WO2019178214A1 - Procédés et compositions liés à la méthylation et à la récurrence chez des patients atteints d'un cancer gastrique - Google Patents

Procédés et compositions liés à la méthylation et à la récurrence chez des patients atteints d'un cancer gastrique Download PDF

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WO2019178214A1
WO2019178214A1 PCT/US2019/022031 US2019022031W WO2019178214A1 WO 2019178214 A1 WO2019178214 A1 WO 2019178214A1 US 2019022031 W US2019022031 W US 2019022031W WO 2019178214 A1 WO2019178214 A1 WO 2019178214A1
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methylation
gastric cancer
cohort
patient
levels
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PCT/US2019/022031
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English (en)
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Ajay Goel
Daisuke IZUMI
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Baylor Research Institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates generally to the fields of molecular biology and oncology. More particularly, it concerns methods and compositions involving biomarkers and cancer prognosis, diagnosis, and treatment.
  • GC Gastric cancer
  • Methods and compositions are provided related to methylation of one or more biomarkers in cancer patients, particularly gastric cancer patients.
  • Methods include one or more steps involving a set of biomarkers and compositions, or kits, involve the set of biomarkers such that they can evaluated for methylation.
  • Methods include but are not limited to the following: methods for measuring methylation of one or more biomarkers, methods for evaluating methylation of one or more biomarkers, methods for detecting methylation of one or more biomarkers, methods for quantifying the level of methylation of one or more biomarkers, methods for comparing methylation of one or more biomarkers, methods for evaluating a gastric cancer patient for recurrence of gastric cancer, methods of identifying a patience at risk for recurrence of gastric cancer, methods for measuring increased levels of methylation in one or more biomarkers as compared to representative levels of methylation in patients who do not experience recurrence of gastric cancer, methods for measuring levels of methylation in one or more biomarkers that are representative of levels of methylation in patients who do not experience recurrence of gastric cancer, methods for measuring decreased levels of methylation in one or more biomarkers as compared to representative levels of methylation in patients who experience recurrence of gastric cancer,
  • Steps for implementing any of the methods above or disclosed elsewhere in the application may include, but are not limited to, measuring the level of methylation in one or more biomarkers, measuring the level of methylation in one or more biomarkers, quantifying the level of methylation in one or more biomarkers, determining the level of methylation in one or more biomarkers, comparing the level of methylation in one or more biomarkers to the level of methylation in a control, determining a risk score for recurrence, calculating a risk score for recurrence, evaluating the patient’s risk for recurrence, identifying the patient as at risk for gastric cancer, diagnosing or prognosing the patient as having an increased risk for gastric cancer recurrence, treating the patient for gastric cancer, not treating the patient for gastric cancer, and/or treating the patient as being at risk for gastric cancer recurrence.
  • there are methods comprising measuring methylation of at least one biomarker from Supplemental table 1 in a biological sample from a gastric cancer patient. In some embodiments, there are methods comprising determining the level of or measuring methylation of, of at least, or of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 biomarker(s) from Supplemental table 1 in a biological sample from a gastric cancer patient. In some embodiments, a method comprises determining the levels of or measuring methylation of all the biomarkers from Supplemental table 1 in the biological sample from the gastric cancer patient.
  • methods involve the level of methylation of, of at least, or of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the following biomarkers: C3orf2l, EPB41L1, GAK, PPP1R1B, SLC12A7, cg03364486, cgl267l030, FANCA, GNPDA2, KCTD1, ONECUT2, PFKB3, PPT2, and/or ZNF638.
  • any of 1, 2, 3, 4, or 5 of biomarkers C3orf2l, EPB41L1, GAK, PPP1R1B, and/or SLC12A7 have a methylation level that is increased as compared to the methylation levels representative of a cohort of patients with nonrecurrent cancer.
  • any of 1, 2, 3, 4, 5, 6, 7, 8, or 9 of biomarkers cg03364486, cgl267l030, FANCA, GNPDA2, KCTD1, ONECUT2, PFKB3, PPT2, and/or ZNF638 have a methylation level that is decreased as compared to the methylation levels representative of a cohort of patients with nonrecurrent cancer.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 biomarkers from Supplemental table 1 are excluded from being determined or measured for methylation in the biological sample from the gastric cancer patient.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 biomarkers from Supplemental table 1 may be differentially methylated (/. ⁇ ? ., methylation levels are increased or decreased) as compared to the level of methylation in a cohort of gastric patients who have recurrent gastric cancer or who do not have recurrent gastric cancer. It is understood that a comparison may be made with respect to a threshold or reference level of methylation for any of the biomarkers discussed herein.
  • there are methods for treating gastric cancer in a patient comprising administering to the patient at least one platinum-based chemotherapeutic agent and at least a 5-fluorouracil-based agent, wherein a biological sample from the patient has been determined to have hypomethylation of, of at least, or of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 biomarkers from Supplemental table 1 (C3orf2l, EPB41F1, GAK, PPP1R1B, SLC12A7, cg03364486, cgl267l030, FANCA, GNPDA2, KCTD1, ONECUT2, PFKB3, PPT2, and ZNF638) in a biological sample from a gastric cancer patient.
  • Supplemental table 1 C3orf2l, EPB41F1, GAK, PPP1R1B, SLC12A7, cg03364486, cgl267l030, FANCA, GNPDA2, KCTD1, ONECUT2,
  • C3orf2l, EPB41L1, GAK, PPP1R1B, and/or SLC12A7 in Supplemental Table 1 have a methylation level that is increased as compared to the methylation levels representative of a cohort of patient with nonrecurrent cancer.
