tau| >30. FIG. 3E shows the association between leu-enkephalin tau score and Cox model survival coefficients for overall survival, recurrence-free survival, and cancerspecific survival.
[0039] FIG. 4 shows reconstructing directed transcriptional networks and master regulators in ccRCC. Directed networks representing relationships between modules (boxed), as well as gene-gene relationships within four separate modules. Each node is outlined based on its module color. Key drivers are represented by large nodes, and shaded key drivers are those with known associations to the opioid pathway.
[0040] FIG. 5 shows top gene ontology pathways enriched for differentially expressed genes in ccRCC. The color of the circle reflects enrichment p value, and the size of the circle reflects the number of differentially expressed genes in the respective gene ontology categories.
[0041 ] FIG. 6 shows comparison of OPRM1, OPRK1, and OPRD1 gene expression between cases and controls. Each point represents gene expression for an individual sample in the cohort.
[0042] FIG. 7 shows that tumors with high and low opioid pathway gene expression have differential pathologic stage in ccRCC. Boxplots illustrate the opioid pathway gene expression distribution across pathologic stage for tumors with high and low gene expression of specific opioid signaling genes. For each opioid signaling gene, the respective top and bottom gene expression quartiles were determined and the sample subset were defined accordingly. Association between gene expression and stage for these high and low expressors were estimated by the Kruskal Wallis test. RSEM normalized gene expression is plotted on the y axis and pathologic stage on the x axis. Kruskal Wallis P values are listed for each gene.
[0043] FIGs. 8A-8C show individual sample expression across module eigengenes. FIG. 8A shows that median module eigengene expression was calculated for each sample and the distribution across all samples was plotted. FIG. 8B shows that the variance of module eigengene expression for each sample was calculated and distribution across all samples plotted. FIG. 8C shows module eigengene correlation heatmap and dendrogram, in which the darker colors reflect greater correlation strength. The diagonal signifies a correlation = 1.
[0044] FIG. 9 shows fisher’s exact test overrepresentation between TCGA and MSKCC ccRCC networks. The blue boxes reflect significant Fisher’s exact test enrichment between specific MSK and KIRC networks (P < 0.05). The darker shades correspond to increased odds ratio. The eight TCGA survival-associated networks are seen to strongly overlap with the set of networks independently derived in the ccRCC-MSKCC cohort.
(0045] FIGs. 10A-10H show correlation between TCGA and MSKCC gene expression for eight survival-associated networks. Gene expression is correlated between the TCGA KIRC and ccRCC-MSKCC for each of these eight networks. Spearman rho and p value listed. [0046] FIGs. 11A-11D show gene network enrichment for microenvironment cell-type specific gene signatures. Each panel represents a different cell type as labeled in the title, with its respective PMID in which the original cell-type specific signature was published. Fisher’s exact test odds ratios are plotted along the y axis, with each respective 95% confidence interval. Significant associations (P < 0.05) are marked with asterisks. Dotted red horizontal line represents odds ratio = 1.
[0047] FIG. 12A-12B show prediction of drug effects on a survival-associated network in bladder cancer (BLCA). FIG. 12A shows that the “pink” network is associated with survival in BLCA. FIG. 12B shows that Tau for pink network hub expression with various drugs is plotted along the x axis. Leu-enkephalin modulates the “pink” network in an anti-survival direction, while naloxone is pro-survival. Positive controls include various chemotherapeutic agents, which are seen to modulate this network in a pro-survival direction. The dotted red (outside two) vertical lines represent |tau| = 90, which represents strong evidence of an effect. The dotted blue (middle two) lines represent |tau| > 30, suggestive evidence of an effect.
|0048] FIGs. 13A-13D show gene expression correlation between immune signature genes (y axis) and four ccRCC master regulator genes (x axis): PLXNB1 (FIG. 13A), CREB5 (FIG. 13B), IL4R (FIG. 13C), CLEC2D (FIG. 13D). One representative correlation plot is depicted here for each master regulator gene. Spearman Rho and P values are reported for each gene-pair comparison. All correlation statistics between master regulators and immune signature genes are listed in FIG. 21.
[0049] FIG. 14 shows mutations of the Reactome opioid signaling pathway in ccRCC.
100501 FIGs. 15A-15B show module membership for each module. FIG. 15A. hub status = 0; FIG. 15B. hub status = 1.
[0051] FIG. 16 shows gene ontology coexpression network enrichment.
[0052] FIGs. 17A-17I show Cox survival analysis for association of the 15 networks and survival endpoints. [0053] FIG. 18 shows clinical and demographic overrepresentation in sample subgroups with high module eigengene expression.
[0054] FIG. 19 shows list of hub genes for the eight cancer-specific survival-associated networks.
|0055] FIG. 20 shows master regulators of the transition from normal to disease state.
[0056] FIG. 21 shows gene expression correlation statistics between immune signature genes and four ccRCC master regulator genes involved in opioid immunomodulation (CREB5, IL4R, CLEC2D, PLXNB1).
DETAILED DESCRIPTION
[0057] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology. It is to be understood that the present disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[0058] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al., eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al., (1991) CT? 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al., (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells,' Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al., eds (1996) Weir ’s Handbook of Experimental Immunology.
[0059] Kidney cancer is the seventh most commonly diagnosed solid tumor in the United States, with clear cell being its most common subtype. Genetic and molecular changes are associated with survival, and gene network expression changes distinguish renal cell carcinoma subtypes (Ricketts CJ, et al., Cell Reports 23: 313-326 (2018)). Opioids play a critical role in perioperative analgesia in oncologic surgery, and recent in vitro evidence implicates opioids in proliferation, invasion and metastasis of ccRCC specifically (Ma et al., Renal Failure. 2016;39:258-64).
[0060] While opioids constitute the major component of perioperative analgesic regimens for surgery in general, a variety of evidence points to an association between perioperative opioid exposure and longer-term oncological outcomes. The mechanistic details underlying these effects are not well understood. The relationship between opioids and cancer outcomes is controversial. Some retrospective studies have failed to replicate earlier findings and reported effect sizes are variable and often specific to cancer subtypes. In some cases, this may point to truly null effects or confounders. It may also indicate that opioids have cancer subtype-specific effects that are masked in larger heterogeneous cohorts. Without prospective experiments, quasi-experimental studies, or a molecular understanding of opioids in humans with cancer, it is difficult to adjudicate between these possibilities.
[0061 ] The present disclosure demonstrates that opioids modulate survival-associated coexpression networks in clear cell renal cell carcinoma (ccRCC). The present disclosure examines RNAseq and cancer-specific outcomes data in both the TCGA KIRC cohort and an independent ccRCC replication cohort who underwent nephrectomy. Undirected and directed gene networks were constructed and each were correlated with recurrence-free survival (RFS), cancer-specific survival (CSS), and overall survival (OS). The expression and network connectivity of opioid- and survival -related pathways between ccRCC and controls were compared and drug-induced transcriptional profiles from in vitro cancer cells were projected onto ccRCC gene networks to characterize pathways through which opioids may influence survival. Eight coexpression networks associated with survival endpoints in ccRCC were identified, and master regulators of the transition from the normal to disease state were inferred, a number of which are linked to opioid pathways. These results are the first to suggest a mechanism for opioid effects on cancer outcomes through modulation of survival-associated coexpression networks. Accordingly, the survival-associated networks disclosed herein may function as potential pharmacogenomic biomarkers, helping to risk- stratify individual patients and predict individual response to a drug of interest. By better modeling underlying cancer biology and its response to pharmacologic perturbations, integrative systems-based models may use individual gene expression profiles to guide personalized anesthetic and analgesic plans and to optimize cancer-specific outcomes for individual cancer patients.
Definitions
[0062] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.
[0063] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
[0064] The term “adapter” refers to a short, chemically synthesized, nucleic acid sequence which can be used to ligate to the end of a nucleic acid sequence in order to facilitate attachment to another molecule. The adapter can be single-stranded or double-stranded. An adapter can incorporate a short (typically less than 50 base pairs) sequence useful for PCR amplification or sequencing.
[0065] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another.
[0066] As used herein, an “alteration” of a gene or gene product (e.g., a marker gene or gene product) refers to the presence of a mutation or mutations within the gene or gene product, e.g., a mutation, which affects the quantity or activity of the gene or gene product, as compared to the normal or wild-type gene. The genetic alteration can result in changes in the quantity, structure, and/or activity of the gene or gene product in a cancer tissue or cancer cell, as compared to its quantity, structure, and/or activity, in a normal or healthy tissue or cell (e.g., a control). For example, an alteration which is associated with cancer, or predictive of responsiveness to an intraoperative analgesic, can have an altered nucleotide sequence (e.g., a mutation), amino acid sequence, chromosomal translocation, intra-chromosomal inversion, copy number, expression level, protein level, protein activity, in a cancer tissue or cancer cell, as compared to a normal, healthy tissue or cell. Exemplary mutations include, but are not limited to, point mutations (e.g., silent, missense, or nonsense), deletions, insertions, inversions, linking mutations, duplications, translocations, inter- and intra- chromosomal rearrangements. Mutations can be present in the coding or non-coding region of the gene.
[0067] As used herein, the terms “amplify” or “amplification” with respect to nucleic acid sequences, refer to methods that increase the representation of a population of nucleic acid sequences in a sample. Nucleic acid amplification methods are well known to the skilled artisan and include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Q-replicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two- step multiplexed amplifications, rolling circle amplification (RCA), recombinase- polymerase amplification (RPA)(TwistDx, Cambridge, UK), transcription mediated amplification, signal mediated amplification of RNA technology, loop-mediated isothermal amplification of DNA, helicase-dependent amplification, single primer isothermal amplification, and self- sustained sequence replication (3 SR), including multiplex versions or combinations thereof. Copies of a particular nucleic acid sequence generated in vitro in an amplification reaction are called “amplicons” or “amplification products.”
[0068] The terms “cancer” or “tumor” are used interchangeably and refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell. As used herein, the term “cancer” includes premalignant, as well as malignant cancers.
[0069] As used herein, the term “cancer-specific survival” or “CSS” refers to the time from the date of diagnosis of a cancer to the date of death from the cancer apart from other causes. Patients who die from causes unrelated to the cancer are not counted in this measurement.
10070 [ The terms “complementary” or “complementarity” as used herein with reference to polynucleotides (z.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) refer to the base-pairing rules. The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3’ end of the other, is in “antiparallel association.” For example, the sequence “5'-A-G-T-3”’ is complementary to the sequence “3’-T-C-A-5.” Certain bases not commonly found in naturally-occurring nucleic acids may be included in the nucleic acids described herein. These include, for example, inosine, 7- deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA). Complementarity need not be perfect; stable duplexes may contain mismatched base pairs, degenerative, or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs. A complement sequence can also be an RNA sequence complementary to the DNA sequence or its complement sequence, and can also be a cDNA.
[0071 ] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.
[0072] A “control nucleic acid sample” or “reference nucleic acid sample” as used herein, refers to nucleic acid molecules from a control or reference sample. In certain embodiments, the reference or control nucleic acid sample is a wild type or a non-mutated DNA or RNA sequence. In certain embodiments, the reference nucleic acid sample is purified or isolated (e.g., it is removed from its natural state). In other embodiments, the reference nucleic acid sample is from a non-tumor sample, e.g., a blood control, a normal adjacent tumor (NAT), or any other non-cancerous sample from the same or a different subject.
[0073] “Detecting” as used herein refers to determining the presence of a mutation or alteration in a nucleic acid of interest in a sample. Detection does not require the method to provide 100% sensitivity. Analysis of nucleic acid markers can be performed using techniques known in the art including, but not limited to, sequence analysis, and electrophoretic analysis. Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al. , Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al., Methods Mol. Cell Biol, 3 :39-42 (1992)), sequencing with mass spectrometry such as matrix- assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al., Nat. Biotechnol, 16:381-384 (1998)), and sequencing by hybridization. Chee et al., Science, 274:610-614 (1996); Drmanac et al., Science, 260: 1649-1652 (1993); Drmanac et al., Nat. Biotechnol, 16:54-58 (1998). Non-limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Additionally, next generation sequencing methods can be performed using commercially available kits and instruments from companies such as the Life Technologies/Ion Torrent PGM or Proton, the Illumina HiSEQ or MiSEQ, and the Roche/454 next generation sequencing system.
[0074] “Detectable label” as used herein refers to a molecule or a compound or a group of molecules or a group of compounds used to identify a nucleic acid or protein of interest. In some embodiments, the detectable label may be detected directly. In other embodiments, the detectable label may be a part of a binding pair, which can then be subsequently detected. Signals from the detectable label may be detected by various means and will depend on the nature of the detectable label. Detectable labels may be isotopes, fluorescent moieties, colored substances, and the like. Examples of means to detect detectable labels include but are not limited to spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluorescence, or chemiluminescence, or any other appropriate means.
[0075] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.
[0076] “Gene” as used herein refers to a DNA sequence that comprises regulatory and coding sequences necessary for the production of an RNA, which may have a non-coding function (e.g., a ribosomal or transfer RNA) or which may include a polypeptide or a polypeptide precursor. The RNA or polypeptide may be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained. Although a sequence of the nucleic acids may be shown in the form of DNA, a person of ordinary skill in the art recognizes that the corresponding RNA sequence will have a similar sequence with the thymine being replaced by uracil, i.e., "T" is replaced with "U."
[0077] The term “hybridize” as used herein refers to a process where two substantially complementary nucleic acid strands (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex through formation of hydrogen bonds between complementary base pairs. Hybridizations are typically and preferably conducted with probe-length nucleic acid molecules, preferably 15- 100 nucleotides in length, more preferably 18-50 nucleotides in length. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, and the thermal melting point (Tm) of the formed hybrid. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, N.J. In some embodiments, specific hybridization occurs under stringent hybridization conditions. An oligonucleotide or polynucleotide (e.g., a probe or a primer) that is specific for a target nucleic acid will “hybridize” to the target nucleic acid under suitable conditions.
[0078] As used herein, the term “library” refers to a collection of nucleic acid sequences, e.g., a collection of nucleic acids derived from whole genomic, subgenomic fragments, cDNA, cDNA fragments, RNA, RNA fragments, or a combination thereof. In one embodiment, a portion or all of the library nucleic acid sequences comprises an adapter sequence. The adapter sequence can be located at one or both ends. The adapter sequence can be useful, e.g., for a sequencing method (e.g., an NGS method), for amplification, for reverse transcription, or for cloning into a vector.
100791 The library can comprise a collection of nucleic acid sequences, e.g., a target nucleic acid sequence (e.g., a tumor nucleic acid sequence), a reference nucleic acid sequence, or a combination thereof. In some embodiments, the nucleic acid sequences of the library can be derived from a single subject. In other embodiments, a library can comprise nucleic acid sequences from more than one subject (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more subjects). In some embodiments, two or more libraries from different subjects can be combined to form a library having nucleic acid sequences from more than one subject.
[0080] A “library nucleic acid sequence” refers to a nucleic acid molecule, e.g., a DNA, RNA, or a combination thereof, that is a member of a library. Typically, a library nucleic acid sequence is a DNA molecule, e.g., genomic DNA or cDNA. In some embodiments, a library nucleic acid sequence is fragmented, e.g., sheared or enzymatically prepared, genomic DNA. In certain embodiments, the library nucleic acid sequences comprise sequence from a subject and sequence not derived from the subject, e.g., adapter sequence, a primer sequence, or other sequences that allow for identification, e.g., “barcode” sequences.
[0081 ] The term “multiplex PCR” as used herein refers to amplification of two or more PCR products or amplicons which are each primed using a distinct primer pair.
[0082] “Next-generation sequencing or NGS” as used herein, refers to any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules (e.g., in single molecule sequencing) or clonally expanded proxies for individual nucleic acid molecules in a high throughput parallel fashion (e.g., greater than 103, 104, 105 or more molecules are sequenced simultaneously). In one embodiment, the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment. Next generation sequencing methods are known in the art, and are described, e.g., in Metzker, M. Nature Biotechnology Reviews 11 :31-46 (2010). [0083] As used herein, “oligonucleotide” refers to a molecule that has a sequence of nucleic acid bases on a backbone comprised mainly of identical monomer units at defined intervals. The bases are arranged on the backbone in such a way that they can bind with a nucleic acid having a sequence of bases that are complementary to the bases of the oligonucleotide. The most common oligonucleotides have a backbone of sugar phosphate units. A distinction may be made between oligodeoxyribonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group at the 2' position.
Oligonucleotides may also include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group. Oligonucleotides of the method which function as primers or probes are generally at least about 10-15 nucleotides long and more preferably at least about 15 to 25 nucleotides long, although shorter or longer oligonucleotides may be used in the method. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including, for example, chemical synthesis, DNA replication, restriction endonuclease digestion of plasmids or phage DNA, reverse transcription, PCR, or a combination thereof. The oligonucleotide may be modified e.g., by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides.
[0084] As used herein, the term “overall survival” or “OS” means the observed length of life from the start of treatment to death or the date of last contact.
[0085] As used herein, the term “perioperative” refers to the time period of a patient's surgical procedure. It commonly includes ward admission, anesthesia, surgery, and recovery. The perioperative period is characterized by a sequence including the time preceding an operation when a patient is being prepared for surgery (“the preoperative period”), followed by the time spent in surgery (“the intraoperative period”), and by the time following an operation when the patient is closely monitored for complications while recovering from the effects of anesthesia (“the postoperative period”).
[0086] As used herein, the term “primer” refers to an oligonucleotide, which is capable of acting as a point of initiation of nucleic acid sequence synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a target nucleic acid strand is induced, i.e., in the presence of different nucleotide triphosphates and a polymerase in an appropriate buffer (“buffer” includes pH, ionic strength, cofactors etc.) and at a suitable temperature. One or more of the nucleotides of the primer can be modified for instance by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. The term primer as used herein includes all forms of primers that may be synthesized including peptide nucleic acid primers, locked nucleic acid primers, phosphorothioate modified primers, labeled primers, and the like. The term “forward primer” as used herein means a primer that anneals to the anti-sense strand of dsDNA. A “reverse primer” anneals to the sense-strand of dsDNA.