  • cg03364486, cgl267l030, FANCA, GNPDA2, KCTD1, ONECUT2, PFKB3, PPT2, and ZNF638 in Supplemental Table 1 have a methylation level that is decreased as compared to the methylation levels representative of a cohort of patient with nonrecurrent cancer.
  • there are methods for treating a gastric cancer patient who is at risk for recurrence comprising administering to the patient at least one platinum-based chemotherapeutic agent and at least a 5-fluorouracil-based agent, wherein a biological sample from the patient has been determined to have hypomethylation of at least ONECUT2 in a biological sample from a gastric cancer patient.
  • the biological sample from the patient has been determined to have hypomethylation of at least one, two, three, four, or five additional biomarkers that include: GNPDA2, PFKFB3, PPP1R1B, SFC12A7, or ZNF638.
  • there are methods for determining the risk of recurrence in a patient with or treated for gastric cancer comprising measuring methylation of at least one biomarker from Supplemental table 1 in a biological sample from the patient, wherein the patient has a level of methylation that is indicative of higher risk of recurrence.
  • the at least one biomarker comprises ONECUT2, GNPDA2, PFKFB3, PPP1R1B, SFC12A7, or ZNF638.
  • methods involve measuring or determining the level of methylation of 1, 2, 3, 4, 5, or 6 of these biomarkers: ONECUT2, GNPDA2, PFKFB3, PPP1R1B, SFC12A7, or ZNF638.
  • the measured level of methylation is reduced compared to the level of methylation for the biomarker in a control sample for nonrecurrent gastric cancer.
  • there are methods of diagnosing or prognosing a patient who has or previously had gastric cancer comprising measuring methylation of at least one biomarker from Supplemental table 1 in a biological sample from the patient, wherein the patient has a level of methylation that is indicative of higher risk of recurrence.
  • the at least one, two, three, four, five, or six biomarkers comprise ONECUT2, GNPDA2, PFKFB3, PPP1R1B, SFC12A7, or ZNF638.
  • methods involve measuring or determining the level of methylation of 1, 2, 3, 4, 5, or 6 of these biomarkers: ONECUT2, GNPDA2, PFKFB3, PPP1R1B, SFC12A7, or ZNF638.
  • Methods may also include a step of comparing a level of methylation to the level of methylation of the biomarker(s) in a control sample or to a control level of methylation.
  • the control sample is from a cohort of recurrent gastric cancer patients or the control level of methylation or reference level of methylation is a range of methylation levels from a cohort of patients who had recurrent gastric cancer.
  • a control sample or control level of methylation is from a cohort of nonrecurrent gastric cancer patients.
  • the control sample is from a cohort of nonrecurrent gastric cancer patients or the control level of methylation or reference level of methylation is a range of methylation levels from a cohort of patients who had recurrent gastric cancer.
  • methods concern measuring, quantifying, detecting, or determining the methylation level of at least ONECUT2.
  • the methylation level of ONECUT2 is measured, quantified, detected, and/or determined.
  • the methylation level of ONECUT2 is within the levels of methylation representative of the cohort of recurrent gastric cancer patients.
  • the methylation level of ONECUT2 is increased as compared to the levels of methylation representative of the cohort of recurrent gastric cancer patients.
  • methods concern measuring, quantifying, detecting, or determining the methylation level of at least GNPDA2.
  • the methylation level of GNPDA2 is measured, quantified, detected, and/or determined.
  • the methylation level of GNPDA2 is within the levels of methylation representative of the cohort of recurrent gastric cancer patients.
  • the methylation level of GNPDA2 is increased as compared to the levels of methylation representative of the cohort of recurrent gastric cancer patients.
  • methods concern measuring, quantifying, detecting, or determining the methylation level of at least PFKFB3.
  • the methylation level of PFKFB3 is measured, quantified, detected, and/or determined.
  • the methylation level of PFKFB3 is within the levels of methylation representative of the cohort of recurrent gastric cancer patients.
  • the methylation level of PFKFB3 is increased as compared to the levels of methylation representative of the cohort of recurrent gastric cancer patients.
  • methods concern measuring, quantifying, detecting, or determining the methylation level of at least ZNF638.
  • the methylation level of ZNF638 is measured, quantified, detected, and/or determined.
  • the methylation level of ZNF638 is within the levels of methylation representative of the cohort of recurrent gastric cancer patients.
  • the methylation level of ZNF638 is increased as compared to the levels of methylation representative of the cohort of recurrent gastric cancer patients.
  • methods concern measuring, quantifying, detecting, or determining the methylation level of at least PPP1R1B.
  • the methylation level of PPP1R1B is measured, quantified, detected, and/or determined.
  • the methylation level of PPP1R1B is within the levels of methylation representative of the cohort of recurrent gastric cancer patients.
  • the methylation level of PPP1R1B is decreased as compared to the levels of methylation representative of the cohort of recurrent gastric cancer patients.
  • methods concern measuring, quantifying, detecting, or determining the methylation level of at least SLC12A7.
  • the methylation level of SLC12A7 is measured, quantified, detected, and/or determined.
  • the methylation level of SLC12A7 is within the levels of methylation representative of the cohort of recurrent gastric cancer patients.
  • the methylation level of SLC12A7 is decreased as compared to the levels of methylation representative of the cohort of recurrent gastric cancer patients.
  • methods concern wherein the methylation level of at least ONECUT2 is measured.
  • the methylation level of ONECUT2 is within the levels of methylation representative of the cohort of nonrecurrent gastric cancer patients.
  • the methylation level of ONECUT2 is decreased as compared to the levels of methylation representative of the cohort of nonrecurrent gastric cancer patients.
  • methods concern wherein the methylation level of at least GNPDA2 is measured.
  • the methylation level of GNPDA2 is within the levels of methylation representative of the cohort of nonrecurrent gastric cancer patients.
  • the methylation level of GNPDA2 is decreased as compared to the levels of methylation representative of the cohort of nonrecurrent gastric cancer patients.
  • methods concern wherein the methylation level of at least PFKFB3 is measured.
  • the methylation level of PFKFB3 is within the levels of methylation representative of the cohort of nonrecurrent gastric cancer patients.
  • the methylation level of PFKFB3 is decreased as compared to the levels of methylation representative of the cohort of nonrecurrent gastric cancer patients.
  • methods concern wherein the methylation level of at least ZNF638 is measured.
  • the methylation level of ZNF638 is within the levels of methylation representative of the cohort of nonrecurrent gastric cancer patients.
  • the methylation level of ZNF638 is decreased as compared to the levels of methylation representative of the cohort of nonrecurrent gastric cancer patients.
  • methods concern wherein the methylation level of at least PPP1R1B is measured.
  • the methylation level of PPP1R1B is within the levels of methylation representative of the cohort of nonrecurrent gastric cancer patients.
  • the methylation level of PPP1R1B is increased as compared to the levels of methylation representative of the cohort of nonrecurrent gastric cancer patients.
  • methods concern wherein the methylation level of at least SLC12A7 is measured.
  • the methylation level of SLC12A7 is within the levels of methylation representative of the cohort of nonrecurrent gastric cancer patients.
  • the methylation level of SLC12A7 is increased as compared to the levels of methylation representative of the cohort of nonrecurrent gastric cancer patients.
  • a cohort comprises at least or at most 50, 100, 200, 300, 400, 500 or more patients (or any range derivable therein).
  • the biological sample was a gastric tissue sample, a blood sample, a stool sample, a saliva sample, a tumor sample, or other tissue sample.
  • the biological sample comprised tumor tissue.
  • the biological sample comprises metastatic tumor tissue or is from the lymph nodes.
  • the size of one or more gastric tumors from the patient is known or considered.
  • the lymph node metastasis status of the patient is known or considered. Some methods comprise evaluating tumor size and/or evaluating lymph node metastasis status.
  • methods include calculating a risk score of recurrence for the patient.
  • the risk score is used to categorize the likelihood a patience will experience recurrence within 3, 6, 9, 12 months and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more of primary cancer treatment.
  • the likelihood can be expressed in any way, such as a chance percentage of, for example, 10, 20, 30, 40, 50, 60, 70, 80, or 90% chance of detecting recurrence with a specified time period.
  • hypomethylation is determined based on comparing methylation to a positive or negative control, wherein the positive control is a level of methylation representative of recurrent gastric cancer and the negative control is a level of methylation representative of nonrecurrent gastric cancer.
  • one or more methylation levels is compared to a control level or representative level indicative of nonrecurrent or recurrent gastric cancer. The comparison may be done with the methylation level determined from a control sample that is similarly processed as the test sample or it may be done with a previously determined representative or threshold level of methylation that corresponds to recurrent gastric cancer or nonrecurrent gastric cancer.
  • methods include treating the patient for gastric cancer after evaluating or measuring methylation in one or more biomarkers. This may or may not be done in conjunction with calculating a risk score for the patient. It may depend on a particular classifier that is used to evaluate methylation and gastric cancer recurrence.
  • treating the patient for gastric cancer comprises administering to the patient at least one platinum-based chemotherapeutic agent and at least a 5-fluorouracil-based agent.
  • methods include identifying the patient as having a low risk for recurrent gastric cancer based on at least the measured methylation level(s) of at least one biomarker. In some embodiments, the measured methylation level(s) of at least one, 2, 3, 4, 5, 6, 7 or more biomarkers indicates hypermethylation. In some embodiments, methods include identifying the patient as having a high risk for recurrent gastric cancer based on at least the measured methylation level(s) of at least one biomarker, or even 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 biomarkers.
  • methods include identifying the patient as having a high risk for recurrent gastric cancer based on at least the measured methylation level(s) of at least one biomarker, or even 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 biomarkers. In some embodiments, the measured methylation level(s) of at least one, 2, 3, 4, 5, or 6 biomarkers or more indicates hypomethylation.
  • treating the patient for gastric cancer comprising administering a platinum-based chemotherapeutic agent and/or a 5-fluorouracil-based chemotherapeutic agent.
  • the patient is treated with both a platinum- based chemotherapeutic agent and a 5-fluorouracil-based chemotherapeutic agent.
  • the patient has previously undergone surgery to remove a gastric tumor.
  • the patient has or had stage II or stage III gastric cancer.
  • methods comprise calculating a risk score of recurrence for the patient based on methylation of one or more biomarker(s), tumor size, and lymph node status.
  • the risk score identifies the patient as having a risk of recurrence that is or is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or higher percentage of having recurrence within a certain time period, such as within 3, 6, 9, 12 months and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years.
  • the kit further comprises one or more agents for measuring or detecting or quantifying methylation in C3orf2l, EPB41L1, GAK, PPP1R1B, SLC12A7, cg03364486, cgl267l030, FANCA, GNPDA2, KCTD1, ONECUT2, PFKB3, PPT2, or ZNF638 or for detecting one or more controls.
  • the kit further comprises reagents for isolating nucleic acids from a biological sample.
  • the reagents are for isolating nucleic acids from a serum sample.
  • the reagents are for isolating nucleic acids from a sample described herein.
  • the term subject or patient may refer to an animal (for example a mammal), including but not limited to humans, non-human primates, rodents, dogs, or pigs.
  • the methods of obtaining provided herein include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy.