[0087] As used herein, “primer pair” refers to a forward and reverse primer pair (i.e., a left and right primer pair) that can be used together to amplify a given region of a nucleic acid of interest.
|0088| “Probe” as used herein refers to nucleic acid that interacts with a target nucleic acid via hybridization. A probe may be fully complementary to a target nucleic acid sequence or partially complementary. The level of complementarity will depend on many factors based, in general, on the function of the probe. A probe or probes can be used, for example to detect the presence or absence of a mutation in a nucleic acid sequence by virtue of the sequence characteristics of the target. Probes can be labeled or unlabeled, or modified in any of a number of ways well known in the art. A probe may specifically hybridize to a target nucleic acid. Probes may be DNA, RNA or a RNA/DNA hybrid. Probes may be oligonucleotides, artificial chromosomes, fragmented artificial chromosome, genomic nucleic acid, fragmented genomic nucleic acid, RNA, recombinant nucleic acid, fragmented recombinant nucleic acid, peptide nucleic acid (PNA), locked nucleic acid, oligomer of cyclic heterocycles, or conjugates of nucleic acid. Probes may comprise modified nucleobases, modified sugar moieties, and modified internucleotide linkages. A probe may be used to detect the presence or absence of a target nucleic acid. Probes are typically at least about 10, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100 nucleotides or more in length. [0089] As used herein, “recurrence-specific survival” or “RSS” means the observed length of life from the time of surgical resection to the time of first recurrence of the cancer, otherwise censored at the time of last follow-up. In RSS, deaths not involving recurrence of cancer are excluded.
100901 As used herein, a “sample” refers to a substance that is being assayed for the presence of a mutation in a nucleic acid of interest. Processing methods to release or otherwise make available a nucleic acid for detection are well known in the art and may include steps of nucleic acid manipulation. A biological sample may be a body fluid or a tissue sample. In some cases, a biological sample may consist of or comprise blood, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and/or chorionic villi, cultured cells, and the like. Fresh, fixed or frozen tissues may also be used. In one embodiment, the sample is preserved as a frozen sample or as formaldehyde- or paraformaldehyde-fixed paraffin- embedded (FFPE) tissue preparation. For example, the sample can be embedded in a matrix, e.g., an FFPE block or a frozen sample. Whole blood samples of about 0.5 to 5 ml collected with EDTA, ACD or heparin as anti-coagulant are suitable.
[0091 ] The term “sensitivity,” as used herein in reference to the methods of the present technology, is a measure of the ability of a method to detect a preselected sequence variant in a heterogeneous population of sequences. A method has a sensitivity of S % for variants of F % if, given a sample in which the preselected sequence variant is present as at least F % of the sequences in the sample, the method can detect the preselected sequence at a preselected confidence of C %, S % of the time. By way of example, a method has a sensitivity of 90% for variants of 5% if, given a sample in which the preselected variant sequence is present as at least 5% of the sequences in the sample, the method can detect the preselected sequence at a preselected confidence of 99%, 9 out of 10 times (F=5%; C=99%; S=90%).
[0092] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes. [0093] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.
[0094] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.
[0095] The term “specific” as used herein in reference to an oligonucleotide primer means that the nucleotide sequence of the primer has at least 12 bases of sequence identity with a portion of the nucleic acid to be amplified when the oligonucleotide and the nucleic acid are aligned. An oligonucleotide primer that is specific for a nucleic acid is one that, under the stringent hybridization or washing conditions, is capable of hybridizing to the target of interest and not substantially hybridizing to nucleic acids which are not of interest. Higher levels of sequence identity are preferred and include at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and more preferably at least 98% sequence identity.
[0096] “Specificity,” as used herein, is a measure of the ability of a method to distinguish a truly occurring preselected sequence variant from sequencing artifacts or other closely related sequences. It is the ability to avoid false positive detections. False positive detections can arise from errors introduced into the sequence of interest during sample preparation, sequencing error, or inadvertent sequencing of closely related sequences like pseudo-genes or members of a gene family. A method has a specificity of X % if, when applied to a sample set of Nrotai sequences, in which
Figure imgf000036_0001
sequences are truly variant and XNottme are not truly variant, the method selects at least X % of the not truly variant as not variant. E.g., a method has a specificity of 90% if, when applied to a sample set of 1,000 sequences, in which 500 sequences are truly variant and 500 are not truly variant, the method selects 90% of the 500 not truly variant sequences as not variant. Exemplary specificities include 90, 95, 98, and 99%. [0097] The term “stringent hybridization conditions” as used herein refers to hybridization conditions at least as stringent as the following: hybridization in 50% formamide, 5xSSC, 50 mM NaHzPC , pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5x Denhart's solution at 42°C overnight; washing with 2x SSC, 0.1% SDS at 45° C; and washing with 0.2x SSC, 0.1% SDS at 45° C. In another example, stringent hybridization conditions should not allow for hybridization of two nucleic acids which differ over a stretch of 20 contiguous nucleotides by more than two bases.
[0098] As used herein, the terms “subject”, “patient”, or “individual” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the subject, patient or individual is a human.
100991 As used herein, the terms “target sequence” and “target nucleic acid sequence” refer to a specific nucleic acid sequence to be detected and/or quantified in the sample to be analyzed.
[0100] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.
[0101] It is also to be appreciated that the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition. Methods for Detecting Polynucleotides Associated with Positive or Negative Responsiveness to Intraoperative Opioid Analgesics
[0102] Polynucleotides associated with responsiveness to intraoperative opioid analgesics may be detected by a variety of methods known in the art. Non-limiting examples of detection methods are described below. The detection assays in the methods of the present technology may include purified or isolated DNA (genomic or cDNA), RNA or protein or the detection step may be performed directly from a biological sample without the need for further DNA, RNA or protein purification/isolation.
Nucleic Acid Amplification and/or Detection
[0103] Polynucleotides associated with responsiveness to intraoperative opioid analgesics can be detected by the use of nucleic acid amplification techniques that are well known in the art. The starting material may be genomic DNA, cDNA, RNA or mRNA. Nucleic acid amplification can be linear or exponential. Specific variants or mutations may be detected by the use of amplification methods with the aid of oligonucleotide primers or probes designed to interact with or hybridize to a particular target sequence in a specific manner, thus amplifying only the target variant.
10.104 [ Non-limiting examples of nucleic acid amplification techniques include polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), digital PCR (dPCR), reverse transcriptase polymerase chain reaction (RT-PCR), nested PCR, ligase chain reaction (see Abravaya, K. et al., Nucleic Acids Res . (1995), 23:675-682), branched DNA signal amplification (see Urdea, M. S. et al., AIDS (1993), 7(suppl 2): S 11- S14), amplifiable RNA reporters, Q-beta replication, transcription-based amplification, boomerang DNA amplification, strand displacement activation, cycling probe technology, isothermal nucleic acid sequence based amplification (NASBA) (see Kievits, T. et al., J Virological Methods (1991), 35:273-286), Invader Technology, next-generation sequencing technology or other sequence replication assays or signal amplification assays.
[0105] Primers'. Oligonucleotide primers for use in amplification methods can be designed according to general guidance well known in the art as described herein, as well as with specific requirements as described herein for each step of the particular methods described. In some embodiments, oligonucleotide primers for cDNA synthesis and PCR are 10 to 100 nucleotides in length, preferably between about 15 and about 60 nucleotides in length, more preferably 25 and about 50 nucleotides in length, and most preferably between about 25 and about 40 nucleotides in length.
|0106| Tm of a polynucleotide affects its hybridization to another polynucleotide (e.g., the annealing of an oligonucleotide primer to a template polynucleotide). In certain embodiments of the disclosed methods, the oligonucleotide primer used in various steps selectively hybridizes to a target template or polynucleotides derived from the target template (i.e., first and second strand cDNAs and amplified products). Typically, selective hybridization occurs when two polynucleotide sequences are substantially complementary (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary). See Kanehisa, M., Polynucleotides Res. (1984), 12:203, incorporated herein by reference. As a result, it is expected that a certain degree of mismatch at the priming site is tolerated. Such mismatch may be small, such as a mono-, di- or tri -nucleotide. In certain embodiments, 100% complementarity exists.
[0107] Probes'. Probes are capable of hybridizing to at least a portion of the nucleic acid of interest or a reference nucleic acid (i.e., wild-type sequence). Probes may be an oligonucleotide, artificial chromosome, fragmented artificial chromosome, genomic nucleic acid, fragmented genomic nucleic acid, RNA, recombinant nucleic acid, fragmented recombinant nucleic acid, peptide nucleic acid (PNA), locked nucleic acid, oligomer of cyclic heterocycles, or conjugates of nucleic acid. Probes may be used for detecting and/or capturing/purifying a nucleic acid of interest.
[0108] Typically, probes can be about 10 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 75 nucleotides, or about 100 nucleotides long. However, longer probes are possible. Longer probes can be about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 750 nucleotides, about 1,000 nucleotides, about 1,500 nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about 3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides, about 5,000 nucleotides, about 7,500 nucleotides, or about 10,000 nucleotides long.
[01 9] Probes may also include a detectable label or a plurality of detectable labels. The detectable label associated with the probe can generate a detectable signal directly. Additionally, the detectable label associated with the probe can be detected indirectly using a reagent, wherein the reagent includes a detectable label, and binds to the label associated with the probe.
[0110] In some embodiments, detectably labeled probes can be used in hybridization assays including, but not limited to Northern blots, Southern blots, microarray, dot or slot blots, and in situ hybridization assays such as fluorescent in situ hybridization (FISH) to detect a target nucleic acid sequence within a biological sample. Certain embodiments may employ hybridization methods for measuring expression of a polynucleotide gene product, such as mRNA. Methods for conducting polynucleotide hybridization assays have been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al. Molecular Cloning: A Laboratory Manual (2nd Ed. Cold Spring Harbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif, 1987);
Young and Davis, PNAS. 80: 1194 (1983).
[0111] Detectably labeled probes can also be used to monitor the amplification of a target nucleic acid sequence. In some embodiments, detectably labeled probes present in an amplification reaction are suitable for monitoring the amount of amplicon(s) produced as a function of time. Examples of such probes include, but are not limited to, the 5'- exonuclease assay (TAQMAN® probes described herein (see also U.S. Pat. No. 5,538,848) various stemloop molecular beacons (see for example, U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, 1996, Nature Biotechnology 14:303- 308), stemless or linear beacons (see, e.g., WO 99/21881), PNA Molecular Beacons™ (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, for example, Kubista et al., 2001, SPIE 4264:53-58), non-FRET probes (see, for example, U.S. Pat. No. 6,150,097), Sunrise®/ Amplifluor™ probes (U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion probes (Solinas et al., 2001, Nucleic Acids Research 29:E96 and U.S. Pat. No. 6,589,743), bulge loop probes (U.S. Pat. No. 6,590,091), pseudo knot probes (U.S. Pat. No. 6,589,250), cyclicons (U.S. Pat. No. 6,383,752), MGB Eclipse™ probe (Epoch Biosciences), hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA) light-up probes, self-assembled nanoparticle probes, and ferrocene-modified probes described, for example, in U.S. Pat. No. 6,485,901 ;
Mhlanga et al., 2001, Methods 25:463-471 ; Whitcombe et al., 1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000, Molecular Cell Probes. 14:321-328; Svanvik et al., 2000, Anal Biochem. 281 :26-35; Wolffs et al., 2001, Biotechniques 766:769-771
; Tsourkas et al., 2002, Nucleic Acids Research. 30:4208-4215; Riccelli et al., 2002, Nucleic Acids Research 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332; Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et a/., 2002, Trends Biotechnol. 20:249- 56; Huang et al., 2002, Chem. Res. Toxicol. 15: 118- 126; and Yu et al., 2001, J. Am. Chem. Soc 14: 11155-11161.
[0112] In some embodiments, the detectable label is a fluorophore. Suitable fluorescent moieties include but are not limited to the following fluorophores working individually or in combination: 4-acetamido-4'-isothiocyanatostilbene- 2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes); 5-(2- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino-l- naphthyl)mal eimide; anthranilamide; Black Hole Quencher™ (BHQ™) dyes (biosearch Technologies); BODIPY dyes: BOD IP Y® R-6G, BOPIPY® 530/550, BODIPY® FL; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4- methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"-dibromopyrogallol- sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'- isothiocyanatophenyl)-4- methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'- disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid; 5- [dimethylamino]naphthalene-l -sulfonyl chloride (DNS, dansyl chloride); 4-(4'- dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'- isothiocyanate (DABITC); Eclipse™ (Epoch Biosciences Inc.); eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin isothiocyanate; ethidium; fluorescein and derivatives: 5- carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)amino fluorescein (DTAF), 2', 7'- dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), hexachloro-6-carboxyfluorescein (HEX), QFITC (XRITC), tetrachlorofluorescem (TET); fiuorescamine; IR144;
IR1446; lanthamide phosphors; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin, R- phycoerythrin; allophycocyanin; o-phthaldialdehyde; Oregon Green®; propidium iodide; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 -pyrene butyrate; QSY® 7; QSY® 9; QSY® 21; QSY® 35 (Molecular Probes); Reactive Red 4 (Cibacron®Brilliant Red 3B-A); rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, riboflavin, rosolic acid, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); terbium chelate derivatives; N,N,N',N'-tetramethyl-6- carb oxy rhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); and VIC®. Detector probes can also comprise sulfonate derivatives of fluorescenin dyes with S03 instead of the carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of CY 5 (commercially available for example from Amersham).
[0113] Detectably labeled probes can also include quenchers, including without limitation black hole quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch).
|01l4| Detectably labeled probes can also include two probes, wherein for example a fluorophore is on one probe, and a quencher is on the other probe, wherein hybridization of the two probes together on a target quenches the signal, or wherein hybridization on the target alters the signal signature via a change in fluorescence.
[0115] In some embodiments, interchelating labels such as ethidium bromide, SYBR® Green I (Molecular Probes), and PicoGreen® (Molecular Probes) are used, thereby allowing visualization in real-time, or at the end point, of an amplification product in the absence of a detector probe. In some embodiments, real-time visualization may involve the use of both an intercalating detector probe and a sequence-based detector probe. In some embodiments, the detector probe is at least partially quenched when not hybridized to a complementary sequence in the amplification reaction, and is at least partially unquenched when hybridized to a complementary sequence in the amplification reaction.
[011 ] In some embodiments, the amount of probe that gives a fluorescent signal in response to an excited light typically relates to the amount of nucleic acid produced in the amplification reaction. Thus, in some embodiments, the amount of fluorescent signal is related to the amount of product created in the amplification reaction. In such embodiments, one can therefore measure the amount of amplification product by measuring the intensity of the fluorescent signal from the fluorescent indicator.