  • the sample is obtained from a biopsy from esophageal, stomach or the muscle tissue, mucosa or submucosa thereof.
  • the sample may be obtained from any of the tissues provided herein that include but are not limited to gall bladder, skin, heart, lung, breast, pancreas, liver, muscle, kidney, smooth muscle, bladder, intestine, brain, prostate, or thyroid tissue.
  • the sample may include but not be limited to blood, serum, sweat, hair follicle, buccal tissue, tears, menses, urine, feces, or saliva.
  • the sample may be a tissue sample, a whole blood sample, a urine sample, a saliva sample, a serum sample, a plasma sample or a fecal sample.
  • the sample is obtained from cystic fluid or fluid derived from a tumor or neoplasm.
  • the cyst, tumor or neoplasm is in the digestive system.
  • any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing.
  • the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.
  • the sample may be a fresh, frozen or preserved sample or a fine needle aspirate.
  • the sample is a formalin-fixed, paraffin- embedded (FFPE) sample.
  • An acquired sample may be placed in short term or long term storage by placing in a suitable medium, excipient, solution, or container. In certain cases storage may require keeping the sample in a refrigerated, or frozen environment. The sample may be quickly frozen prior to storage in a frozen environment. In certain instances the frozen sample may be contacted with a suitable cryopreservation medium or compound.
  • cryopreservation mediums or compounds include but are not limited to: glycerol, ethylene glycol, sucrose, or glucose.
  • Some embodiments further involve isolating nucleic acids such as ribonucleic or RNA from a biological sample or in a sample of the patient.
  • Other steps may or may not include amplifying a nucleic acid in a sample and/or hybridizing one or more probes to an amplified or non-amplified nucleic acid.
  • the methods may further comprise assaying nucleic acids in a sample.
  • a microarray may be used to measure or assay the level of biomarker expression in a sample.
  • the methods may further comprise recording the biomarker expression level in a tangible medium or reporting the expression level to the patient, a health care payer, a physician, an insurance agent, or an electronic system.
  • a difference between or among weighted coefficients ore expression levels or between or among the weighted comparisons may be, be at least or be at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0.
  • determination of calculation of a diagnostic, prognostic, or risk score is performed by applying classification algorithms based on the expression values of biomarkers with differential expression p values of about, between about, or at most about 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.020, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.040, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.050, 0.051, 0.052, 0.053, 0.054, 0.055, 0.056,
  • any of the methods described herein may be implemented on tangible computer- readable medium comprising computer-readable code that, when executed by a computer, causes the computer to perform one or more operations.
  • a tangible computer-readable medium comprising computer-readable code that, when executed by a computer, causes the computer to perform operations comprising: a) receiving information corresponding to an expression level of a biomarkers in a sample from a patient; and b) determining a difference value in the expression levels using the information corresponding to the expression levels in the sample compared to a control or reference expression level for the gene.
  • tangible computer-readable medium further comprise computer- readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising making recommendations comprising: wherein the patient in the step a) is under or after a first treatment for cancer, administering the same treatment as the first treatment to the patient if the patient does not have increased expression level; administering a different treatment from the first treatment to the patient if the patient has increased expression level.
  • receiving information comprises receiving from a tangible data storage device information corresponding to the expression levels from a tangible storage device.
  • the medium further comprises computer-readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising: sending information corresponding to the difference value to a tangible data storage device, calculating a prognosis score for the patient, treating the patient with a traditional therapy if the patient does not have expression levels, and/or or treating the patient with an alternative esophageal therapy if the patient has increased expression levels.
  • the tangible, computer-readable medium further comprise computer-readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising calculating a prognosis score for the patient.
  • the operations may further comprise making recommendations comprising: administering a treatment to a patient that is determined to have a decreased expression level.
  • the terms “or” and“and/or” are utilized to describe multiple components in combination or exclusive of one another.
  • “x, y, and/or z” can refer to“x” alone,“y” alone,“z” alone,“x, y, and z,”“(x and y) or z,”“x or (y and z),” or“x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.
  • any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention.
  • any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
  • Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.
  • FIG. 1A-D Genome-wide methylation profiling to predict GC recurrence.
  • A Heatmap illustrates the methylation levels of the 14 CpG probes differentially methylated between Rec and Non-Rec in the TCGA cohort.
  • B Lasso-Cox regression analysis showed selected 14 differentially methylated probes between Rec and Non-Rec in the TCGA cohort.
  • C ROC curve shows the predictive performance of the 14 candidate probes for discriminating Rec from Non-rec patients in TCGA cohort.
  • Kaplan-Meier curve shows the RFS and OS of stage II and III GC patients based on risk score determined by logistic regression analysis using 14 candidate probes in TCGA cohort (Log-rank test).
  • FIG. 2A-B Evaluation of the identified 14 CGIs/DMRs in two independent clinical sample cohorts.
  • A Forest plots show AUC values and hazard ratio for discriminating Rec of 6 CpG methylation probes identified by backward stepwise logistic regression analysis.
  • B Kaplan-Meier curves show the RFS and OS of stage II and III GC patients stratified by ONECUT2 methylation level in the training and validation cohorts (Log-rank test).
  • FIG. 3A-C Develop and validation for CpG methylation-based recurrence prediction nomogram.
  • A CpG methylation-based recurrence prediction nomogram was established using multivariate logistic regression analysis for predicting recurrence using the methylation level of ONECUT2, tumor size and lymph node metastasis status in the training cohort.
  • B ROC curves show the predictive performance of the CMRPN in the training and validation cohorts.
  • (C) Kaplan-Meier curves show the RFS and OS of stage II and III GC patients stratified by the risk score determined by CMRPN in the training and validation cohorts (Log-rank test).
  • FIG. 4 The graph shows the result of the backward stepwise logistic regression analysis for predicting recurrence in the training cohort.
  • Certain aspects of the invention provide a test that could assist physicians to select the optimal therapy for a patient from several alternative treatment options.
  • a major clinical challenge in cancer treatment is to identify the subset of patients who will benefit from a therapeutic regimen, both in metastatic and adjuvant settings.
  • the number of anti-cancer drugs and multi-drug combinations has increased substantially in the past decade, however, treatments continue to be applied empirically using a trial- and-error approach.
  • methods and compositions are provided to diagnose patients to determine the optimal treatment option for cancer patients.
  • the inventors Using a systematic and comprehensive biomarker discovery, prioritization and validation approach, the inventors have established a novel methylation-based recurrence prediction nomogram that can assist clinicians in post-surgical decision-making in gastric cancer patients.
  • the term“substantially the same”,“not significantly different”, or“within the range” refers to a level of expression that is not significantly different than what it is compared to.
  • the term substantially the same refers to a level of expression that is less than 2, 1.5, or 1.25 fold different than the expression level it is compared to or less than 20, 15, 10, or 5% difference in expression.
  • subject or“patient” is meant any single subject for which therapy is desired, including humans, cattle, dogs, guinea pigs, rabbits, chickens, and so on. Also intended to be included as a subject are any subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects used as controls.
  • primer or“probe” as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded and/or single- stranded form, although the single-stranded form is preferred.
  • a probe may also refer to a nucleic acid that is capable of hybridizing by base complementarity to a nucleic acid of a gene of interest or a fragment thereof.
  • “increased” or“elevated” or“decreased” refers to methylation level of a biomarker in the subject’s sample as compared to a reference level representing the same biomarker or a different biomarker.
  • the reference level may be a reference level of expression from a non-cancerous tissue from the same subject.
  • the reference level may be a reference level of expression from a different subject or group of subjects.
  • the reference level of expression may be an expression level obtained from a sample (e.g., a tissue, fluid or cell sample) of a subject or group of subjects without cancer, with gastric cancer, or an expression level obtained from a non-cancerous tissue of a subject or group of subjects with cancer.
  • the reference level may be a single value or may be a range of values.
  • the reference level of expression can be determined using any method known to those of ordinary skill in the art.
  • the reference level may also be depicted graphically as an area on a graph.
  • a reference level is a normalized level.
  • CpG island refers to regions of DNA with a high G/C content and a high frequency of CpG dinucleotides relative to the whole genome of an organism of interest. Also used interchangeably in the art is the term "CG island.” The 'r' in “CpG island” refers to the phosphodiester bond between the cytosine and guanine nucleotides. Methylation may refer to methylation and/or hydroxymethylation of DNA.
  • Methods and compositions may be provided for treating gastric cancer with particular applications of biomarkers. Based on a profile of methylation markers, different treatments may be prescribed or recommended for different cancer patients and patient populations.
  • Gastric cancer also known as stomach cancer tends to develop slowly over many years. Before a true cancer develops, pre-cancerous changes often occur in the inner lining (mucosa) of the stomach. These early changes rarely cause symptoms and therefore often go undetected.
  • Cancers starting in different sections of the stomach may cause different symptoms and tend to have different outcomes.
  • the cancer’s location can also affect the treatment options. For example, cancers that start at the gastroesophageal (GE) junction are staged and treated the same as cancers of the esophagus.
  • GE gastroesophageal
  • a cancer that starts in the cardia of the stomach but then grows into the GE junction is also staged and treated like a cancer of the esophagus.
  • Stomach cancers can spread (metastasize) in different ways. They can grow through the wall of the stomach and invade nearby organs. They can also spread to the lymph vessels and nearby lymph nodes. Lymph nodes are bean-sized structures that help fight infections.
  • the stomach has a very rich network of lymph vessels and nodes. As the stomach cancer becomes more advanced, it can travel through the bloodstream and spread to organs such as the liver, lungs, and bones. If cancer has spread to the lymph nodes or to other organs, the patient’s outlook is not as good.
  • stomach cancer includes: adenocarcinomas, lymphomas, gastrointestinal stromal tumor (GIST), and carcinoid tumor. Squamous cell carcinoma, small cell carcinoma, and leiomyosarcoma, can also start in the stomach, but these cancers are very rare.
  • the most common staging system is the TNM (for tumors/nodes/metastases) system, from the American Joint Committee on Cancer (AJCC).
  • the TNM system assigns a number based on three categories.“T” denotes the degree of invasion of the intestinal wall, “N” the degree of lymphatic node involvement, and“M” the degree of metastasis.
  • T denotes the degree of invasion of the intestinal wall
  • N the degree of lymphatic node involvement
  • M the degree of metastasis.
  • the broader stage of a cancer is usually quoted as a number I, II, III, IV derived from the TNM value grouped by prognosis; a higher number indicates a more advanced cancer and likely a worse outcome. Details of this system are in the tables below:
  • The“cancer” referred to in the methods described herein may include or exclude any of the above stages or TNM categories.
  • the cancer may be or may exclude Stage 0, Stage I-A, Stage I-B, Stage II-A, Stage II-B, Stage III-A, Stage III-B, Stage III-C, or Stage
  • the patient may be one that has and/or has been determined to have Stage 0, Stage I-A, Stage I-B, Stage II-A, Stage II-B, Stage III-A, Stage III-B, Stage III-C, or Stage IV cancer.