(0117] Primers or probes may be designed to selectively hybridize to any portion of a nucleic acid sequence encoding a polypeptide selected from among:
|0118] A1CF, ABAT, ABCB1, ABCB9, ABCC2, ABCC6P1, ABCG1, ABHD6, ABLIM3, ABP1, ACAD11, ACADL, ACAT1, ACBD4, ACO2, ACOT4, ACOX2, ACSL1, ACSM5, ACY1, ADAMTS3, ADH6, AGPAT3, AGT, AIFM1, AKR1C1, AKR1C2, AKR7A2, AKR7A3, ALAD, ALDH1A1, ALDH1A2, ALDH1L1, ALDH2, ALDH3A2, ALDH4A1, ALDH7A1, ALDH8A1, ALDOB, ALPK2, ALPL, AMDHD1, ANK3, ANKRD56, ANPEP, ANXA13, ANXA2P2, ANXA2, A0X1, APITD1, AQP3, AQP9, ARHGAP1, ARHGAP24, ARL4C, ASB13, ASRGL1, ASTN2, ATP6V0A1, AUH, AXL, AZGP1, B3GNT7, B4GALNT1, B4GALT1, BACE2, BAIAP2L1, BAIAP2L2, BAMBI, BARX2, BCAT1, BCMO1, BDH2, BDKRB2, BEND3, BHMT2, BHMT, BNIP3, BPHL, BTG3, C10orfl08, Cl lorf45, Cl lorf52, C14orf64, C14orf73, C17orf51, C18orfl8, C19orf77, C1RL, C1R, CIS, Clorfl l5, Clorf201, Clorf203, Clorf210, Clorf21, Clorf89, Clorf96, C21orf7, C22orf45, C2orf24, C2orf67, C3, C4orfl9, C5orf23, C6, C7orfl0, C8orf47, C9orfl25, CABCI, CABLES 1, CADM3, CALML4, CBR4, CBWD1, CBX7, CCDC146, CCDC64, CCDC68, CD276, CD44, CD47, CD55, CD82, CDADC1, CDC42SE2, CDCA2, CDCP1, CDH16, CDH2, CDHR2, CDHR5, CDK18, CDK20, CDON, CEACAM1, CES2, CFB, CGREF1, CHDH, CHI3L1, CHPF2, CHST13, CIDEB, CISH, CIT, CKAP4, CLDN10, CLDN2, CLEC18A, CLEC18B, CLEC18C, CLIC6, CLPTM1L, CMBL, CNDP2, CNNM1, COBL, COL22A1, COL23A1, COL4A5, COL8A2, COLEC12, COPG2, CPT2, CRAT, CRB3, CREB3L3, CRY2, CRYM, CRYZ, CTHRC1, CYB5A, CYB5D1, CYP1B1, CYP27A1, CYP2J2, CYP4V2, CYS1, DAB2, DAPK2, DCBLD2, DDAH1, DEPDC7, DGKG, DHDH, DHTKD1, DIRAS2, DLG5, DMGDH, DMRTA1, DPF3, DPYS, DSEL, ECHDC3, EFNA5, EGOT, ELFN2, EMX1, EMX2OS, EMX2, ENAM, ENPEP, ENPP3, EPB49, EPHA7, EPHX2, ERBB3, ETFA, ETFDH, ETNK2, ETV1, ETV6, EZR, FAAH, FABP3, FAHD1, FAM149A, FAM164C, FAM60A, FAM69A, FANCC, FBP1, FBXL16, FBXO32, FCAMR, FGFR1OP, FGFR3, FGGY, FHL2, FLJ23867, FLJ36031, FLNC, FMO4, FNDC4, FREM2, FTCD, GAL3ST1, GALM, GALNT2, GALNT7, GATM, GATS, GBAS, GDA, GFPT2, GGT3P, GJB1, GK, GLB1L, GLIS1, GLRB, GLT25D1, GOT1, GPD1, GPER, GPT, GPX3, GPX8, GRAMD1C, GRTP1, GSTA1, GSTA2, GXYLT2, GYLTL1B, HAAO, HABP2, HABP4, HGD, HHLA2, HIBCH, HIGD1A, HLF, HMGCL, HMOX1, HNF4A, HOXCIO, HSDL2, HSP90B1, HSPA2, HSPB8, HYAL1, HYOU1, IDH1, IGDCC4, IGF2BP2, IL17RB, IL1R2, IL22RA1, IMPA2, IMPDH1, IRS2, ITGB6, JPH2, KCNIP3, KCNJ15, KCNJ16, KCNS3, KCTD17, KCTD1, KIAA1543, KLC4, KLF15, KLHDC7A, KL, KRT19, KRT80, KSR1, LAD1, LAMA3, LAMB1, LAMB3, LAMC2, LDHD, LEF1, LIMK2, LNP1, LOC100126784, LOC100131551, LOC151534, LOC388387, LOC389332, LOC723809, LRG1, LRRC19, LRRC8E, LYG1, MAF, MAGED1, MAN1C1, MAOB, MAP7, MAPK8IP1, MAPT, MARVELD3, METTL7A, METTL7B, MFI2, MINA, MLYCD, MMD, MME, MMP14, MMP7, MMP9, MOBKL2B, MOSC2, MPI, MPV17L, MPZL1, MSRA, MTHFD1L, MTHFD2, MUC1, MXRA8, MYO1E, MYO3A, MYO7B, MYOM3, NAMPT, NAP1L1, NAPSA, NEFL, NGEF, NHEJ1, NIPSNAP1, NOMO1, NOMO3, NPR3, NRXN2, NTN4, NUDT6, OPN3, 0SCP1, OSTalpha, PANK1, PAPP A, PAQR7, PARD6B, PBLD, PBX3, PCBD2, PCCA, PCK1, PCOLCE2, PC, PDE10A, PDIA3P, PDIA4, PDIA5, PDK2, PDXP, PDZD3, PDZK1P1, PECI, PECR, PEPD, PER3, PGPEP1, PHYH, PIGT, PIPOX, PKHD1, PLA2G4C, PL AU, PLIN2, PLIN3, PLOD2, PLTP, PMAIP1, PNMA6A, PON2, PPFIBP2, PPL, PPP1R14C, PRODH2, PRODH, PSD3, PTGER2, PTGFRN, PTGR2, PTH1R, PTH2R, PVR, PXMP2, QDPR, QRFPR, QSOX1, RAB17, RAB3IP, RAB7L1, RAI2, RARRES1, RCN1, RGN, RGS14, RHOBTB1, RIT1, RND3, RNF5P1, RORC, RPN2, RUNDC3B, RUNX1, RUNX2, SAMD5, SATB2, SCARB1, SCD, SCGN, SCLY, SCNN1A, SEMA3C, SEMA3D, SEMA4B, SEMA4F, SEMA6A, SEPHS2, SEPSECS, SERPINA3, SERPINA6, SERPINF1, SERPINF2, SGSM1, SH3BGRL2, SH3PXD2B, SH3YL1, SHISA4, SHMT1, SLC10A2, SLC12A7, SLC16A12, SLC16A13, SLC16A4, SLC16A5, SLC17A4, SLC1A1, SLC22A2, SLC22A4, SLC22A5, SLC23A1, SLC25A23, SLC25A34, SLC25A42, SLC25A44, SLC26A1, SLC28A1, SLC2A2, SLC2A5, SLC2A9, SLC38A5, SLC3A1, SLC46A1, SLC5A12, SLC5A1, SLC5A8, SLC5A9, SLC6A12, SLC6A19, SLC6A3, SLC7A5, SLC9A1, SLC9A3R1, SLC04C1, SLITRK2, SLITRK4, SLPI, SMPDL3A, SMTNL2, SPATA18, SPATS2L, SPNS2, SP0CK1, SPON2, SQLE, STAMBPL1, STEAP3, STK17A, STK32B, STK39, STON2, STX3, SULF2, SYBU, SYT12, SYT9, SYTL2, TBC1D2, TCEA3, TCFL5, TCN2, TCTA, TEF, TFEC, TFPI2, TGFBI, THAP9, THSD4, TLN2, TMCC1, TMCO4, TMEM125, TMEM130, TMEM139, TMEM140, TMEM164, TMEM171, TMEM176A, TMEM176B, TMEM195, TMEM26, TMEM37, TMEM45A, TMPRSS3, TNFAIP6, TNFRSF10C, TNFRSF21, TPBG, TPMT, TPST2, TRAP1, TRIM55, TRPV4, TSGA14, TSPAN1, TTC39C, TUBB3, UBA5, UGT1A6, UGT1A9, UGT2A3, UGT2B7, UGT3A1, USP2, VCAM1, VIL1, WDR72, WDR81, WFDC2, WFS1, WNT5A, WNT5B, ZBTB7C, ZFAT, ZFHX4, ZNF385B, ZNF711, ZSCAN2, ABCC6P2, ABCC6, ACAA2, ACE2, ACMSD, ACSM2A, ACSM2B, ACY3, ADM2, AGMAT, AGXT2, AMN, ANKS4B, APOM, ASP A, BBOX1, Cllorf54, C9orf66, CLCN5, CLRN3, CRYL1, CUBN, CYP4A11, DDC, EHHADH, FMO1, FUT6, GBA3, GIPC2, GLYATL1, GLYAT, HAO2, HNF1A, HRSP12, KHK, LGALS2, LRP2, MIOX, NAT8, PDZK1, PHYHIPL, PKLR, RBP5, SLC13A1, SLC16A9, SLC17A1, SLC17A3, SLC22A11, SLC22A12, SLC23A3, SLC27A2, SLC37A4, SLC39A5, SLC47A1, SLC5A10, SLC6A13, SLC7A9, TINAG, TM4SF5, TMEM27, TRIM10, TRIM15, TRPM3, TTC38, UPB1, USH1C,
[0119] AJAP1, ANLN, ARHGAP11A, ASF1B, ASPM, ATAD2, AURKA, BRCA1, BUB1, C10orf2, C13orf34, C15orf23, C16orf75, CACNA2D4, CCDC99, CCNA2, CCNB1, CCNF, CDC6, CDCA7, CDK1, CDT1, CENPE, CENPF, CENPH, CENPL, CENPN, CENPO, CHAF1A, CHAF1B, CHEK1, CPOX, CTSF, DBF4, DERL1, DHFR, DIAPH3, DTL, E2F1, ECT2, EPR1, ESPL1, EZH2, FAM11 IB, FANCA, FANCD2, FANCI, FASN, FEN1, GGH, GINS1, GINS2, GINS3, GPRIN1, GPSM2, HELLS, HMMR, HPS3, IQGAP3, KIAA0101, KIF11, KIF18B, KIF20A, KIF23, KIF24, KIF2C, KIF4A, KIFC1, KPNA2, LMNB1, LMNB2, LRP8, MAD2L1, MCM2, MCM5, MCM6, MCM7, MELK, MKI67, MLF1IP, MTMR4, MYBL2, NCAPD2, NCAPH, NDC80, NEB, NUSAP1, PAQR4, PBXIP1, PLK4, PPARG, PPRC1, PRC1, PTTG1, RACGAP1, RAD51AP1, RFC4, RIPK2, RRP12, SG0L2, SHCBP1, SMC4, SPATA7, STIL, STMN1, TACC3, TCF19, TIMELESS, TK1, TMEM25, TRIM59, TRIP13, TUBB, UBE2C, UHRF1, WDR4, WDR67, WSCD1, ZWILCH, ZWINT, BUB IB, CCNB2, CDC20, CDCA5, CDCA8, CEP55, FOXM1, GTSE1, HJURP, NCAPG, PLK1, RRM2, TOP2A, TPX2,
[0120] ABCA6, ACTA2, ACTC1, ACTG2, ACTN1, ADAM12, ADAM19, ADAMTS2, ADH1B, ADRA2A, AEBP1, AG2, ALDH1L2, AMIGO2, ANGPTL1, ATP10A, ATP8B2, AXIN2, BAG2, Cl lorf41, C14orfl32, Clorfl98, C2orf40, C7, C9orfl l0, CACNA1H, CAMK1G, CAND2, CCDC3, CCL19, CCL21, CDH11, CES1, CFH, CLEC11A, CNN1, COL11A1, COL12A1, COL16A1, COL6A1, COL6A2, COL6A3, CORO2B, CPXM1, CPXM2, CPZ, CREB3L1, CRYBG3, CSRP1, CTGF, CTSK, CYBRD1, CYTSB, DBN1, DCN, DES, DIO2, ECM1, EFEMP1, ELN, ENAH, F3, FAM109B, FAM126A, FAM129A, FAM129B, FAM83D, FBLN1, FBLN5, FBLN7, FBN1, FEZ1, FGD1, FGF14, FGF1, FGF7, FHL3, FLRT2, FMO3, FMOD, FN1, FNDC1, FOXP1, FOXS1, FZD7, GALNTL1, GAS1, GATA6, GEFT, GEM, GLT8D2, GNAO1, GPC3, GREM1, GRID1, H19, HDGFRP3, HOPX, HTR2B, IGF2, IGFBP2, IGFBP7, IL18R1, IL1R1, INHBA, INMT, ITGBL1, ITPRIPL2, KCNMB1, KIAA1199, KIAA1211, KIAA1755, KRT7, LAMA2, LARGE, LDB3, LEPREL2, LGI2, LIPC, LMCD1, LOC145820, LOC399959, LOC401093, LOXL1, LPHN1, LPHN3, LRFN3, LTBP1, LTBP4, LUM, LYNX1, MAGED4B, MAP1A, MAP6, MARK1, MARVELD1, MDGA1, MEIS3, MEST, MFAP2, MFAP4, MFGE8, MLPH, MMP11, MOXD1, MRC2, MRGPRF, MRVI1, MSRB3, MUSTN1, MYH10, MYH11, MYL9, MYO10, MYOID, NACAD, NAP1L3, NAV3, NDRG4, NEXN, NFASC, NOV, NPTXR, NR2F2, NT5DC2, NXN, OXTR, PCDH7, PCOLCE, PDE1A, PDE5A, PDLIM3, PDLIM7, PGR, PHYHD1, PID1, PLEKHA4, PLN, POSTN, PPP1R12B, PPP1R14A, PRR15L, PRRX1, PTGIR, PTGIS, PTK7, PVRL1, RAB23, RAB31, RAMP1, RBP1, RGMA, RGS16, RGS4, RICH2, ROR2, SCARF2, SCG5, SETMAR, SFRP1, SFRP2, SFRP4, SGCD, SHISA3, SLC20A2, SLC24A3, SLC2A10, SLIT2, SLIT3, SMOC2, SNAI1, SOBP, SOD3, S0RCS3, SPAG1, SPEG, SRPX2, SRPX, SSC5D, ST5, ST6GALNAC6, SULF1, SVEP1, SYT17, TACSTD2, TCF21, TGFB3, THBS2, TIMP2, TMEM117, TMEM119, TMEM30B, TMEM90B, TNC, TNFAIP8L3, TNXB, TPM2, TPM4, TRAM2, TSPAN2, VANGL2, VCL, VGLL3, WFDC1, ZAK, ZBTB47, ZCCHC24, ZNF469, ZNF503, ZNF703, ZNF853, ANTXR1, ASPN, CCDC80, COL14A1, COL1A1, COL1A2, COL3A1, COL5A1, DACT1, DACT3, EMILIN1, FAP, FBLIM1, GGT5, HSPB6, ISLR, ITGA11, LHFP, LMOD1, LTBP2, MGP, MICAL2, PALLD, PDGFRA, PODN, PRELP, SYNPO2, and TAGLN.
[0121] In some embodiments, detection can occur through any of a variety of mobility dependent analytical techniques based on the differential rates of migration between different nucleic acid sequences. Exemplary mobility-dependent analysis techniques include electrophoresis, chromatography, mass spectroscopy, sedimentation, gradient centrifugation, field-flow fractionation, multi-stage extraction techniques, and the like. In some embodiments, mobility probes can be hybridized to amplification products, and the identity of the target nucleic acid sequence determined via a mobility dependent analysis technique of the eluted mobility probes, as described in Published PCT Applications WO04/46344 and WOO 1/92579. In some embodiments, detection can be achieved by various microarrays and related software such as the Applied Biosystems Array System with the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer and other commercially available array systems available from Affymetrix, Agilent, Illumina, and Amersham Biosciences, among others (see also Gerry et al., J. Mol. Biol. 292:251-62, 1999; De Bellis et al., Minerva Biotec 14:247-52, 2002; and Stears et al., Nat. Med. 9:14045, including supplements, 2003).
[ 01221 It is also understood that detection can comprise reporter groups that are incorporated into the reaction products, either as part of labeled primers or due to the incorporation of labeled dNTPs during an amplification, or attached to reaction products, for example but not limited to, via hybridization tag complements comprising reporter groups or via linker arms that are integral or attached to reaction products. In some embodiments, unlabeled reaction products may be detected using mass spectrometry.
NGS Platforms
[0123] In some embodiments, high throughput, massively parallel sequencing employs sequencing-by-synthesis with reversible dye terminators. In other embodiments, sequencing is performed via sequencing-by-ligation. In yet other embodiments, sequencing is single molecule sequencing. Examples of Next Generation Sequencing techniques include, but are not limited to pyrosequencing, Reversible dye-terminator sequencing, SOLiD sequencing, Ion semiconductor sequencing, Helioscope single molecule sequencing etc.
[0124] The Ion Torrent™ (Life Technologies, Carlsbad, CA) amplicon sequencing system employs a flow-based approach that detects pH changes caused by the release of hydrogen ions during incorporation of unmodified nucleotides in DNA replication. For use with this system, a sequencing library is initially produced by generating DNA fragments flanked by sequencing adapters. In some embodiments, these fragments can be clonally amplified on particles by emulsion PCR. The particles with the amplified template are then placed in a silicon semiconductor sequencing chip. During replication, the chip is flooded with one nucleotide after another, and if a nucleotide complements the DNA molecule in a particular microwell of the chip, then it will be incorporated. A proton is naturally released when a nucleotide is incorporated by the polymerase in the DNA molecule, resulting in a detectable local change of pH. The pH of the solution then changes in that well and is detected by the ion sensor. If homopolymer repeats are present in the template sequence, multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.
[0125] The 454TM GS FLX ™ sequencing system (Roche, Germany), employs a light-based detection methodology in a large-scale parallel pyrosequencing system. Pyrosequencing uses DNA polymerization, adding one nucleotide species at a time and detecting and quantifying the number of nucleotides added to a given location through the light emitted by the release of attached pyrophosphates. For use with the 454™ system, adapter-ligated DNA fragments are fixed to small DNA-capture beads in a water-in-oil emulsion and amplified by PCR (emulsion PCR). Each DNA-bound bead is placed into a well on a picotiter plate and sequencing reagents are delivered across the wells of the plate. The four DNA nucleotides are added sequentially in a fixed order across the picotiter plate device during a sequencing run. During the nucleotide flow, millions of copies of DNA bound to each of the beads are sequenced in parallel. When a nucleotide complementary to the template strand is added to a well, the nucleotide is incorporated onto the existing DNA strand, generating a light signal that is recorded by a CCD camera in the instrument. [0126] Sequencing technology based on reversible dye-terminators: DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed. Four types of reversible terminator bases (RT -bases) are added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. A camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle.
[0127] Helicos's single-molecule sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface. At each cycle, DNA polymerase and a single species of fluorescently labeled nucleotide are added, resulting in template-dependent extension of the surface-immobilized primer-template duplexes. The reads are performed by the Helioscope sequencer. After acquisition of images tiling the full array, chemical cleavage and release of the fluorescent label permits the subsequent cycle of extension and imaging.
[0128] Sequencing by synthesis (SBS), like the "old style" dye-termination electrophoretic sequencing, relies on incorporation of nucleotides by a DNA polymerase to determine the base sequence. A DNA library with affixed adapters is denatured into single strands and grafted to a flow cell, followed by bridge amplification to form a high-density array of spots onto a glass chip. Reversible terminator methods use reversible versions of dye-terminators, adding one nucleotide at a time, detecting fluorescence at each position by repeated removal of the blocking group to allow polymerization of another nucleotide. The signal of nucleotide incorporation can vary with fluorescently labeled nucleotides, phosphate-driven light reactions and hydrogen ion sensing having all been used. Examples of SBS platforms include Illumina GA and HiSeq 2000. The MiSeq® personal sequencing system (Illumina, Inc.) also employs sequencing by synthesis with reversible terminator chemistry.
(0129] In contrast to the sequencing by synthesis method, the sequencing by ligation method uses a DNA ligase to determine the target sequence. This sequencing method relies on enzymatic ligation of oligonucleotides that are adjacent through local complementarity on a template DNA strand. This technology employs a partition of all possible oligonucleotides of a fixed length, labeled according to the sequenced position. Oligonucleotides are annealed and ligated and the preferential ligation by DNA ligase for matching sequences results in a dinucleotide encoded color space signal at that position (through the release of a fluorescently labeled probe that corresponds to a known nucleotide at a known position along the oligo). This method is primarily used by Life Technologies’ SOLiD™ sequencers. Before sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing only copies of the same DNA molecule, are deposited on a solid planar substrate.
10.1301 SMRT™ sequencing is based on the sequencing by synthesis approach. The DNA is synthesized in zero-mode wave-guides (ZMWs)-small well-like containers with the capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labeled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring at the bottom of the well is detected. The fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.
Methods for Selecting Intraoperative Analgesic Regimen in Urological Cancer Patients Undergoing Tumor Resection Surgery
[0131] In one aspect, the present disclosure provides a method for selecting a cancer patient undergoing tumor resection surgery for renal cancer for treatment with an intraoperative opioid analgesic comprising (a) detecting expression levels of at least one survival-associated gene expression network in a biological sample obtained from the cancer patient that are either comparable or decreased relative to a control sample obtained from a healthy subject or a predetermined threshold, wherein the at least one survival-associated gene expression network is a NRF2-dependent macrophage network and/or a Th2 immune network; and (b) administering to the cancer patient an effective amount of an intraoperative opioid analgesic during the tumor resection surgery. The expression levels of the at least one survival-associated gene expression network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH). In some embodiments, the biological sample comprises genomic DNA, cDNA, RNA, and/or mRNA. [0132] In one aspect, the present disclosure provides a method for prolonging survival of a cancer patient undergoing tumor resection surgery for renal cancer comprising administering to the cancer patient an effective amount of an intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a NRF2-dependent macrophage network and/or a Th2 immune network in a biological sample obtained from the cancer patient are either comparable or decreased relative to a control sample obtained from a healthy subject or a predetermined threshold. Additionally or alternatively, in some embodiments, expression levels of the NRF2-dependent macrophage network and/or the Th2 immune network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
[0133] In any of the preceding embodiments of methods disclosed herein, the intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. The effective amount of the intraoperative opioid analgesic may range from about 1 MME to about 200 MMEs. In certain embodiments, the effective amount of the intraoperative opioid analgesic is about 1 MME to about 20 MMEs, about 20 MMEs to about 45 MMEs, or about 45 MMEs to about 200 MMEs. In some embodiments, the effective amount of the intraoperative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35 MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45 MMEs, about 45-50 MMEs, about 50-55 MMEs, about 55-60 MMEs, about 60-65 MMEs, about 65-70 MMEs, about 70-75 MMEs, about 75-80 MMEs, about 80-85 MMEs, about 85-90 MMEs, about 90-95 MMEs, about 95-100 MMEs, about 100-110 MMEs, about 110-120 MMEs, about 120-130 MMEs, about 130-140 MMEs, about 140-150 MMEs, about 150-160 MMEs, about 160-170 MMEs, about 170-180 MMEs, about 180-190 MMEs, or about 190-200 MMEs. Additionally or alternatively, in some embodiments, the effective amount of the intraoperative opioid analgesic is administered as a series of bolus doses or as a continuous infusion during the tumor resection surgery. In certain embodiments, the effective amount of the intraoperative opioid analgesic is administered to the cancer patient prior to incision. Additionally or alternatively, in some embodiments, the effective amount of the intraoperative opioid analgesic is administered intravenously.
[0134] Additionally or alternatively, in some embodiments, the methods of the present technology further comprise administering to the cancer patient an effective amount of a local anesthetic solution via an epidural catheter before, during and/or after the tumor resection surgery. The effective amount of the local anesthetic solution may range from about 0.05%- 4% local anesthetic solution in a volume of 1-10 ml per hour when administered via an epidural catheter. In some embodiments, the effective amount of the local anesthetic solution is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about 1.2 %, about
1.3 %, about 1.4 %, about 1.5 %, about 1.6 %, about 1.7 %, about 1.8 %, about 1.9 %, about
2.0 %, about 2.1 %, about 2.2 %, about 2.3 %, about 2.4 %, about 2.5 %, about 2.6 %, about
2.7 %, about 2.8 %, about 2.9 %, about 3.0 %, about 3.1 %, about 3.2 %, about 3.3 %, about
3.4 %, about 3.5 %, about 3.6 %, about 3.7 %, about 3.8 %, about 3.9 %, or about 4.0 % local anesthetic solution in a volume of about 1 ml per hour, about 1.5 ml per hour, about 2 ml per hour, about 2.5 ml per hour, about 3 ml per hour, about 3.5 ml per hour, about 4 ml per hour, about 4.5 ml per hour, about 5 ml per hour, about 5.5 ml per hour, about 6 ml per hour, about
6.5 ml per hour, about 7 ml per hour, about 7.5 ml per hour, about 8 ml per hour, about 8.5 ml per hour, about 9 ml per hour, about 9.5 ml per hour, or about 10 ml per hour when administered via an epidural catheter. Additionally or alternatively, in some embodiments, the effective amount of the local anesthetic solution is administered as a series of bolus doses or as a continuous infusion during the tumor resection surgery. Examples of suitable local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine.
[0135] In other embodiments, the effective amount of the local anesthetic solution may be administered before, during and/or after the tumor resection surgery using any regional anesthesia technique directed at nerves innervating the thorax and chest wall (e.g., via serratus plane nerve block, intercostal nerve block, or paravertebral block). The effective amount of the local anesthetic solution may range from about 0.05%-4% local anesthetic solution in a volume of 10-40 ml when administered using any regional anesthesia technique directed at nerves innervating the thorax and chest wall (e.g., via serratus plane nerve block, intercostal nerve block, or paravertebral block). In some embodiments, the effective amount of the local anesthetic solution is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about 1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6 %, about 1.7 %, about 1.8 %, about 1.9 %, about 2.0 %, about 2.1 %, about 2.2 %, about 2.3 %, about 2.4 %, about 2.5 %, about 2.6 %, about 2.7 %, about 2.8 %, about 2.9 %, about 3.0 %, about 3.1 %, about 3.2 %, about 3.3 %, about 3.4 %, about 3.5 %, about 3.6 %, about 3.7 %, about 3.8 %, about 3.9 %, or about 4.0 % local anesthetic solution in a volume of about 10 ml, about 12.5 ml, about 15 ml, about 17.5 ml, about 20 ml, about 22.5 ml, about 25 ml, about 27.5 ml, about 30 ml, about 32.5 ml, about 35 ml, about 37.5 ml, or about 40 ml when administered using any regional anesthesia technique directed at nerves innervating the thorax and chest wall (e.g., via serratus plane nerve block, intercostal nerve block, or paravertebral block). Examples of suitable local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine.
[0136] Additionally or alternatively, in certain embodiments, the local anesthetic solution may further comprise an opioid (e.g., fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil). In some embodiments, the local anesthetic solution may comprise 0.5 mcg/ml-50 mcg/ml opioid. In certain embodiments, the local anesthetic solution may comprise about 0.5 mcg/ml, about 0.6 mcg/ml, about 0.7 mcg/ml, about 0.8 mcg/ml, about 0.9 mcg/ml, about 1.0 mcg/ml, about 1.5 mcg/ml, about 2.0 mcg/ml, about 2.5 mcg/ml, about 3.0 mcg/ml, about 3.5 mcg/ml, about 4.0 mcg/ml, about 4.5 mcg/ml, about 5.0 mcg/ml, about 5.5 mcg/ml, about 6.0 mcg/ml, about 6.5 mcg/ml, about 7.0 mcg/ml, about 7.5 mcg/ml, about 8.0 mcg/ml, about 8.5 mcg/ml, about 9.0 mcg/ml, about 10 mcg/ml, about 15 mcg/ml, about 20 mcg/ml, about 25 mcg/ml, about 30 mcg/ml, about 35 mcg/ml, about 40 mcg/ml, about 45 mcg/ml, or about 50 mcg/ml.
[0137] Additionally or alternatively, in certain embodiments, the methods of the present technology further comprise administering to the cancer patient an effective amount of a post-operative opioid analgesic after the tumor resection surgery. Examples of postoperative opioid analgesics include, but are not limited to, fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. In some embodiments, the post-operative opioid analgesic and the intraoperative opioid analgesic are the same opioid analgesic or different opioid analgesics. In other embodiments, the effective amount of the post-operative opioid analgesic and the effective amount of the intraoperative opioid analgesic are the same or different. Additionally or alternatively, in some embodiments, the effective amount of the post-operative opioid analgesic is administered to the cancer patient as a bolus of about 0.005 mg to about 100 mg. In some embodiments, the effective amount of the post-operative opioid analgesic is administered to the cancer patient as a bolus of about 0.005 mg, about 0.006 mg, about 0.007 mg, about 0.008 mg, about 0.009 mg, about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1-5 mg, about 5-10 mg, about 1-5 mg, about 5-10 mg, about 1-5 mg, about 5-10 mg, about 1-5 mg, about 5-10 mg, about 10-15 mg, about 15-20 mg, about 20-25 mg, about 25-30 mg, about 30-35 mg, about 35-40 mg, about 40-45 mg, about 45-50 mg, about 50-55 mg, about 55-60 mg, about 60-65 mg, about 65-70 mg, about 70-75 mg, about 75-80 mg, about 80-85 mg, about 85-90 mg, about 90-95 mg, or about 95-100 mg. In other embodiments, the effective amount of the post-operative opioid analgesic may be continuously delivered to the cancer patient at a per hour rate of about 0.01 mg/hr to about 10 mg/hr. In certain embodiments, the effective amount of the post-operative opioid analgesic is continuously delivered to the cancer patient at a per hour rate of about 0.01 mg/hr, about 0.02 mg/hr, about 0.03 mg/hr, about 0.04 mg/hr, about 0.05 mg/hr, about 0.06 mg/hr, about 0.07 mg/hr, about 0.08 mg/hr, about 0.09 mg/hr, about 0.1 mg/hr, about 0.2 mg/hr, about 0.3 mg/hr, about 0.4 mg/hr, about 0.5 mg/hr, about 0.6 mg/hr, about 0.7 mg/hr, about 0.8 mg/hr, about 0.9 mg/hr, about 1 mg/hr, about 1.5 mg/hr, about 2 mg/hr, about 2.5 mg/hr, about 3 mg/hr, about 3.5 mg/hr, about 4 mg/hr, about 4.5 mg/hr, about 5 mg/hr, about 5.5 mg/hr, about 6 mg/hr, about 6.5 mg/hr, about 7 mg/hr, about 7.5 mg/hr, about 8 mg/hr, about 8.5 mg/hr, about 9 mg/hr, about 9.5 mg/hr, or about 10 mg/hr. Additionally or alternatively, in some embodiments, the effective amount of the post-operative opioid analgesic is administered intravenously, orally, or transdermally.
[0138] In one aspect, the present disclosure provides a method for selecting a cancer patient undergoing tumor resection surgery for renal cancer for treatment with an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic comprising (a) detecting expression levels of at least one survival-associated gene expression network in a biological sample obtained from the cancer patient that are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold, wherein the at least one survival-associated gene expression network is a NRF2-dependent macrophage network and/or a Th2 immune network; and (b) administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery. The expression levels of the at least one survival-associated gene expression network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH). In some embodiments, the biological sample comprises genomic DNA, cDNA, RNA, and/or mRNA.
[0139] In one aspect, the present disclosure provides a method for prolonging survival of a cancer patient undergoing tumor resection surgery for renal cancer comprising administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a NRF2-dependent macrophage network and/or a Th2 immune network in a biological sample obtained from the cancer patient are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold. Additionally or alternatively, in some embodiments, expression levels of the NRF2-dependent macrophage network and/or the Th2 immune network may be detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
[0140| In any of the preceding embodiments of methods disclosed herein, the low- dose intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. Additionally or alternatively, in some embodiments, the effective amount of the low-dose intraoperative opioid analgesic is about 1 MME to about 50 MMEs. In some embodiments, the effective amount of the low- dose intraoperative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25
MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30
MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35
MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45
MMEs, or about 45-50 MMEs.
101411 Additionally or alternatively, in some embodiments of the methods disclosed herein, the opioid-free intraoperative analgesic is an amide-type local anesthetic or an ester- type local anesthetic. Examples of amide-type local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, or levobupivacaine. Examples of ester-type local anesthetics include, but are not limited to, cocaine, procaine, tetracaine, chloroprocaine, or benzocaine. The opioid-free intraoperative analgesic may be administered via an epidural catheter. Additionally or alternatively, in some embodiments of the methods disclosed herein, the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 1-10 ml per hour when administered via an epidural catheter. In certain embodiments, the effective amount of the opioid-free intraoperative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about 1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6 %, about 1.7 %, about 1.8 %, about 1.9 %, about 2.0 %, about 2.1 %, about 2.2 %, about 2.3 %, about 2.4 %, about 2.5 %, about 2.6 %, about 2.7 %, about 2.8 %, about 2.9 %, about 3.0 %, about 3.1 %, about 3.2 %, about 3.3 %, about 3.4 %, about 3.5 %, about 3.6 %, about 3.7 %, about 3.8 %, about 3.9 %, or about 4.0 % amide-type or ester-type local anesthetic solution in a volume of about 1 ml per hour, about 1.5 ml per hour, about 2 ml per hour, about 2.5 ml per hour, about 3 ml per hour, about 3.5 ml per hour, about 4 ml per hour, about 4.5 ml per hour, about 5 ml per hour, about 5.5 ml per hour, about 6 ml per hour, about 6.5 ml per hour, about 7 ml per hour, about 7.5 ml per hour, about 8 ml per hour, about 8.5 ml per hour, about 9 ml per hour, about 9.5 ml per hour, or about 10 ml per hour when administered via an epidural catheter.
[0142] In certain embodiments, the opioid-free intraoperative analgesic may be administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block. In some embodiments, the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 10-40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block. In some embodiments, the effective amount of the opioid-free intraoperative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about 1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6
%, about 1.7 %, about 1.8 %, about 1.9 %, about 2.0 %, about 2.1 %, about 2.2 %, about 2.3
%, about 2.4 %, about 2.5 %, about 2.6 %, about 2.7 %, about 2.8 %, about 2.9 %, about 3.0
%, about 3.1 %, about 3.2 %, about 3.3 %, about 3.4 %, about 3.5 %, about 3.6 %, about 3.7
%, about 3.8 %, about 3.9 %, or about 4.0 % amide-type or ester-type local anesthetic solution in a volume of about 10 ml, about 12.5 ml, about 15 ml, about 17.5 ml, about 20 ml, about 22.5 ml, about 25 ml, about 27.5 ml, about 30 ml, about 32.5 ml, about 35 ml, about 37.5 ml, or about 40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
[0143] Additionally or alternatively, in certain embodiments of the methods disclosed herein, the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic may be administered as a series of bolus doses, or as a continuous infusion during the tumor resection surgery.
10144] Additionally or alternatively, in some embodiments, the methods of the present technology further comprise administering to the cancer patient an effective amount of an opioid-free post-operative analgesic or a low-dose post-operative opioid analgesic after the tumor resection surgery. Examples of suitable opioid-free post-operative analgesics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine. Examples of suitable low-dose post-operative opioid analgesics include, but are not limited to, fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. In some embodiments, the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same analgesic or different analgesics. In other embodiments, the effective amount of the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same or different. [0145] In some embodiments, the effective amount of the low-dose post-operative opioid analgesic is about 1 MME to about 50 MMEs. In some embodiments, the effective amount of the low-dose post-operative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35 MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45 MMEs, or about 45-50 MMEs.
[0146] In some embodiments, the effective amount of the opioid-free post-operative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about
1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6 %, about 1.7 %, about 1.8 %, about
1.9 %, about 2.0 %, about 2.1 %, about 2.2 %, about 2.3 %, about 2.4 %, about 2.5 %, about
2.6 %, about 2.7 %, about 2.8 %, about 2.9 %, about 3.0 %, about 3.1 %, about 3.2 %, about
3.3 %, about 3.4 %, about 3.5 %, about 3.6 %, about 3.7 %, about 3.8 %, about 3.9 %, or about 4.0 % amide-type or ester-type local anesthetic solution in a volume of about 10 ml, about 12.5 ml, about 15 ml, about 17.5 ml, about 20 ml, about 22.5 ml, about 25 ml, about 27.5 ml, about 30 ml, about 32.5 ml, about 35 ml, about 37.5 ml, or about 40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
[0147] In any and all of the preceding embodiments of the methods disclosed herein, the NRF2-dependent macrophage network comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 410, at least 415, at least 420, at least 425, at least 430, at least 435, at least 440, at least 445, at least 450, at least 455, at least 460, at least 465, at least 470, at least 475, at least 480, at least 485, at least 490, at least 495, at least 500, at least 510, at least 515, at least 520, at least 525, at least 530, at least 535, at least 540, at least 545, at least 550, at least 555, at least 560, at least 565, at least 570, at least 575, at least 580, at least 585, at least 590, at least 595, at least 600, at least 610, at least 615, at least 620, at least 625, at least 630, at least 635, at least 640, at least 645, or at least 650 or more genes selected from among A1CF, AB AT, ABCB1, ABCB9, ABCC2, ABCC6P1, ABCG1, ABHD6, ABLIM3, ABP1, ACAD11, ACADL, ACAT1, ACBD4, ACO2, ACOT4, ACOX2, ACSL1, ACSM5, ACY1, ADAMTS3, ADH6, AGPAT3, AGT, AIFM1, AKR1C1, AKR1C2, AKR7A2, AKR7A3, ALAD, ALDH1A1, ALDH1A2, ALDH1L1, ALDH2, ALDH3A2, ALDH4A1, ALDH7A1, ALDH8A1, ALDOB, ALPK2, ALPL, AMDHD1, ANK3, ANKRD56, ANPEP, ANXA13, ANXA2P2, ANXA2, AOX1, APITD1, AQP3, AQP9, ARHGAP1, ARHGAP24, ARL4C, ASB13, ASRGL1, ASTN2, ATP6V0A1, AUH, AXL, AZGP1, B3GNT7, B4GALNT1, B4GALT1, BACE2, BAIAP2L1, BAIAP2L2, BAMBI, BARX2, BCAT1, BCMO1, BDH2, BDKRB2, BEND3, BHMT2, BHMT, BNIP3, BPHL, BTG3, C10orfl08, Cl lorf45, Cl lorf52, C14orf64, C14orf73, C17orf51, C18orfl8, C19orf77, C1RL, C1R, CIS, Clorfl l5, Clorf201, Clorf203, Clorf210, Clorf21, Clorf89, Clorf96, C21orf7, C22orf45, C2orf24, C2orf67, C3, C4orfl9, C5orf23, C6, C7orfl0, C8orf47, C9orfl25, CABCI, CABLES1, CADM3, CALML4, CBR4, CBWD1, CBX7, CCDC146, CCDC64, CCDC68, CD276, CD44, CD47, CD55, CD82, CDADC1, CDC42SE2, CDCA2, CDCP1, CDH16, CDH2, CDHR2, CDHR5, CDK18, CDK20, CDON, CEACAM1, CES2, CFB, CGREF1, CHDH, CHI3L1, CHPF2, CHST13, CIDEB, CISH, CIT, CKAP4, CLDN10, CLDN2, CLEC18A, CLEC18B, CLEC18C, CLIC6, CLPTM1L, CMBL, CNDP2, CNNM1, COBL, COL22A1, COL23A1, COL4A5, COL8A2, COLEC12, COPG2, CPT2, CRAT, CRB3, CREB3L3, CRY2, CRYM, CRYZ, CTHRC1, CYB5A, CYB5D1, CYP1B1, CYP27A1, CYP2J2, CYP4V2, CYS1, DAB2, DAPK2, DCBLD2, DDAH1, DEPDC7, DGKG, DHDH, DHTKD1, DIRAS2, DLG5, DMGDH, DMRTA1, DPF3, DPYS, DSEL, ECHDC3, EFNA5, EGOT, ELFN2, EMX1, EMX2OS, EMX2, ENAM, ENPEP, ENPP3, EPB49, EPHA7, EPHX2, ERBB3, ETFA, ETFDH, ETNK2, ETV1, ETV6, EZR, FAAH, FABP3, FAHD1, FAM149A, FAM164C, FAM60A, FAM69A, FANCC, FBP1, FBXL16, FBXO32, FCAMR, FGFR1OP, FGFR3, FGGY, FHL2, FLJ23867, FLJ36031, FLNC, FMO4, FNDC4, FREM2, FTCD, GAL3ST1, GALM, GALNT2, GALNT7, GATM, GATS, GBAS, GDA, GFPT2, GGT3P, GJB1, GK, GLB1L, GLIS1, GLRB, GLT25D1, GOT1, GPD1, GPER, GPT, GPX3, GPX8, GRAMD1C, GRTP1, GSTA1, GSTA2, GXYLT2, GYLTL1B, HAAO, HABP2, HABP4, HGD, HHLA2, HIBCH, HIGD1A, HLF, HMGCL, HMOX1, HNF4A, HOXCIO, HSDL2, HSP90B1, HSPA2, HSPB8, HYAL1, HYOU1, IDH1, IGDCC4, IGF2BP2, IL17RB, IL1R2, IL22RA1, IMPA2, IMPDH1, IRS2, ITGB6, JPH2, KCNIP3, KCNJ15, KCNJ16, KCNS3, KCTD17, KCTD1, KIAA1543, KLC4, KLF15, KLHDC7A, KL, KRT19, KRT80, KSR1, LAD1, LAMA3, LAMB1, LAMB3, LAMC2, LDHD, LEF1, LIMK2, LNP1, LOC100126784, LOC100131551, LOC151534, LOC388387, LOC389332, LOC723809, LRG1, LRRC19, LRRC8E, LYG1, MAF, MAGED1, MAN1C1, MAOB, MAP7, MAPK8IP1, MAPT, MARVELD3, METTL7A, METTL7B, MFI2, MINA, MLYCD, MMD, MME, MMP14, MMP7, MMP9, MOBKL2B, MOSC2, MPI, MPV17L, MPZL1, MSRA, MTHFD1L, MTHFD2, MUC1, MXRA8, MYO1E, MYO3A, MYO7B, MYOM3, NAMPT, NAP1L1, NAPSA, NEFL, NGEF, NHEJ1, NIPSNAP1, NOMO1, NOMO3, NPR3, NRXN2, NTN4, NUDT6, OPN3, 0SCP1, OSTalpha, PANK1, PAPP A, PAQR7, PARD6B, PBLD, PBX3, PCBD2, PCCA, PCK1, PCOLCE2, PC, PDE10A, PDIA3P, PDIA4, PDIA5, PDK2, PDXP, PDZD3, PDZK1P1, PECI, PECR, PEPD, PER3, PGPEP1, PHYH, PIGT, PIPOX, PKHD1, PLA2G4C, PL AU, PLIN2, PLIN3, PLOD2, PLTP, PMAIP1, PNMA6A, PON2, PPFIBP2, PPL, PPP1R14C, PRODH2, PRODH, PSD3, PTGER2, PTGFRN, PTGR2, PTH1R, PTH2R, PVR, PXMP2, QDPR, QRFPR, QSOX1, RAB17, RAB3IP, RAB7L1, RAI2, RARRES1, RCN1, RGN, RGS14, RHOBTB1, RIT1, RND3, RNF5P1, RORC, RPN2, RUNDC3B, RUNX1, RUNX2, SAMD5, SATB2, SCARB1, SCD, SCGN, SCLY, SCNN1A, SEMA3C, SEMA3D, SEMA4B, SEMA4F, SEMA6A, SEPHS2, SEPSECS, SERPINA3, SERPINA6, SERPINF1, SERPINF2, SGSM1, SH3BGRL2, SH3PXD2B, SH3YL1, SHISA4, SHMT1, SLC10A2, SLC12A7, SLC16A12, SLC16A13, SLC16A4, SLC16A5, SLC17A4, SLC1A1, SLC22A2, SLC22A4, SLC22A5, SLC23A1, SLC25A23, SLC25A34, SLC25A42, SLC25A44, SLC26A1, SLC28A1, SLC2A2, SLC2A5, SLC2A9, SLC38A5, SLC3A1, SLC46A1, SLC5A12, SLC5A1, SLC5A8, SLC5A9, SLC6A12, SLC6A19, SLC6A3, SLC7A5, SLC9A1, SLC9A3R1, SLC04C1, SLITRK2, SLITRK4, SLPI, SMPDL3A, SMTNL2, SPATAI 8, SPATS2L, SPNS2, SP0CK1, SPON2, SQLE, STAMBPL1, STEAP3, STK17A, STK32B, STK39, STON2, STX3, SULF2, SYBU, SYT12, SYT9, SYTL2, TBC1D2, TCEA3, TCFL5, TCN2, TCTA, TEF, TFEC, TFPI2, TGFBI, THAP9, THSD4, TLN2, TMCC1, TMCO4, TMEM125, TMEM130, TMEM139, TMEM140, TMEM164, TMEM171, TMEM176A, TMEM176B, TMEM195, TMEM26, TMEM37, TMEM45A, TMPRSS3, TNFAIP6, TNFRSF10C, TNFRSF21, TPBG, TPMT, TPST2, TRAP1, TRIM55, TRPV4, TSGA14, TSPAN1, TTC39C, TUBB3, UBA5, UGT1A6, UGT1A9, UGT2A3, UGT2B7, UGT3A1, USP2, VCAM1, VIL1, WDR72, WDR81, WFDC2, WFS1, WNT5A, WNT5B, ZBTB7C, ZFAT, ZFHX4, ZNF385B, ZNF711, ZSCAN2, ABCC6P2, ABCC6, ACAA2, ACE2, ACMSD, ACSM2A, ACSM2B, ACY3, ADM2, AGMAT, AGXT2, AMN, ANKS4B, APOM, ASP A, BBOX1, Cl lorf54, C9orf66, CLCN5, CLRN3, CRYL1, CUBN, CYP4A11, DDC, EHHADH, FMO1, FUT6, GBA3, GIPC2, GLYATL1, GLYAT, HAO2, HNF1A, HRSP12, KHK, LGALS2, LRP2, MIOX, NAT8, PDZK1, PHYHIPL, PKLR, RBP5, SLC13A1, SLC16A9, SLC17A1, SLC17A3, SLC22A11, SLC22A12, SLC23A3, SLC27A2, SLC37A4, SLC39A5, SLC47A1, SLC5A10, SLC6A13, SLC7A9, TINAG, TM4SF5, TMEM27, TRIM10, TRIM15, TRPM3, TTC38, UPB1, and USH1C.