  • the cancer may be stage NO and/or M0; Tl, NO, and/or M0; Tl, Nl, and/or M0; T2, NO, and/or M0; Tl, N2, and/or M0; T2, Nl, and/or M0; T3, NO, and/or M0; Tl, N3, and/or MO; T2, N2, and/or MO; T3, Nl, and/or MO; T4a, NO, and/or MO; T2, N3, and/or MO; T3, N2, and/or MO; T4a, Nl, and/or MO; T3, N3, and/or MO; T4a, Nl, and/or MO; T3, N3, and/or MO; T4a, Nl, and/or MO; T3, N3, and/
  • therapies that may be included in method embodiments of the disclosure include the following therapies described in this section as well as other therapies described throughout the disclosure.
  • the methods described herein may include or exclude any of the cancer therapies described in the disclosure.
  • the methods include endoscopic mucosal resection. This is the removal of the tumor with an endoscope.
  • the methods include surgery to remove the part of the stomach with cancer and nearby lymph nodes.
  • the surgery comprises a subtotal or partial gastrectomy. In a partial gastrectomy, the surgeon connects the remaining part of the stomach to the esophagus or small intestine.
  • the methods include surgery plus chemotherapy or chemotherapy and radiation therapy.
  • Embodiments include a subtotal gastrectomy or a total gastrectomy, which is the removal of all of the stomach. During a total gastrectomy, the surgeon attaches the esophagus directly to the small intestine. Regional lymph nodes may also be removed during surgery. Accordingly, embodiments of the disclosure may include a lymphadenectomy .
  • Radiation therapy is the use of high-energy x-rays or other particles to destroy cancer cells.
  • a radiation therapy regimen may comprise a specific number of treatments given over a set period of time.
  • the radiation therapy comprises external-beam radiation therapy, which is radiation given from a machine outside the body. Radiation therapy may be used before surgery to shrink the size of the tumor or after surgery to destroy any remaining cancer cells.
  • chemotherapy is administered to the patient.
  • the patient is one that has received a prior chemotherapeutic treatment.
  • the chemotherapy may be administered systemically, intravenously, or orally.
  • a chemotherapy regimen may comprise a specific number of cycles given over a set period of time.
  • a patient may receive 1 drug at a time or combinations of different drugs at the same time.
  • the goal of chemotherapy can be to destroy cancer remaining after surgery, slow the tumor’s growth, or reduce cancer-related symptoms.
  • the patient is administered chemotherapy in combination with radiation therapy.
  • chemotherapeutic regimens include, for example, the combination of fluorouracil (5-FU, Adrucil) and cisplatin (Platinol).
  • Drugs similar to 5-FU such as capecitabine (Xeloda), and similar to cisplatin, such as oxaliplatin (Eloxatin), may be used in some embodiments.
  • Further embodiments include chemotherapeutic s such as docetaxel (Docefrez, Taxotere), epirubicin (Ellence), irinotecan (Camptosar), and paclitaxel (Taxol).
  • Antimetabolites can be used in cancer treatment, as they interfere with DNA production and therefore cell division and the growth of tumors. Because cancer cells spend more time dividing than other cells, inhibiting cell division harms tumor cells more than other cells. Anti-metabolites masquerade as a purine (azathioprine, mercaptopurine) or a pyrimidine, chemicals that become the building-blocks of DNA. They prevent these substances becoming incorporated in to DNA during the S phase (of the cell cycle), stopping normal development and division. They also affect RNA synthesis.
  • azathioprine azathioprine, mercaptopurine
  • pyrimidine chemicals that become the building-blocks of DNA. They prevent these substances becoming incorporated in to DNA during the S phase (of the cell cycle), stopping normal development and division. They also affect RNA synthesis.
  • thymidine is used in DNA but not in RNA (where uracil is used instead)
  • inhibition of thymidine synthesis via thymidylate synthase selectively inhibits DNA synthesis over RNA synthesis. Due to their efficiency, these drugs are the most widely used cytostatics. In the ATC system, they are classified under L01B.
  • Thymidylate synthase inhibitors are chemical agents which inhibit the enzyme thymidylate synthase and have potential as an anticancer chemotherapy.
  • thymidylate synthetase can be inhibited by the thymidylate synthase inhibitors such as fluorinated pyrimidine fluorouracil, or certain folate analogues, the most notable one being raltitrexed (trade name Tomudex).
  • Additional agents include pemetrexed, nolatrexed, ZD9331, and GS7904L.
  • prodrugs that can be converted to thymidylate synthase inhibitors in the body, such as Capecitabine (INN), an orally- administered chemotherapeutic agent used in the treatment of numerous cancers.
  • Capecitabine is a prodrug, that is enzymatically converted to 5-fluorouracil in the body.
  • Chemotherapy agents for this condition may include capecitabine, fluorouracil, irinotecan, leucovorin, oxaliplatin and UFT.
  • Another type of agent that is sometimes used are the epidermal growth factor receptor inhibitors.
  • cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments.
  • Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, famesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate,
  • CDDP cisplatin
  • carboplatin carboplatin
  • procarbazine mechlorethamine
  • the chemotherapy is used in a neoadjuvant setting. In some embodiments, chemotherapy is used in an adjuvant and neoadjuvant setting.
  • radiotherapy can be used in the neoadjuvant and adjuvant setting for some stages of gastric cancer.
  • Targeted therapy may also be used in the methods described herein.
  • Targeted therapy is a treatment that targets the cancer’s specific genes, proteins, or the tissue environment that contributes to cancer growth and survival. This type of treatment blocks the growth and spread of cancer cells while limiting damage to healthy cells.
  • the doctor may run tests to identify the genes, proteins, and other factors in your tumor. This helps doctors better match each patient with the most effective treatment whenever possible.
  • the methods further comprise testing a biological sample from the patient for HER2 expression.