[0148] In any and all of the preceding embodiments of the methods disclosed herein, the Th2 immune network comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 115, at least 120, at least 125, or at least 130 or more genes selected from among AJAP1, ANLN, ARHGAP11 A, ASF1B, ASPM, ATAD2, AURKA, BRCA1, BUB1, C10orf2, C13orf34, C15orf23, C16orf75, CACNA2D4, CCDC99, CCNA2, CCNB1, CCNF, CDC6, CDCA7, CDK1, CDT1, CENPE, CENPF, CENPH, CENPL, CENPN, CENPO, CHAF1A, CHAF1B, CHEK1, CPOX, CTSF, DBF4, DERL1, DHFR, DIAPH3, DTL, E2F1, ECT2, EPR1, ESPL1, EZH2, FAM111B, FANCA, FANCD2, FANCI, FASN, FEN1, GGH, GINS1, GINS2, GINS3, GPRIN1, GPSM2, HELLS, HMMR, HPS3, IQGAP3, KIAA0101, KIF11, KIF18B, KIF20A, KIF23, KIF24, KIF2C, KIF4A, KIFC1, KPNA2, LMNB1, LMNB2, LRP8, MAD2L1, MCM2, MCM5, MCM6, MCM7, MELK, MKI67, MLF1IP, MTMR4, MYBL2, NCAPD2, NCAPH, NDC80, NEB, NUSAP1, PAQR4, PBXIP1, PLK4, PPARG, PPRC1, PRC1, PTTG1, RACGAP1, RAD51AP1, RFC4, RIPK2, RRP12, SG0L2, SHCBP1, SMC4, SPATA7, STIL, STMN1, TACC3, TCF19, TIMELESS, TK1, TMEM25, TRIM59, TRIP13, TUBB, UBE2C, UHRF1, WDR4, WDR67, WSCD1, ZWILCH, ZWINT, BUB IB, CCNB2, CDC20, CDCA5, CDCA8, CEP55, FOXM1, GTSE1, HJURP, NCAPG, PLK1, RRM2, TOP2A, and TPX2.
[0149] Additionally or alternatively, in some embodiments, the tumor resection surgery comprises nephrectomy. The renal cancer may have a histologic subtype selected from among clear cell renal cell carcinomas (ccRCC), papillary renal cell carcinomas (pRCC), chromophobe renal cell carcinomas (crRCC), multilocular cystic RCC, collecting duct carcinoma, medullary carcinoma, mucinous tubular and spindle cell carcinoma, neuroblastoma-associated RCC, Xpl l.2 translocation-TFE3 carcinoma, and unclassified lesions. Additionally or alternatively, in certain embodiments, the cancer patient exhibits stage I, stage II, stage III, or stage IV renal cancer.
[0150] In another aspect, the present disclosure provides a method for selecting a cancer patient undergoing tumor resection surgery for bladder cancer for treatment with an opioid- free intraoperative analgesic or a low-dose intraoperative opioid analgesic comprising (a) detecting expression levels of a survival-associated gene expression network in a biological sample obtained from the cancer patient that are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold; and (b) administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery, wherein the survival- associated gene expression network comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, or at least 275 or more genes selected from among ABCA6, ACTA2, ACTC1, ACTG2, ACTN1, ADAM12, ADAM I 9, ADAMTS2, ADH1B, ADRA2A, AEBP1, AG2, ALDH1L2, AMIGO2, ANGPTL1, ATP10A, ATP8B2, AXIN2, BAG2, Cl lorf41, C14orfl32, Clorfl98, C2orf40, C7, C9orfl l0, CACNA1H, CAMK1G, CAND2, CCDC3, CCL19, CCL21, CDH11, CES1, CFH, CLEC11A, CNN1, COL11A1, COL12A1, COL16A1, COL6A1, COL6A2, COL6A3, CORO2B, CPXM1, CPXM2, CPZ, CREB3L1, CRYBG3, CSRP1, CTGF, CTSK, CYBRD1, CYTSB, DBN1, DCN, DES, DI02, ECM1, EFEMP1, ELN, ENAH, F3, FAM109B, FAM126A, FAM129A, FAM129B, FAM83D, FBLN1, FBLN5, FBLN7, FBN1, FEZ1, FGD1, FGF14, FGF1, FGF7, FHL3, FLRT2, FMO3, FMOD, FN1, FNDC1, FOXP1, FOXS1, FZD7, GALNTL1, GAS1, GATA6, GEFT, GEM, GLT8D2, GNAO1, GPC3, GREM1, GRID1, H19, HDGFRP3, HOPX, HTR2B, IGF2, IGFBP2, IGFBP7, IL18R1, IL1R1, INHBA, INMT, ITGBL1, ITPRIPL2, KCNMB1, KIAA1199, KIAA1211, KIAA1755, KRT7, LAMA2, LARGE, LDB3, LEPREL2, LGI2, LIPC, LMCD1, LOC145820, LOC399959, LOC401093, LOXL1, LPHN1, LPHN3, LRFN3, LTBP1, LTBP4, LUM, LYNX1, MAGED4B, MAP1A, MAP6, MARK1, MARVELD1, MDGA1, MEIS3, MEST, MFAP2, MFAP4, MFGE8, MLPH, MMP11, MOXD1, MRC2, MRGPRF, MRVI1, MSRB3, MUSTN1, MYH10, MYH11, MYL9, MYO10, MYOID, NACAD, NAP1L3, NAV3, NDRG4, NEXN, NFASC, NOV, NPTXR, NR2F2, NT5DC2, NXN, OXTR, PCDH7, PCOLCE, PDE1A, PDE5A, PDLIM3, PDLIM7, PGR, PHYHD1, PID1, PLEKHA4, PLN, POSTN, PPP1R12B, PPP1R14A, PRR15L, PRRX1, PTGIR, PTGIS, PTK7, PVRL1, RAB23, RAB31, RAMP1, RBP1, RGMA, RGS16, RGS4, RICH2, ROR2, SCARF2, SCG5, SETMAR, SFRP1, SFRP2, SFRP4, SGCD, SHISA3, SLC20A2, SLC24A3, SLC2A10, SLIT2, SLIT3, SMOC2, SNAI1, SOBP, SOD3, SORCS3, SPAG1, SPEG, SRPX2, SRPX, SSC5D, ST5, ST6GALNAC6, SULF1, SVEP1, SYT17, TACSTD2, TCF21, TGFB3, THBS2, TIMP2, TMEM117, TMEM119, TMEM30B, TMEM90B, TNC, TNFAIP8L3, TNXB, TPM2, TPM4, TRAM2, TSPAN2, VANGL2, VCL, VGLL3, WFDC1, ZAK, ZBTB47, ZCCHC24, ZNF469, ZNF503, ZNF703, ZNF853, ANTXR1, ASPN, CCDC80, COL14A1, COL1A1, COL1A2, COL3A1, COL5A1, DACT1, DACT3, EMILIN1, FAP, FBLIM1, GGT5, HSPB6, ISLR, ITGA11, LHFP, LMOD1, LTBP2, MGP, MICAL2, PALLD, PDGFRA, PODN, PRELP, SYNPO2, and TAGLN. In some embodiments, the expression levels of the survival-associated gene expression network are detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligationdependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
10.1511 In another aspect, the present disclosure provides a method for prolonging survival of a cancer patient undergoing tumor resection surgery for bladder cancer comprising administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a survival-associated gene expression network in a biological sample obtained from the cancer patient are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold, wherein the survival-associated gene expression network comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, or at least 275 or more genes selected from among ABCA6, ACTA2, ACTC1, ACTG2, ACTN1, ADAM12, ADAM I 9, ADAMTS2, ADH1B, ADRA2A, AEBP1, AG2, ALDH1L2, AMIG02, ANGPTL1, ATP10A, ATP8B2, AXIN2, BAG2, Cl lorf41, C14orfl32, Clorfl98, C2orf40, C7, C9orfl l0, CACNA1H, CAMK1G, CAND2, CCDC3, CCL19, CCL21, CDH11, CES1, CFH, CLEC11A, CNN1, COL11A1, COL12A1, COL16A1, COL6A1, COL6A2, COL6A3, CORO2B, CPXM1, CPXM2, CPZ, CREB3L1, CRYBG3, CSRP1, CTGF, CTSK, CYBRD1, CYTSB, DBN1, DCN, DES, DIO2, ECM1, EFEMP1, ELN, ENAH, F3, FAM109B, FAM126A, FAM129A, FAM129B, FAM83D, FBLN1, FBLN5, FBLN7, FBN1, FEZ1, FGD1, FGF14, FGF1, FGF7, FHL3, FLRT2, FMO3, FMOD, FN1, FNDC1, FOXP1, FOXS1, FZD7, GALNTL1, GAS1, GATA6, GEFT, GEM, GLT8D2, GNAO1, GPC3, GREM1, GRID1, H19, HDGFRP3, HOPX, HTR2B, IGF2, IGFBP2, IGFBP7, IL18R1, IL1R1, INHBA, INMT, ITGBL1, ITPRIPL2, KCNMB1, KIAA1199, KIAA1211, KIAA1755, KRT7, LAMA2, LARGE, LDB3, LEPREL2, LGI2, LIPC, LMCD1, LOC145820, LOC399959, LOC401093, LOXL1, LPHN1, LPHN3, LRFN3, LTBP1, LTBP4, LUM, LYNX1, MAGED4B, MAP1A, MAP6, MARK1, MARVELD1, MDGA1, MEIS3, MEST, MFAP2, MFAP4, MFGE8, MLPH, MMP11, MOXD1, MRC2, MRGPRF, MRVI1, MSRB3, MUSTN1, MYH10, MYH11, MYL9, MYO10, MYOID, NACAD, NAP1L3, NAV3, NDRG4, NEXN, NFASC, NOV, NPTXR, NR2F2, NT5DC2, NXN, OXTR, PCDH7, PCOLCE, PDE1A, PDE5A, PDLIM3, PDLIM7, PGR, PHYHD1, PID1, PLEKHA4, PLN, POSTN, PPP1R12B, PPP1R14A, PRR15L, PRRX1, PTGIR, PTGIS, PTK7, PVRL1, RAB23, RAB31, RAMP1, RBP1, RGMA, RGS16, RGS4, RICH2, ROR2, SCARF2, SCG5, SETMAR, SFRP1, SFRP2, SFRP4, SGCD, SHISA3, SLC20A2, SLC24A3, SLC2A10, SLIT2, SLIT3, SMOC2, SNAI1, SOBP, SOD3, S0RCS3, SPAG1, SPEG, SRPX2, SRPX, SSC5D, ST5, ST6GALNAC6, SULF1, SVEP1, SYT17, TACSTD2, TCF21, TGFB3, THBS2, TIMP2, TMEM117, TMEM119, TMEM30B, TMEM90B, TNC, TNFAIP8L3, TNXB, TPM2, TPM4, TRAM2, TSPAN2, VANGL2, VOL, VGLL3, WFDC1, ZAK, ZBTB47, ZCCHC24, ZNF469, ZNF503, ZNF703, ZNF853, ANTXR1, ASPN, CCDC80, COL14A1, COL1A1, COL1A2, COL3A1, COL5A1, DACT1, DACT3, EMILIN1, FAP, FBLIM1, GGT5, HSPB6, ISLR, ITGA11, LHFP, LMOD1, LTBP2, MGP, MICAL2, PALLD, PDGFRA, PODN, PRELP, SYNPO2, and TAGLN. In certain embodiments, the expression levels of the survival-associated gene expression network are detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligationdependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
[0152] In any of the preceding embodiments of methods disclosed herein, the low- dose intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. Additionally or alternatively, in some embodiments, the effective amount of the low-dose intraoperative opioid analgesic is about 1 MME to about 50 MMEs. In some embodiments, the effective amount of the low- dose intraoperative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35 MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45 MMEs, or about 45-50 MMEs.
[0153] Additionally or alternatively, in some embodiments of the methods disclosed herein, the opioid-free intraoperative analgesic is an amide-type local anesthetic or an ester- type local anesthetic. Examples of amide-type local anesthetics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, or levobupivacaine. Examples of ester-type local anesthetics include, but are not limited to, cocaine, procaine, tetracaine, chloroprocaine, or benzocaine. The opioid-free intraoperative analgesic may be administered via an epidural catheter. Additionally or alternatively, in some embodiments of the methods disclosed herein, the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 1-10 ml per hour when administered via an epidural catheter. In certain embodiments, the effective amount of the opioid-free intraoperative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about 1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6 %, about 1.7 %, about 1.8 %, about 1.9 %, about 2.0 %, about 2.1 %, about 2.2 %, about 2.3 %, about 2.4 %, about 2.5 %, about 2.6 %, about 2.7 %, about 2.8 %, about 2.9 %, about 3.0 %, about 3.1 %, about 3.2 %, about 3.3 %, about 3.4 %, about 3.5 %, about 3.6 %, about 3.7 %, about 3.8 %, about 3.9 %, or about 4.0 % amide-type or ester-type local anesthetic solution in a volume of about 1 ml per hour, about 1.5 ml per hour, about 2 ml per hour, about 2.5 ml per hour, about 3 ml per hour, about 3.5 ml per hour, about 4 ml per hour, about 4.5 ml per hour, about 5 ml per hour, about 5.5 ml per hour, about 6 ml per hour, about 6.5 ml per hour, about 7 ml per hour, about 7.5 ml per hour, about 8 ml per hour, about 8.5 ml per hour, about 9 ml per hour, about 9.5 ml per hour, or about 10 ml per hour when administered via an epidural catheter.
[0154| In certain embodiments, the opioid-free intraoperative analgesic may be administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block. In some embodiments, the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 10-40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block. In some embodiments, the effective amount of the opioid-free intraoperative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about 1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6
%, about 1.7 %, about 1.8 %, about 1.9 %, about 2.0 %, about 2.1 %, about 2.2 %, about 2.3
%, about 2.4 %, about 2.5 %, about 2.6 %, about 2.7 %, about 2.8 %, about 2.9 %, about 3.0
%, about 3.1 %, about 3.2 %, about 3.3 %, about 3.4 %, about 3.5 %, about 3.6 %, about 3.7
%, about 3.8 %, about 3.9 %, or about 4.0 % amide-type or ester-type local anesthetic solution in a volume of about 10 ml, about 12.5 ml, about 15 ml, about 17.5 ml, about 20 ml, about 22.5 ml, about 25 ml, about 27.5 ml, about 30 ml, about 32.5 ml, about 35 ml, about 37.5 ml, or about 40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
[0155] Additionally or alternatively, in certain embodiments of the methods disclosed herein, the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic may be administered as a series of bolus doses, or as a continuous infusion during the tumor resection surgery.
[0156] Additionally or alternatively, in some embodiments, the methods of the present technology further comprise administering to the cancer patient an effective amount of an opioid-free post-operative analgesic or a low-dose post-operative opioid analgesic after the tumor resection surgery. Examples of suitable opioid-free post-operative analgesics include, but are not limited to, lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine. Examples of suitable low-dose post-operative opioid analgesics include, but are not limited to, fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil. In some embodiments, the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same analgesic or different analgesics. In other embodiments, the effective amount of the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same or different.
[0157] In some embodiments, the effective amount of the low-dose post-operative opioid analgesic is about 1 MME to about 50 MMEs. In some embodiments, the effective amount of the low-dose post-operative opioid analgesic is about 1 MME, about 2 MMEs, about 3 MMEs, about 4 MMEs, about 5 MMEs, about 6 MMEs, about 7 MMEs, about 8 MMEs, about 9 MMEs, about 10 MMEs, about 11 MMEs, about 12 MMEs, about 13 MMEs, about 14 MMEs, about 15 MMEs, about 16 MMEs, about 17 MMEs, about 18 MMEs, about 19 MMEs, about 20 MMEs, about 21 MMEs, about 22 MMEs, about 23 MMEs, about 24 MMEs, about 25 MMEs, about 26 MMEs, about 27 MMEs, about 28 MMEs, about 29 MMEs, about 30 MMEs, about 31 MMEs, about 32 MMEs, about 33 MMEs, about 34 MMEs, about 35 MMEs, about 36 MMEs, about 37 MMEs, about 38 MMEs, about 39 MMEs, about 40-45 MMEs, or about 45-50 MMEs.
[0158] In some embodiments, the effective amount of the opioid-free post-operative analgesic is about 0.05 %, about 0.06 %, about 0.07 %, about 0.08 %, about 0.09 %, about 0.1 %, about 0.15 %, about 0.2 %, about 0.25 %, about 0.3 %, about 0.35 %, about 0.4 %, about 0.45 %, about 0.5 %, about 0.55 %, about 0.6 %, about 0.65 %, about 0.7 %, about 0.75 %, about 0.8 %, about 0.85 %, about 0.9 %, about 0.95 %, about 1.0 %, about 1.1 %, about
1.2 %, about 1.3 %, about 1.4 %, about 1.5 %, about 1.6 %, about 1.7 %, about 1.8 %, about
1.9 %, about 2.0 %, about 2.1 %, about 2.2 %, about 2.3 %, about 2.4 %, about 2.5 %, about
2.6 %, about 2.7 %, about 2.8 %, about 2.9 %, about 3.0 %, about 3.1 %, about 3.2 %, about
3.3 %, about 3.4 %, about 3.5 %, about 3.6 %, about 3.7 %, about 3.8 %, about 3.9 %, or about 4.0 % amide-type or ester-type local anesthetic solution in a volume of about 10 ml, about 12.5 ml, about 15 ml, about 17.5 ml, about 20 ml, about 22.5 ml, about 25 ml, about 27.5 ml, about 30 ml, about 32.5 ml, about 35 ml, about 37.5 ml, or about 40 ml when administered via any regional anesthesia technique directed at nerves innervating the thorax and chest wall, such as via a serratus plane nerve block, or via an intercostal nerve block, or via a paravertebral block.
[0159] Additionally or alternatively, in some embodiments, the tumor resection surgery comprises cystectomy. Additionally or alternatively, in certain embodiments, the cancer patient exhibits stage I, stage II, stage III, or stage IV bladder cancer.
[0160] In any and all embodiments of the methods disclosed herein, the patient is human.
[0161] In any and all embodiments of the methods disclosed herein, the biological sample obtained from the cancer patient comprises biopsied tumor tissue, whole blood, plasma, or serum.
[0162] Administration of any of the intraoperative or postoperative analgesics disclosed herein can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. EXAMPLES
[0163] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. The examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the methods of the present technology. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects, or embodiments of the present technology described above. The variations, aspects, or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present technology.
Example 1. Materials and Methods
[0164] Measuring gene expression and clinical outcomes in human cohorts. This study uses data from publicly available TCGA cohort, as well as an independent cohort who underwent nephrectomy at Memorial Sloan Kettering Cancer Center. The Firebrowse and LinkedOmics portals were used to access TCGA-KIRC data (Vasaikar SV, et al., Nucleic Acids Res. 46: 956-63 (2017)). Level 3 normalized RSEM gene expression was extracted for cases and controls, as well as relevant clinical measures and metadata. For the MSKCC validation cohort, gene expression was measured using RNA-sequencing. Cancer-specific and recurrence-free survival were calculated by extracting relevant clinical data from internal clinical records at MSKCC and linking them with previous data contributions to the larger TCGA cohort.
101651 Measuring gene expression in validation (MSKCC) cohort. RNAseq raw read sequences were aligned against human genome assembly hgl9 by STAR 2-pass alignment (Dobin A, et al., Bioinform OxfEngl. 29: 15-21 (2012)). RNAseq gene level count values were computed by using the R package GenomicAlignments (Lawrence M, et al., Pios Comput Biol. 9: el 003118 (2013)) over aligned reads with UCSC KnownGene (Rosenbloom KR, et al., Nucleic Acids Res. 43: 670-81 (2014)) in hgl9 as the base gene model. The Union counting mode was used and only mapped paired reads were considered. Fragments per kilobase million (FPKM) values were then computed from gene level counts by using fpkm function from the R package “DESeq2”( Love MI, et al., Genome Biol. 15: 550 (2014)).
[0166] Analyzing gene and network level variation in RNAseq data. Differential gene expression was calculated in a subpopulation (N=72) for which ccRCC and neighboring healthy renal tissue were biopsied and sequenced. Median expression was calculated for each gene and genes in the bottom 20% were filtered out to remove bias from genes with low gene expression. Voom was used to estimate differential expression. The Kruskal Wallis test was used to associate pathologic stage and opioid pathway gene expression for samples with high and low opioid pathway expression. Samples with high and low opioid gene expression were defined for each gene as those samples in the top and bottom quartile of gene expression and expression-stage associations were determined for these sample subsets.
[0167] To estimate gene coexpression in the TCGA KIRC and BLCA cohorts, gene expression data was log2 transformed, and linear regression was used to correct gene expression for age, race, gender, and tumor purity. Genes in the bottom 20% percentile in variance and median expression were filtered to reduce noise, and samples with an interarray correlation greater than two standard deviations away from the mean were considered outliers and removed. Weighted gene coexpression analysis was used to determine correlation gene networks. The first principal component of each module was calculated (“module eigengene”) and univariate and multivariate Cox model were used to correlate eigengene expression with overall survival, cancer-specific survival, and recurrence-free survival. For multivariate testing, two models were used for the sake of comprehensiveness: one that included age, race, gender, and tumor purity, and a second model that included the aforementioned covariates, as well as stage.
[0168] Calculating differential gene expression and gene coexpression. For differential gene expression, TCGA RSEM estimated counts were input directly into voom, and a paired sample design was used for analysis (Law CW, et al., Genome Biol. 15: R29 (2014); Ritchie ME, et al., Nucleic Acids Res. 43: e47 (2015)). Genes were considered differentially expressed when adjusted P values < 0.05. Geneshot was used to identify opioid-related genes by calculating the top 100 genes most relevant to the search term “opioid” (Lachmann A, et al., Nucleic Acids Res. 47: W571-7 (2019)), and genes in the Reactome opioid receptor signaling pathway were extracted from MSigDB (Liberzon A, et al, Bioinform OxfEngl. 27: 1739-40 (2011)).
[0169] To calculate gene coexpression, Pearson correlation was calculated between each gene in the final corrected and filtered gene expression matrix. The Pearson correlation matrix was raised to a power, B - a soft threshold calculated empirically - and converted to an adjacency matrix (Zhang B, et al., Stat Appl Genet Mol A'. Articlel7 (2005)). A further quadratic transformation estimated the topological overlap matrix, capturing nearest neighbor association in the network (Ravasz E, et al., Science 297: 1551-1555 (2002)). Dynamic hierarchical clustering was used to identify independent coexpression modules, each named by an arbitrary color (Langfelder P, et al., Bioinformatics 24: 719-20 (2007)).
10.1.701 Internal and external validation of gene networks. The robustness of each module was empirically calculated by repeatedly splitting the gene expression data in training and tests sets and calculating a module preservation score between each new network (Langfelder P, et al., Pios ComputBiol. 7: el001057 (2011)). A composite preservation statistic (Z) was calculated by integrating several measures of connectivity and network preservation and previously characterized threshold (Z>10) was used to assess for preservation. Empirical p- values were also calculated and Bonferroni p-value threshold < 0.05 was used to confirm these results. Previous evidence suggests that Z scores and p values have a strong inverse correlation, so this approach simply utilizes two statistics that reflect the same measure of robustness. As an external validation, gene expression was measured in an independent cohort and corrected for batch, sex, and age using linear regression. Gene coexpression was calculated independently in this cohort as previously described, without explicit reference or parameterization from the TCGA population. Module membership was directly compared between TCGA-KIRC and MSKCC-KIRC networks, and Fisher’s exact test was used to calculate enrichment. Modules were considered preserved if enrichment odds ratio > 1 and Bonferroni p-value < 0.05.
[0171 ] Calculating enrichment and connectivity measures in differentially expressed genes and networks. The R package, goSeq, was used to estimate biologic pathway enrichment for differentially expressed genes, with gene length bias correction and multiple testing correction. The anRichment R package was used to estimate gene ontology enrichment for each module, and p-values were corrected with the Benjamini -Hochberg method. Enrichr was used to calculate module overrepresentation for experimental datasets, including ChlP- seq and gene knockout data, for a variety of cell lines and animal models, and Q values were calculated to account for multiple hypothesis testing (Chen EY, et al., Bmc Bioinformatics 14: 128 (2013)). Previously published immune cell type signatures were also used for enrichment testing (Ricketts CJ, et al., Cell Reports 23: 313-326 (2018)). Module enrichment for differentially expressed genes, immune signatures, and previously published ccRCC networks was estimated with Fisher’s exact test and p-values were corrected with the Bonferroni method when appropriate. ICGC Data Portal was used to identify mutational burden and frequency in the Reactome pathway (Zhang J, et al., Database 2011 : bar026).
[0172] Differential network connectivity was calculated by comparing the mean intramodular connectivity for each disease network with those same network genes in the control cohort (Zhang B, et al., Cell 153: 707-720 (2013)). The ratio of average network connectivity between cases and controls was used as an estimate of differential connectivity. For example, a measure of 2 signifies that the average correlation strength for a group of genes in a network is two times greater in disease than in controls. Two separate false discovery rates (FDR) were estimated by randomly shuffling samples and genes of disease and control networks. Shuffling samples creates networks with random edges and shuffling genes creates networks with random nodes. The final FDR was quantified by selecting the larger estimate and a conservative FDR threshold was used to assess significance (FDR < 0.001).
[01731 Projecting drug-induced transcriptional profiles onto survival networks.
Connectivity scores were calculated between network hubs and drug-induced transcriptional profiles for leu-enkephalin, naloxone, and the VEGF-inhibitor class. The drug profiles were catalogued by Connectivity Map and the CLUE Research platform was used to calculate connectivity scores (Lamb J, et al., Science 313: 1929-35 (2006)). Connectivity Map has catalogued gene expression profiles for thousands of chemical and genetic perturbations across nine cell lines, and connectivity scores between all reference perturbations were calculated based on a weighted Kolmogorov Smirnov statistic, normalized for cell line and perturbation type (Subramanian A, et al., Proc National Acad Sci. 102: 15545-50 (2005)). A non-parametric weighted connectivity score and an enrichment score, T, was then calculated for each module-drug pair of interest, ranging from -100 to +100 (Subramanian A, et al., Cell 171 : 1437-1452 (2017)). T measures the fraction of reference connectivity scores greater than the tested module-drug pair. A positive score shows that hub expression and drug-induced expression are in the same direction, while negative scores reflect expression in the opposite direction. A score of 90 indicates that only 10% of the reference set had a stronger score. Unlike a null distribution generated by random permutation, this empirical test avoids strong assumptions about the distribution of gene expression data under perturbed conditions. Instead, it tests module-drug connectivity directly against an expansive and diverse gene set under biologic and pharmacologic perturbation and provides a useful corresponding effect size. Empirical validation has demonstrated that T > |90| also pass p-value and FDR thresholds < 0.05 based on permutation-based null distribution methods, but lower T estimates may also pass those thresholds (Subramanian A, et al., Cell 171 : 1437-1452 (2017)). Hubs positively correlated with survival were considered upregulated and hubs negatively correlated with survival were considered downregulated. Each module was studied independently and each gene in the corresponding hub set was considered as a binary, either upregulated or downregulated. Drug-hub pairs with T > +90 indicate pro-survival relationship in hubs positively correlated with survival. Drug-hub pairs with T > +90 indicate anti-survival relationship in hubs negatively correlated with survival.
[0174] Calculating master regulators of directed sene-sene networks. Directed transcriptional relationships were retrieved using the “aracne. network” R library, derived from the ARACNe algorithm. ARACNe first calculates pairwise gene expression mutual information to identify candidate relationship and then uses data processing inequality to trim edges representing indirect relationships between genes that are strongly co-regulated without being directly dependent (Margolin AA, Nemenman I, et al., Bmc Bioinformatics 7: S7 (2004)). This two-step procedure recovers gene expression dependencies with high fidelity. Directed networks describing relationships between modules were constructed by calculating edges between genes in different modules. For each pair of modules, edge weight was calculated by summing the total number of edges between the module pair, scaled by the product of their respective module sizes. Permutation testing (N=1000) was performed to calculate a null distribution of edge weights between each pair of modules, and an edge was only kept if Benjamini -Hochberg p value < 0.05. Within each module, master regulators were inferred using MARINa, leveraging a phenotype transition signature derived from t-test analysis comparing gene expression between cases and controls (Lefebvre C, et al., Mol Syst Biol. 6: 377(2010)).
Example 2. Characterizing Opioid Pathway Gene Expression Changes in Clear Cell Renal Cell Carcinoma.
101751 Gene expression changes associated with ccRCC across the transcriptome were characterized. Using TCGA data, tissue from ccRCC were compared with adjacent normal renal tissue in 72 individuals (FIG. 1A). Six thousand three hundred twenty one genes (6,321) were upregulated and six thousand three hundred sixty six (6,366) genes were downregulated (P < 0.05). Cell migration, biologic adhesion, and immune regulation gene ontology pathways are most robustly overrepresented in these differentially expressed genes (P < 0.05, FIG. 5), consistent with previous literature. Notably, many genes involved in opioid metabolism, regulation, and signaling are also differentially expressed in ccRCC (FIGs. 1B-1F), including IL4R, OGFR, OPRL1, OGFRL1, ARRB1, ARRB2, POMC, FOS, CYP3A4, PTGS2, and BDNF (p < 0.05). Given the differential expression of several opioid- related genes, enrichment testing for the Reactome opioid signaling pathway was specifically investigated and suggestive evidence was found for its overrepresentation in differentially expressed genes (Fisher’s exact test, nominal P = 0.03, OR = 1.7(1.04-2.81)). Further analysis also demonstrates that sixty-two of the top one hundred genes predicted to be functionally associated with opioids (Lachmann A, et al., Nucleic Acids Res. 47: 571-7 (2019)) are also differentially expressed. Opioid signaling is not nearly as overrepresented as large pathways more proximal to pathogenesis, like immune regulation and cell migration, but these analyses provide evidence for its association with ccRCC. Canonical opioid receptor genes 0PRM1, OPRD1, OPRK1 are all poorly expressed in renal tissue and there is no evidence in this analysis that they are differentially expressed in ccRCC, though the expression of each shows greater variability in the disease state (FIG. 6). At the same time, OPRL1 (nociception receptor), OGFR, and TLR4 receptor genes are upregulated in ccRCC (FIGs. 1C-1F). These receptors are known to bind opioids and are implicated in ccRCC progression, in which OGFR signaling is likely protective and TLR4 contributes to cancer pathogenesis. [0176] An analysis of mutational burden in ccRCC in the TCGA cohort provides further orthogonal evidence of the relationship between ccRCC and opioid signaling. This study identified 149 mutations across 76% (67/88) of opioid signaling pathways genes in ccRCC (FIGs. 14), but the functional impact and clinical significance of most of these mutations are unknown. Further analysis showed that pathologic stage is associated with whether the sample is high or low expression of particular Reactrome opioid signaling pathway genes (P < 0.05, FIG. 7), suggesting tumor heterogeneity in opioid pathway expression may be associated with clinical features.