  • the patients with HER2-positive stomach cancer are treated with trastuzumab (Herceptin) In some embodiments, this is in combination with chemotherapy.
  • Herceptin is one type of HER2-targeted therapy.
  • ASCO HER2 positive
  • ASCP ASCP
  • CAP recommend a combination of chemotherapy and HER2-targeted therapy. If the cancer is HER2 negative, HER2-targeted therapy is not a treatment option for you, and your doctor will give you other options for treating the cancer.
  • Ramucirumab is a type of targeted therapy called an anti- angiogenic. It is focused on stopping angiogenesis, which is the process of making new blood vessels. Because a tumor needs the nutrients delivered by blood vessels to grow and spread, the goal of anti-angiogenesis therapies is to“starve” the tumor.
  • Immunotherapies that are designed to boost the body’s natural defenses to fight the cancer may also be used.
  • Immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells. Immune therapy methods are further described below:
  • Embodiments of the disclosure may include administration of immune checkpoint inhibitors, which are further described below.
  • PD-l can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-l and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-l is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-l and/or PDL1 activity.
  • Alternative names for“PD-l” include CD279 and SLEB2.
  • Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H.
  • Alternative names for“PDL2” include B7- DC, Btdc, and CD273.
  • PD-l, PDL1, and PDL2 are human PD-l, PDL1 and PDL2.
  • the PD-l inhibitor is a molecule that inhibits the binding of PD-l to its ligand binding partners.
  • the PD-l ligand binding partners are PDL1 and/or PDL2.
  • a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners.
  • PDL1 binding partners are PD-l and/or B7-1.
  • the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners.
  • a PDL2 binding partner is PD-l.
  • the inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference.
  • Other PD-l inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014/0294898, US 2014/022021, and US2011/0008369, all incorporated herein by reference.
  • the PD-l inhibitor is an anti-PD-l antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody).
  • the anti-PD- 1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab.
  • the PD-l inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-l binding portion of PDL1 or PDL2 fused to a constant region (e.g. , an Fc region of an immunoglobulin sequence).
  • the PDL1 inhibitor comprises AMP- 224.
  • Nivolumab also known as MDX- 1106-04, MDX- 1106, ONO-4538, BMS-936558, and OPDIVO ® , is an anti-PD-l antibody described in W 02006/121168.
  • Pembrolizumab also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA ® , and SCH-900475, is an anti-PD-l antibody described in W02009/114335.
  • Pidilizumab also known as CT-011, hBAT, or hBAT-l, is an anti-PD-l antibody described in W02009/101611.
  • AMP-224 also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342.
  • Additional PD-l inhibitors include MEDI0680, also known as AMP-514, and REGN2810.
  • the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof.
  • the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgMl2B7.
  • the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab.
  • the antibody competes for binding with and/or binds to the same epitope on PD-l, PDL1, or PDL2 as the above- mentioned antibodies.
  • the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • CD 152 cytotoxic T-lymphocyte-associated protein 4
  • the complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006.
  • CTLA-4 is found on the surface of T cells and acts as an“off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells.
  • CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells.
  • CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells.
  • CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal.
  • Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.
  • Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and/or B7-2 activity. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the CTLA-4 and B7-2 interaction.
  • the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • an anti-CTLA-4 antibody e.g., a human antibody, a humanized antibody, or a chimeric antibody
  • an antigen binding fragment thereof e.g., an immunoadhesin, a fusion protein, or oligopeptide.
  • Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.
  • art recognized anti-CTLA-4 antibodies can be used.
  • the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz el al, 1998; can be used in the methods disclosed herein.
  • the teachings of each of the aforementioned publications are hereby incorporated by reference.
  • Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used.
  • a humanized CTLA-4 antibody is described in International Patent Application No. W 02001/014424, W02000/037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.
  • a further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WOO 1/14424).
  • the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab.
  • the antibody competes for binding with and/or binds to the same epitope on PD-l, B7-1, or B7-2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.
  • the methods comprise administration of a cancer immunotherapy.
  • Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer.
  • Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumour-associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates).
  • TAAs tumour-associated antigens
  • Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs.
  • Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines. Immumotherapies are known in the art, and some are described below.
  • the immunotherapy comprises an inhibitor of a co stimulatory molecule.
  • the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof.
  • Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.
  • Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen.
  • Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting.
  • APCs antigen presenting cells
  • One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.
  • One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses.
  • adjuvants include proteins or other chemicals that attract and/or activate dendritic cells, such as granulocyte macrophage colony- stimulating factor (GM-CSF).
  • Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.
  • Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body.
  • the dendritic cells are activated in the presence of tumor antigens, which may be a single tumor- specific peptide/protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.
  • Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.
  • Chimeric antigen receptors are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources.
  • CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.
  • CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions.
  • the general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells.
  • scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells.
  • CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signalling molecule which in turn activates T cells.
  • the extracellular ligand recognition domain is usually a single-chain variable fragment (scFv).
  • CAR-T cell therapy An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells.
  • the specificity of CAR-T cells is determined by the choice of molecule that is targeted.
  • Exemplary CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta).
  • the CAR-T therapy targets CD 19.
  • Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.
  • Interferons are produced by the immune system. They are usually involved in anti viral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IFNk).
  • Interleukins have an array of immune system effects.
  • IF-2 is an exemplary interleukin cytokine therapy.
  • Adoptive T cell therapy is a form of passive immunization by the transfusion of T- cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically they activate when the T-cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TIFs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumour death. [60]
  • APCs antigen presenting cells
  • T-cells specific to a tumor antigen can be removed from a tumor sample (TIFs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens.
  • a cancer treatment may exclude any of the cancer treatments described herein.