Example 3. Calculating ccRCC Gene Networks Relevant to Survival
[0177] Gene expression is organized into networks that can respond to genetic, pharmacologic, and environmental perturbations. Gene networks in renal cell carcinoma was identified (using the N=533 individuals in the TCGA KIRC data set) using weighted gene coexpression network analysis (Zhang B, et al., StatAppl Genet Mol. 4:Articlel7 (2005)). This analysis revealed 15 distinct gene networks, each labeled by an arbitrary color (FIG. 2A). Module membership is reported in FIG. 15. A resampling technique was used to confirm that these networks were internally robust and reproducible (FIG. 2B). Lastly, these gene networks map onto known functional pathways, confirming their biologic coherence (FIG. 16)
[0178] Next, it was hypothesized that a subset of these networks may be associated with survival. To test this hypothesis, the relationship between recurrence-free survival, cancerspecific survival, and overall survival, and pathologic characteristics to the first principal component (“eigengene”) of each module, which captures the predominant variation of gene expression of each respective network, was estimated (FIGs. 2C and 2D). Each individual sample exhibited very high or low expression in relatively few modules, and eigengenes show modest correlation across networks (FIGs. 8A-8C). Increased or decreased expression in a gene network may be related to the length of survival. These data was analyzed using the Cox proportional hazard model, accounting for multiple hypothesis testing. Univariate and multivariate modeling showed that eight networks were associated with survival (P < 0.05), seven of which were also associated with cancer-specific survival (P < 0.05) and recurrence-free survival (P < 0.05) (FIGs. 2D-2J, FIG. 17). Notably, many of these networks were also associated with tumor stage and grade as expected (FIG. 2C) and Fisher’s exact testing of sample subgroups corroborated this finding, showing samples with high eigengene expression were associated with stage (FIG. 18). These results reflect the well-known strong correlation between stage, grade, and survival in ccRCC. Each of these 8 networks was reproduced in an independent cohort of individuals (N=34) with ccRCC from Memorial -Sloan Kettering Cancer Center (ccRCC-MSKCC) (FIG. 9 and FIGs. 10A-10H).
Example 4. Tumor microenvironment and onco enesis pathways associated with survival- associated sene networks
[0179] To examine the relationship between ccRCC pathogenesis and survival-related networks, the network analysis was integrated with publicly available experimental genomic, ChlP-seq, gene expression, and gene ontology data (Chen EY, et al., Bmc Bioinformatics 14: 128 (2013)). Survival networks include modules overrepresented for gene signatures related to T helper type 2 cells (“tan”), angiogenesis (“yellow”), fatty acid metabolism (“green”), and mitochondrial ATP synthesis (“turquoise”), pathways significantly altered in ccRCC. CSS- and RFS-networks are also downstream targets for known ccRCC transcriptional regulators, including NRF2 (“green”, q=5.4x10-7, OR=4.6), JUN (“black”, q=l.7x10-4, OR=1.6), JAK2 (“brown”, q=1.6xl0-4, OR=2.3), and MET (“blue”, q=4.9xl0- 4, OR=1.83). Survival-related networks also include approximately 40% (9 out of 22) of the intOGen RCC mutational driver genes (Gonzalez-Perez A, et al., Nat Methods 10: 1081-2 (2013)). Notably, all survival networks are strongly overrepresented for differentially expressed genes, and four networks (black, brown, salmon, yellow) also gain stronger connections in ccRCC compared to controls (FIG. 3A). Together, these results show that the survival-associated networks are intimately tied to oncogenesis, progression, and pathophysiology in ccRCC. Tossible localization of these networks within the tumor microenvironment was further probed by examining each network for enrichment of 24 empirically derived cell-type specific gene markers (Ricketts CJ, et al., Cell Reports 23: 313- 326 (2018)). These analyses revealed that the “green” network is strongly associated with macrophage-specific signatures, the “tan” network is overrepresented with genes specific to Th2 cells, and the yellow network is associated with B cells T follicular helper cells (P < 0 05, FIGs. 11A-11D) Example 5. Leu-enkephalin modulates gene networks relevant to cancer-specific survival
[0180] Next, whether opioid receptor agonism and antagonism affect the expression of these survival -relevant networks was examined. First, intramodular connectivity was calculated and hub genes for each of the eight networks were identified (FIG. 19). Hub genes are the most highly connected nodes in each network, making them potent targets and important predictors of disease. It is hypothesized that leu-enkephalin, a non-selective opioid receptor agonist, would downregulate pro-survival gene network hubs and upregulate anti-survival network hubs. To perform this analysis, gene expression changes induced by all compounds catalogued by Connectivity Map were projected onto each network (Lamb J, et al., Science 313 : 1929-35 (2006)). This analysis showed that leu-enkephalin has significant anti-survival effects on seven survival -related networks, upregulating modules negatively correlated with survival and downregulating modules positively correlated with survival (FIG. 3B). Leu- enkephalin most strongly modulates the Th2 immune network (“tan”) and NRF2-dependent macrophage network (“green”) (T >= |90|).
[01811 Next, the molecular effects of naloxone was examined, with the hypothesis that opioid receptor antagonism would shift networks towards pro-survival expression (FIG. 3C). This analysis showed that the effect of naloxone on survival -related networks opposes that of leu- enkephalin. It most strongly influences angiogenesis (“yellow”), NRF2-dependent macrophage network (“green”), and hemopoesis (“salmon”) networks and drives them towards pro-survival expression patterns, though naloxone’s effect doesn’t pass strict statistical threshold. As a positive control, the effect of VEGF receptor inhibitors, a pharmacologic class shown to have some survival benefits in renal cell carcinoma patients, was investigated. As anticipated, it has strong pro-survival effects on Th2 immunity (“tan”, T >= |90|) and hemopoesis (“salmon”, T >= |90|), and a moderate effect on NRF2-dependent macrophage network (“green”, T > |80|). Interestingly, it also has a strong anti-survival effect on the brown network (T >= |90|) and minimal effect on three networks, pointing to a potential mechanism for its therapeutic limitations (FIG. 3D).
|0182[ Lastly, the hypothesis that leu-enkephalin preferentially affects networks more strongly associated with survival was tested (FIG. 3E). The leu-enkephalin effect size is positively correlated with Cox model regression coefficients for survival -associated networks and significantly associated with recurrence-free survival (rho=0.93, P=0.007). This suggests that the clinical relationship between opioids and ccRCC survival may depend on the preferential effect of opioids on networks most strongly associated with survival.
[0183] In order to further validate these results, this methodology was applied to bladder cancer, where evidence also suggests that opioids promote tumor progression (Chipollini J, et al., Bmc Anesthesiol 18: 157 (2018)). Bladder cancer is a relevant comparison to ccRCC given that it is also a urological cancer where opioid excretion in urine could enable direct effects on tumorigenesis. Similar analysis in the TCGA BLCA cohort identified that one network (“pink”) was associated with overall survival. Leu-enkephalin showed anti-survival expression effects on the pink network, while naloxone and numerous chemotherapeutic drugs and classes showed pro-survival expression effects (FIGs. 12A-12B).
Example 6. Reconstructins directed transcriptional networks and master regulators of RFS, CSS, and OS
[0184] Finally, it was reasoned that causal networks regulators may provide mechanistic and functional insight into the downstream transcriptional effects of opioids. An information theoretic approach was used to calculate direct transcriptional relationships between genes, and master regulators (MRs) of the transition from normal to disease state were inferred (Margolin AA, Nemenman I, et al., Bmc Bioinformatics 7: S7 (2004); Lefebvre C, et al., Mol SystBiol. 6: 377(2010)). This analysis revealed 211 MRs (FDR < 0.05) across the transcriptional network (FIG. 20). The NRF2-dependent macrophage network (“green”) and Th2 (“tan”) networks, most robustly influenced by leu-enkephalin, had 27 MRs (FIG. 4). Several of their regulators have been experimentally validated drivers of oncogenesis, tumor progression, and metastasis in ccRCC, suggesting possible transcriptional mechanisms through which opioids may affect ccRCC development and metastasis. Notably, CDH2, the MR with the strongest enrichment score, is known to be involved in the opioid pathway, and CDH2 variants influence methadone response. MRs associated with other survival -related networks also are tightly linked with opioid pathway (FIG. 4), including genes involved in the opioid signaling cascade (GIT2, PLD2, RALGDS) and opioid-related modulation of the immune system (CREB5, IL4R, CLEC2D, PLXNB1). Given that opioid-related immunomodulation is generally considered central to opioid effects in cancer, correlation of gene expression of these four latter MRs with expression of immune signature genes were further examined (FIGs. 13A-13D and FIG. 21) Taken together, the evidence suggests that ccRCC progression and opioid regulation converge onto these survival networks and provides a biologic rationale for how these seemingly divergent processes may transcriptionally influence each other in humans.
EQUIVALENTS
[0185] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[0186] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
[0187] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.
Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.
[0188] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all FIGs. and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Claims

WHAT IS CLAIMED IS:
1. A method for selecting a cancer patient undergoing tumor resection surgery for renal cancer for treatment with an intraoperative opioid analgesic comprising
(a) detecting expression levels of at least one survival-associated gene expression network in a biological sample obtained from the cancer patient that are either comparable or decreased relative to a control sample obtained from a healthy subject or a predetermined threshold, wherein the at least one survival-associated gene expression network is a NRF2- dependent macrophage network and/or a Th2 immune network; and
(b) administering to the cancer patient an effective amount of an intraoperative opioid analgesic during the tumor resection surgery.
2. The method of claim 1, wherein the expression levels of the at least one survival- associated gene expression network are detected via next-generation sequencing, PCR, realtime quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
3. A method for prolonging survival of a cancer patient undergoing tumor resection surgery for renal cancer comprising administering to the cancer patient an effective amount of an intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a NRF2-dependent macrophage network and/or a Th2 immune network in a biological sample obtained from the cancer patient are either comparable or decreased relative to a control sample obtained from a healthy subject or a predetermined threshold.
4. The method of any one of claims 1-3, wherein the intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
5. The method of any one of claims 1-4, wherein the effective amount of the intraoperative opioid analgesic is about 1 MME to about 20 MMEs.
6. The method of any one of claims 1-4, wherein the effective amount of the intraoperative opioid analgesic is about 20 MMEs to about 45 MMEs.
7. The method of any one of claims 1-4, wherein the effective amount of the intraoperative opioid analgesic is about 45 MMEs to about 200 MMEs.
8. The method of any one of claims 1-7, wherein the effective amount of the intraoperative opioid analgesic is administered as a series of bolus doses or as a continuous infusion during the tumor resection surgery.
9. The method of any one of claims 1-8, wherein the effective amount of the intraoperative opioid analgesic is administered to the cancer patient prior to incision.
10. The method of any one of claims 1-9, further comprising administering to the cancer patient an effective amount of a local anesthetic solution that comprises one or more of lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, or benzocaine, and optionally an opioid.
11. The method of claim 10, wherein the local anesthetic solution is administered via an epidural catheter, via a serratus plane nerve block or via an intercostal nerve block before, during and/or after the tumor resection surgery.
12. The method of any one of claims 1-11, further comprising administering to the cancer patient an effective amount of a post-operative opioid analgesic after the tumor resection surgery.
13. The method of claim 12, wherein the post-operative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
14. The method of claim 12 or 13, wherein the post-operative opioid analgesic and the intraoperative opioid analgesic are the same or different.
15. The method of any one of claims 12-14, wherein the effective amount of the postoperative opioid analgesic and the effective amount of the intraoperative opioid analgesic are the same or different.
16. A method for selecting a cancer patient undergoing tumor resection surgery for renal cancer for treatment with an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic comprising
(a) detecting expression levels of at least one survival-associated gene expression network in a biological sample obtained from the cancer patient that are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold, wherein the at least one survival-associated gene expression network is a NRF2-dependent macrophage network and/or a Th2 immune network; and
(b) administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery.
17. The method of claim 16, wherein the expression levels of the at least one survival- associated gene expression network are detected via next-generation sequencing, PCR, realtime quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
18. A method for prolonging survival of a cancer patient undergoing tumor resection surgery for renal cancer comprising administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a NRF2-dependent macrophage network and/or a Th2 immune network in a biological sample obtained from the cancer patient are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold.
19. The method of any one of claims 16-18, wherein the low-dose intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
20. The method of any one of claims 16-19, wherein the effective amount of the low-dose intraoperative opioid analgesic is about 1 MME to about 20 MMEs.
21. The method of any one of claims 16-18, wherein the opioid-free intraoperative analgesic is an amide-type local anesthetic or an ester-type local anesthetic.
22. The method of claim 21, wherein the amide-type local anesthetic is lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, or levobupivacaine.
23. The method of claim 21, wherein the ester-type local anesthetic is cocaine, procaine, tetracaine, chloroprocaine, or benzocaine.
24. The method of any one of claims 16-18 or 21-23, wherein the opioid-free intraoperative analgesic is administered via an epidural catheter.
25. The method of claim 24, wherein the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 1-10 ml per hour when administered via the epidural catheter.
26. The method of any one of claims 16-25, wherein the effective amount of the opioid- free intraoperative analgesic or the low-dose intraoperative opioid analgesic is administered as a series of bolus doses, or as a continuous infusion during the tumor resection surgery.
27. The method of any one of claims 16-18 or 21-26, wherein the opioid-free intraoperative analgesic is administered via a serratus plane nerve block, or via an intercostal nerve block.
28. The method of any one of claims 16-27, further comprising administering to the cancer patient an effective amount of an opioid-free post-operative analgesic or a low-dose post-operative opioid analgesic after the tumor resection surgery.
29. The method of claim 28, wherein the opioid-free post-operative analgesic is selected from the group consisting of lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine.
30. The method of claim 28, wherein the low-dose post-operative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
31. The method of any one of claims 28-30, wherein the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same or different.
32. The method of any one of claims 28-31, wherein the effective amount of the opioid- free post-operative analgesic or the low-dose post-operative opioid analgesic and the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same or different.
33. The method of any one of claims 1-32, wherein the NRF2-dependent macrophage network comprises five or more genes selected from among A1CF, AB AT, ABCB1, ABCB9, ABCC2, ABCC6P1, ABCG1, ABHD6, ABLIM3, ABP1, ACAD11, ACADL, ACAT1, ACBD4, ACO2, ACOT4, ACOX2, ACSL1, ACSM5, ACY1, ADAMTS3, ADH6, AGPAT3, AGT, AIFM1, AKR1C1, AKR1C2, AKR7A2, AKR7A3, ALAD, ALDH1A1, ALDH1A2, ALDH1L1, ALDH2, ALDH3A2, ALDH4A1, ALDH7A1, ALDH8A1, ALDOB, ALPK2, ALPL, AMDHD1, ANK3, ANKRD56, ANPEP, ANXA13, ANXA2P2, ANXA2, A0X1, APITD1, AQP3, AQP9, ARHGAP1, ARHGAP24, ARL4C, ASB13, ASRGL1, ASTN2, ATP6V0A1, AUH, AXL, AZGP1, B3GNT7, B4GALNT1, B4GALT1, BACE2, BAIAP2L1, BAIAP2L2, BAMBI, BARX2, BCAT1, BCMO1, BDH2, BDKRB2, BEND3, BHMT2, BHMT, BNIP3, BPHL, BTG3, C10orfl08, Cllorf45, Cllorf52, C14orf64, C14orf73, C17orf51, C18orfl8, C19orf77, C1RL, C1R, CIS, Clorfll5, Clorf201, Clorf203, Clorf210, Clorf21, Clorf89, Clorf96, C21orf7, C22orf45, C2orf24, C2orf67, C3, C4orfl9, C5orf23, C6, C7orfl0, C8orf47, C9orfl25, CABCI, CABLES1, CADM3, CALML4, CBR4, CBWD1, CBX7, CCDC146, CCDC64, CCDC68, CD276, CD44, CD47, CD55, CD82, CDADC1, CDC42SE2, CDCA2, CDCP1, CDH16, CDH2, CDHR2, CDHR5, CDK18, CDK20, CDON, CEACAM1, CES2, CFB, CGREF1, CHDH, CHI3L1, CHPF2, CHST13, CIDEB, CISH, CIT, CKAP4, CLDN10, CLDN2, CLEC18A, CLEC18B, CLEC18C, CLIC6, CLPTM1L, CMBL, CNDP2, CNNM1, COBL, COL22A1, COL23A1, COL4A5, COL8A2, COLEC12, COPG2, CPT2, CRAT, CRB3, CREB3L3, CRY2, CRYM, CRYZ, CTHRC1, CYB5A, CYB5D1, CYP1B1, CYP27A1, CYP2J2, CYP4V2, CYS1, DAB2, DAPK2, DCBLD2, DDAH1, DEPDC7, DGKG, DHDH, DHTKD1, DIRAS2, DLG5, DMGDH, DMRTA1, DPF3, DPYS, DSEL, ECHDC3, EFNA5, EGOT, ELFN2, EMX1, EMX2OS, EMX2, ENAM, ENPEP, ENPP3, EPB49, EPHA7, EPHX2, ERBB3, ETFA, ETFDH, ETNK2, ETV1, ETV6, EZR, FAAH, FABP3, FAHD1, FAM149A, FAM164C, FAM60A, FAM69A, FANCC, FBP1, FBXL16, FBXO32, FCAMR, FGFR10P, FGFR3, FGGY, FHL2, FLJ23867, FLJ36031, FLNC, FMO4, FNDC4, FREM2, FTCD, GAL3ST1, GALM, GALNT2, GALNT7, GATM, GATS, GBAS, GDA, GFPT2, GGT3P, GJB1, GK, GLB1L, GLIS1, GLRB, GLT25D1, GOT1, GPD1, GPER, GPT, GPX3, GPX8, GRAMD1C, GRTP1, GSTA1, GSTA2, GXYLT2, GYLTL1B, HAAO, HABP2, HABP4, HGD, HHLA2, HIBCH, HIGD1A, HLF, HMGCL, HMOX1, HNF4A, HOXCIO, HSDL2, HSP90B1, HSPA2, HSPB8, HYAL1, HYOU1, IDH1, IGDCC4, IGF2BP2, IL17RB, IL1R2, IL22RA1, IMPA2, IMPDH1, IRS2, ITGB6, JPH2, KCNIP3, KCNJ15, KCNJ16, KCNS3, KCTD17, KCTD1, KIAA1543, KLC4, KLF15, KLHDC7A, KL, KRT19, KRT80, KSR1, LAD1, LAMA3, LAMB1, LAMB3, LAMC2, LDHD, LEF1, LIMK2, LNP1, LOC100126784, LOC100131551, LOC151534, LOC388387, LOC389332, LOC723809, LRG1, LRRC19, LRRC8E, LYG1, MAF, MAGED1, MAN1C1, MAOB, MAP7, MAPK8IP1, MAPT, MARVELD3, METTL7A, METTL7B, MFI2, MINA, MLYCD, MMD, MME, MMP14, MMP7, MMP9, MOBKL2B, MOSC2, MPI, MPV17L, MPZL1, MSRA, MTHFD1L, MTHFD2, MUC1, MXRA8, MYO1E, MYO3A, MYO7B, MYOM3, NAMPT, NAP1L1, NAPSA, NEFL, NGEF, NHEJ1, NIPSNAP1, NOMO1, NOMO3, NPR3, NRXN2, NTN4, NUDT6, OPN3, 0SCP1, OSTalpha, PANK1, PAPP A, PAQR7, PARD6B, PBLD, PBX3, PCBD2, PCCA, PCK1, PCOLCE2, PC, PDE10A, PDIA3P, PDIA4, PDIA5, PDK2, PDXP, PDZD3, PDZK1P1, PECI, PECR, PEPD, PER3, PGPEP1, PHYH, PIGT, PIPOX, PKHD1, PLA2G4C, PL AU, PLIN2, PLIN3, PLOD2, PLTP, PMAIP1, PNMA6A, PON2, PPFIBP2, PPL, PPP1R14C, PRODH2, PRODH, PSD3, PTGER2, PTGFRN, PTGR2, PTH1R, PTH2R, PVR, PXMP2, QDPR, QRFPR, QSOX1, RAB17, RAB3IP, RAB7L1, RAI2, RARRES1, RCN1, RGN, RGS14, RHOBTB1, RIT1, RND3, RNF5P1, RORC, RPN2, RUNDC3B, RUNX1, RUNX2, SAMD5, SATB2, SCARB1, SCD, SCGN, SCLY, SCNN1A, SEMA3C, SEMA3D, SEMA4B, SEMA4F, SEMA6A, SEPHS2, SEPSECS, SERPINA3, SERPINA6, SERPINF1, SERPINF2, SGSM1, SH3BGRL2, SH3PXD2B, SH3YL1, SHISA4, SHMT1, SLC10A2, SLC12A7, SLC16A12, SLC16A13, SLC16A4, SLC16A5, SLC17A4, SLC1A1, SLC22A2, SLC22A4, SLC22A5, SLC23A1, SLC25A23, SLC25A34, SLC25A42, SLC25A44, SLC26A1, SLC28A1, SLC2A2, SLC2A5, SLC2A9, SLC38A5, SLC3A1, SLC46A1, SLC5A12, SLC5A1, SLC5A8, SLC5A9, SLC6A12, SLC6A19, SLC6A3, SLC7A5, SLC9A1, SLC9A3R1, SLC04C1, SLITRK2, SLITRK4, SLPI, SMPDL3A, SMTNL2, SPATA18, SPATS2L, SPNS2, SP0CK1, SPON2, SQLE, STAMBPL1, STEAP3, STK17A, STK32B, STK39, STON2, STX3, SULF2, SYBU, SYT12, SYT9, SYTL2, TBC1D2, TCEA3, TCFL5, TCN2, TCTA, TEF, TFEC, TFPI2, TGFBI, THAP9, THSD4, TLN2, TMCC1, TMCO4, TMEM125, TMEM130, TMEM139, TMEM140, TMEM164, TMEM171, TMEM176A, TMEM176B, TMEM195, TMEM26, TMEM37, TMEM45A, TMPRSS3, TNFAIP6, TNFRSF10C, TNFRSF21, TPBG, TPMT, TPST2, TRAP1, TRIM55, TRPV4, TSGA14, TSPAN1, TTC39C, TUBB3, UBA5, UGT1A6, UGT1A9, UGT2A3, UGT2B7, UGT3A1, USP2, VCAM1, VIL1, WDR72, WDR81, WFDC2, WFS1, WNT5A, WNT5B, ZBTB7C, ZFAT, ZFHX4, ZNF385B, ZNF711, ZSCAN2, ABCC6P2, ABCC6, ACAA2, ACE2, ACMSD, ACSM2A, ACSM2B, ACY3, ADM2, AGMAT, AGXT2, AMN, ANKS4B, APOM, ASP A, BBOX1, Cllorf54, C9orf66, CLCN5, CLRN3, CRYL1, CUBN, CYP4A11, DDC, EHHADH, FMO1, FUT6, GBA3, GIPC2, GLYATL1, GLYAT, HAO2, HNF1A, HRSP12, KHK, LGALS2, LRP2, MIOX, NAT8, PDZK1, PHYHIPL, PKLR, RBP5, SLC13A1, SLC16A9, SLC17A1, SLC17A3, SLC22A11, SLC22A12, SLC23A3, SLC27A2, SLC37A4, SLC39A5, SLC47A1, SLC5A10, SLC6A13, SLC7A9, TINAG, TM4SF5, TMEM27, TRIM10, TRIM15, TRPM3, TTC38, UPB1, and USH1C.
34. The method of any one of claims 1-33, wherein the Th2 immune network comprises five or more genes selected from among AJAP1, ANLN, ARHGAP11A, ASF IB, ASPM, ATAD2, AURKA, BRCA1, BUB1, C10orf2, C13orf34, C15orf23, C16orf75, CACNA2D4, CCDC99, CCNA2, CCNB1, CCNF, CDC6, CDCA7, CDK1, CDT1, CENPE, CENPF, CENPH, CENPL, CENPN, CENPO, CHAF1A, CHAF1B, CHEK1, CPOX, CTSF, DBF4, DERL1, DHFR, DIAPH3, DTL, E2F1, ECT2, EPR1, ESPL1, EZH2, FAM11 IB, FANCA, FANCD2, FANCI, FASN, FEN1, GGH, GINS1, GINS2, GINS3, GPRIN1, GPSM2, HELLS, HMMR, HPS3, IQGAP3, KIAA0101, KIF11, KIF18B, KIF20A, KIF23, KIF24, KIF2C, KIF4A, KIFC1, KPNA2, LMNB1, LMNB2, LRP8, MAD2L1, MCM2, MCM5, MCM6, MCM7, MELK, MKI67, MLF1IP, MTMR4, MYBL2, NCAPD2, NCAPH, NDC80, NEB, NUSAP1, PAQR4, PBXIP1, PLK4, PPARG, PPRC1, PRC1, PTTG1, RACGAP1, RAD51AP1, RFC4, RIPK2, RRP12, SGOL2, SHCBP1, SMC4, SPATA7, STIL, STMN1, TACC3, TCF19, TIMELESS, TK1, TMEM25, TRIM59, TRIP13, TUBB, UBE2C, UHRF1, WDR4, WDR67, WSCD1, ZWILCH, ZWINT, BUB IB, CCNB2, CDC20, CDCA5, CDCA8, CEP55, FOXM1, GTSE1, HJURP, NCAPG, PLK1, RRM2, TOP2A, and TPX2.
35. The method of any one of claims 1-34, wherein the tumor resection surgery comprises nephrectomy.
36. The method of any one of claims 1-35, wherein the renal cancer has a histologic subtype selected from among clear cell renal cell carcinomas (ccRCC), papillary renal cell carcinomas (pRCC), chromophobe renal cell carcinomas (crRCC), multilocular cystic RCC, collecting duct carcinoma, medullary carcinoma, mucinous tubular and spindle cell carcinoma, neuroblastoma-associated RCC, Xpl l.2 translocation-TFE3 carcinoma, and unclassified lesions.
37. The method of any one of claims 1-36, wherein the cancer patient exhibits stage I, stage II, stage III, or stage IV renal cancer.
38. The method of any one of claims 1-37, wherein the biological sample obtained from the cancer patient comprises biopsied tumor tissue, whole blood, plasma, or serum.
39. A method for selecting a cancer patient undergoing tumor resection surgery for bladder cancer for treatment with an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic comprising
(a) detecting expression levels of a survival-associated gene expression network in a biological sample obtained from the cancer patient that are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold; and
(b) administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery, wherein the survival-associated gene expression network comprises five or more genes selected from among ABCA6, ACTA2, ACTC1, ACTG2, ACTN1, ADAM12, ADAM I 9, ADAMTS2, ADH1B, ADRA2A, AEBP1, AG2, ALDH1L2, AMIG02, ANGPTL1, ATP10A, ATP8B2, AXIN2, BAG2, Cl lorf41, C14orfl32, Clorfl98, C2orf40, C7, C9orfl l0, CACNA1H, CAMK1G, CAND2, CCDC3, CCL19, CCL21, CDH11, CES1, CFH, CLEC11A, CNN1, COL11A1, COL12A1, COL16A1, COL6A1, COL6A2, COL6A3, CORO2B, CPXM1, CPXM2, CPZ, CREB3L1, CRYBG3, CSRP1, CTGF, CTSK, CYBRD1, CYTSB, DBN1, DCN, DES, DIO2, ECM1, EFEMP1, ELN, ENAH, F3, FAM109B, FAM126A, FAM129A, FAM129B, FAM83D, FBLN1, FBLN5, FBLN7, FBN1, FEZ1, FGD1, FGF14, FGF1, FGF7, FHL3, FLRT2, FMO3, FMOD, FN1, FNDC1, FOXP1, FOXS1, FZD7, GALNTL1, GAS1, GATA6, GEFT, GEM, GLT8D2, GNAO1, GPC3, GREM1, GRID1, H19, HDGFRP3, HOPX, HTR2B, IGF2, IGFBP2, IGFBP7, IL18R1, IL1R1, INHBA, INMT, ITGBL1, ITPRIPL2, KCNMB1, KIAA1199, KIAA1211, KIAA1755, KRT7, LAMA2, LARGE, LDB3, LEPREL2, LGI2, LIPC, LMCD1, LOC145820, LOC399959, LOC401093, LOXL1, LPHN1, LPHN3, LRFN3, LTBP1, LTBP4, LUM, LYNX1, MAGED4B, MAP1A, MAP6, MARK1, MARVELD1, MDGA1, MEIS3, MEST, MFAP2, MFAP4, MFGE8, MLPH, MMP11, MOXD1, MRC2, MRGPRF, MRVI1, MSRB3, MUSTN1, MYH10, MYH11, MYL9, MYO10, MYOID, NACAD, NAP1L3, NAV3, NDRG4, NEXN, NFASC, NOV, NPTXR, NR2F2, NT5DC2, NXN, OXTR, PCDH7, PCOLCE, PDE1A, PDE5A, PDLIM3, PDLIM7, PGR, PHYHD1, PID1, PLEKHA4, PLN, POSTN, PPP1R12B, PPP1R14A, PRR15L, PRRX1, PTGIR, PTGIS, PTK7, PVRL1, RAB23, RAB31, RAMP1, RBP1, RGMA, RGS16, RGS4, RICH2, ROR2, SCARF2, SCG5, SETMAR, SFRP1, SFRP2, SFRP4, SGCD, SHISA3, SLC20A2, SLC24A3, SLC2A10, SLIT2, SLIT3, SMOC2, SNAI1, SOBP, SOD3, S0RCS3, SPAG1, SPEG, SRPX2, SRPX, SSC5D, ST5, ST6GALNAC6, SULF1, SVEP1, SYT17, TACSTD2, TCF21, TGFB3, THBS2, TIMP2, TMEM117, TMEM119, TMEM30B, TMEM90B, TNC, TNFAIP8L3, TNXB, TPM2, TPM4, TRAM2, TSPAN2, VANGL2, VOL, VGLL3, WFDC1, ZAK, ZBTB47, ZCCHC24, ZNF469, ZNF503, ZNF703, ZNF853, ANTXR1, ASPN, CCDC80, COL14A1, COL1A1, COL1A2, COL3A1, COL5A1, DACT1, DACT3, EMILIN1, FAP, FBLIM1, GGT5, HSPB6, ISLR, ITGA11, LHFP, LMOD1, LTBP2, MGP, MICAL2, PALLD, PDGFRA, PODN, PRELP, SYNPO2, and TAGLN.
40. The method of claim 39, wherein the expression levels of the survival-associated gene expression network are detected via next-generation sequencing, PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, dot or slot blots, Multiplex ligation-dependent probe amplification (MLP A), Southern blotting, microarrays, SNP arrays, Molecular inversion probes (MIPs) assay, in situ hybridization (ISH), or fluorescent in situ hybridization (FISH).
41. A method for prolonging survival of a cancer patient undergoing tumor resection surgery for bladder cancer comprising administering to the cancer patient an effective amount of an opioid-free intraoperative analgesic or a low-dose intraoperative opioid analgesic during the tumor resection surgery, wherein expression levels of a survival-associated gene expression network in a biological sample obtained from the cancer patient are elevated compared with a control sample obtained from a healthy subject or a predetermined threshold, wherein the survival-associated gene expression network comprises five or more genes selected from among ABCA6, ACTA2, ACTC1, ACTG2, ACTN1, ADAM12, ADAM19, ADAMTS2, ADH1B, ADRA2A, AEBP1, AG2, ALDH1L2, AMIG02, ANGPTL1, ATP10A, ATP8B2, AXIN2, BAG2, Cllorf41, C14orfl32, Clorfl98, C2orf40, C7, C9orfl l0, CACNA1H, CAMK1G, CAND2, CCDC3, CCL19, CCL21, CDH11, CES1, CFH, CLEC11A, CNN1, COL11A1, COL12A1, COL16A1, COL6A1, COL6A2, COL6A3, CORO2B, CPXM1, CPXM2, CPZ, CREB3L1, CRYBG3, CSRP1, CTGF, CTSK, CYBRD1, CYTSB, DBN1, DCN, DES, DIO2, ECM1, EFEMP1, ELN, ENAH, F3, FAM109B, FAM126A, FAM129A, FAM129B, FAM83D, FBLN1, FBLN5, FBLN7, FBN1, FEZ1, FGD1, FGF14, FGF1, FGF7, FHL3, FLRT2, FMO3, FMOD, FN1, FNDC1, FOXP1, FOXS1, FZD7, GALNTL1, GAS1, GATA6, GEFT, GEM, GLT8D2, GNAO1, GPC3, GREM1, GRID1, H19, HDGFRP3, HOPX, HTR2B, IGF2, IGFBP2, IGFBP7, IL18R1, IL1R1, INHBA, INMT, ITGBL1, ITPRIPL2, KCNMB1, KIAA1199, KIAA1211, KIAA1755, KRT7, LAMA2, LARGE, LDB3, LEPREL2, LGI2, LIPC, LMCD1, LOC145820, LOC399959, LOC401093, LOXL1, LPHN1, LPHN3, LRFN3, LTBP1, LTBP4, LUM, LYNX1, MAGED4B, MAP1A, MAP6, MARK1, MARVELD1, MDGA1, MEIS3, MEST, MFAP2, MFAP4, MFGE8, MLPH, MMP11, M0XD1, MRC2, MRGPRF, MRVI1, MSRB3, MUSTN1, MYH10, MYH11, MYL9, MYO10, MYOID, NACAD, NAP1L3, NAV3, NDRG4, NEXN, NFASC, NOV, NPTXR, NR2F2, NT5DC2, NXN, OXTR, PCDH7, PCOLCE, PDE1A, PDE5A, PDLIM3, PDLIM7, PGR, PHYHD1, PID1, PLEKHA4, PLN, POSTN, PPP1R12B, PPP1R14A, PRR15L, PRRX1, PTGIR, PTGIS, PTK7, PVRL1, RAB23, RAB31, RAMP1, RBP1, RGMA, RGS16, RGS4, RICH2, ROR2, SCARF2, SCG5, SETMAR, SFRP1, SFRP2, SFRP4, SGCD, SHISA3, SLC20A2, SLC24A3, SLC2A10, SLIT2, SLIT3, SMOC2, SNAI1, SOBP, SOD3, SORCS3, SPAG1, SPEG, SRPX2, SRPX, SSC5D, ST5, ST6GALNAC6, SULF1, SVEP1, SYT17, TACSTD2, TCF21, TGFB3, THBS2, TIMP2, TMEM117, TMEM119, TMEM30B, TMEM90B, TNC, TNFAIP8L3, TNXB, TPM2, TPM4, TRAM2, TSPAN2, VANGL2, VCL, VGLL3, WFDC1, ZAK, ZBTB47, ZCCHC24, ZNF469, ZNF503, ZNF703, ZNF853, ANTXR1, ASPN, CCDC80, COL14A1, COL1A1, COL1A2, COL3A1, COL5A1, DACT1, DACT3, EMILIN1, FAP, FBLIM1, GGT5, HSPB6, ISLR, ITGA11, LHFP, LMOD1, LTBP2, MGP, MICAL2, PALLD, PDGFRA, PODN, PRELP, SYNPO2, and TAGLN.
42. The method of any one of claims 39-41, wherein the low-dose intraoperative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
43. The method of any one of claims 39-42, wherein the effective amount of the low-dose intraoperative opioid analgesic is about 1 MME to about 20 MMEs.
44. The method of any one of claims 39-41, wherein the opioid-free intraoperative analgesic is an amide-type local anesthetic or an ester-type local anesthetic.
45. The method of claim 44, wherein the amide-type local anesthetic is lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, or levobupivacaine.
46. The method of claim 44, wherein the ester-type local anesthetic is cocaine, procaine, tetracaine, chloroprocaine, or benzocaine.
47. The method of any one of claims 39-41 or 44-46, wherein the opioid-free intraoperative analgesic is administered via an epidural catheter.
48. The method of claim 47, wherein the effective amount of the opioid-free intraoperative analgesic is about 0.05%-4% amide-type or ester-type local anesthetic solution in a volume of 1-10 ml per hour when administered via the epidural catheter.
49. The method of any one of claims 39-48, wherein the effective amount of the opioid- free intraoperative analgesic or the low-dose intraoperative opioid analgesic is administered as a series of bolus doses, or as a continuous infusion during the tumor resection surgery.
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50. The method of any one of claims 39-41 or 44-49, wherein the opioid-free intraoperative analgesic is administered via a serratus plane nerve block, or via an intercostal nerve block.
51. The method of any one of claims 39-50, further comprising administering to the cancer patient an effective amount of an opioid-free post-operative analgesic or a low-dose post-operative opioid analgesic after the tumor resection surgery.
52. The method of claim 51, wherein the opioid-free post-operative analgesic is selected from the group consisting of lidocaine, mepivacaine, prilocaine, bupivacaine, etidocaine, ropivacaine, levobupivacaine, cocaine, procaine, tetracaine, chloroprocaine, and benzocaine.
53. The method of claim 51, wherein the low-dose post-operative opioid analgesic is fentanyl, hydromorphone, morphine, oxycodone, hydrocodone, codeine, meperidine, remifentanil, or sufentanil.
54. The method of any one of claims 51-53, wherein the opioid-free post-operative analgesic or the low-dose post-operative opioid analgesic and the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same or different.
55. The method of any one of claims 51-54, wherein the effective amount of the opioid- free post-operative analgesic or the low-dose post-operative opioid analgesic and the effective amount of the opioid-free intraoperative analgesic or the low-dose intraoperative opioid analgesic are the same or different.
56. The method of any one of claims 39-55, wherein the cancer patient exhibits stage I, stage II, stage III, or stage IV bladder cancer.
57. The method of any one of claims 1-56, wherein the biological sample obtained from the cancer patient comprises biopsied tumor tissue, whole blood, plasma, or serum.
58. The method of any one of claims 1-57, wherein the tumor resection surgery comprises cystectomy.
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PCT/US2021/062771 2020-12-11 2021-12-10 Methods for tailoring analgesic regimen in cancer patient's based on tumor transcriptomics WO2022125871A1 (en)

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