  • embodiments of the disclosure include patients that have been previously treated for a therapy described herein, are currently being treated for a therapy described herein, or have not been treated for a therapy described herein.
  • the patient is one that has been determined to be resistant to a therapy described herein.
  • the patient is one that has been determined to be sensitive to a therapy described herein.
  • the method may be combined with one or more other gastric cancer diagnosis or screening tests at increased frequency if the patient is determined to be at high risk for recurrence or have a poor prognosis based on the biomarkers.
  • the monitoring protocol may include any methods known in the art.
  • the monitoring include obtaining a sample and testing the sample for diagnosis.
  • the monitoring may include endoscopy, biopsy, endoscopic ultrasound, X-ray, barium swallow, a Ct scan, a MRI, a PET scan, laparoscopy, or HER2 testing.
  • a receiver operating characteristic (ROC), or ROC curve, is a graphical plot that illustrates the performance of a binary classifier system as its discrimination threshold is varied. The curve is created by plotting the true positive rate against the false positive rate at various threshold settings.
  • the true-positive rate is also known as sensitivity in biomedical informatics, or recall in machine learning.
  • the false-positive rate is also known as the fall-out and can be calculated as 1 - specificity).
  • the ROC curve is thus the sensitivity as a function of fall-out.
  • the ROC curve can be generated by plotting the cumulative distribution function (area under the probability distribution from -infinity to + infinity) of the detection probability in the y- axis versus the cumulative distribution function of the false-alarm probability in x-axis.
  • ROC analysis provides tools to select possibly optimal models and to discard suboptimal ones independently from (and prior to specifying) the cost context or the class distribution. ROC analysis is related in a direct and natural way to cost/benefit analysis of diagnostic decision making.
  • ROC curve was first developed by electrical engineers and radar engineers during World War II for detecting enemy objects in battlefields and was soon introduced to psychology to account for perceptual detection of stimuli. ROC analysis since then has been used in medicine, radiology, biometrics, and other areas for many decades and is increasingly used in machine learning and data mining research.
  • the ROC is also known as a relative operating characteristic curve, because it is a comparison of two operating characteristics (TPR and FPR) as the criterion changes.
  • ROC analysis curves are known in the art and described in Metz CE (1978) Basic principles of ROC analysis. Seminars in Nuclear Medicine 8:283-298; Youden WJ (1950) An index for rating diagnostic tests. Cancer 3:32-35; Zweig MH, Campbell G (1993) Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clinical Chemistry 39:561-577; and Greiner M, Pfeiffer D, Smith RD (2000) Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. Preventive Veterinary Medicine 45:23-41, which are herein incorporated by reference in their entirety.
  • a ROC analysis may be used to create cut-off values for prognosis and/or diagnosis purposes.
  • a meta-analysis of expression or activity can be performed.
  • a meta-analysis combines the results of several studies that address a set of related research hypotheses. This is normally done by identification of a common measure of effect size, which is modeled using a form of meta-regression.
  • three types of models can be distinguished in the literature on meta- analysis: simple regression, fixed effects meta regression and random effects meta-regression. Resulting overall averages when controlling for study characteristics can be considered meta-effect sizes, which are more powerful estimates of the true effect size than those derived in a single study under a given single set of assumptions and conditions.
  • a meta- methylation level value in this context, is to be understood as being the median of the normalized level of methylation of a biomarker. Normalization of the methylation of a biomarker can be achieved by dividing the expression level of the individual biomarker to be normalized by the respective individual median methylation of these biomarkers, wherein said median methylation can be calculated from multiple measurements of the respective biomarkers in a sufficiently large cohort of test individuals.
  • the test cohort may comprises at least 3, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 individuals or more including all values and ranges thereof. Dataset-specific bias can be removed or minimized allowing multiple datasets to be combined for meta-analyses (See Sims et al. BMC Medical Genomics (1:42), 1-14, 2008, which is incorporated herein by reference in its entirety).
  • the calculation of a meta-gene expression value is performed by: (i) determining the methylation level value of at least two, preferably more genes (ii) "normalizing" the gene expression value of each individual gene by dividing the expression value with a coefficient which is approximately the median methylation level value of the respective gene in a representative recurrent gastric cancer cohort (iii) calculating the median of the group of normalized gene expression values. The same could occur in a nonrecurrent gastric cancer cohort.
  • a suitable threshold level of methylation is first determined for a biomarker.
  • the suitable threshold level can be determined from measurements of the biomarker methylation in multiple individuals from a test cohort. The median methylation of the biomarker in said multiple methylation measurements is taken as the suitable threshold value.
  • Comparison of multiple biomarkers with a threshold level can be performed as follows:
  • the methylation levels of one or more biomarkers are within a predetermined amount of the mean methylation levels of the one or more biomarkers, on a biomarker-by-biomarker basis, in the biological samples from a cohort of patients having a recurrent gastric cancer or a cohort of patients not having recurrent gastric cancer within a certain time period, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.
  • the mean levels may be determined by measuring the methylation levels of biomarkers in samples from patients in the cohort and calculating a mean methylation level for each biomarker.
  • the patients are patients having gastric cancer.
  • Classification of a metastasis may be done by comparing the measured methylation levels of biomarkers to reference methylation levels of the same biomarkers.
  • the reference methylation levels may be identified as the mean methylation levels in a cohort of patients recurrent gastric cancer or nonrecurrent gastric cancer.
  • the reference methylation levels of such cohorts, and of any patient cohorts described herein, may be established by measuring the methylation levels in biological samples of at least, at most, or exactly 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 subjects in the cohort, or any range derivable therein.
  • the cohort of patients comprises a representative sample of gastric cancer patients who experience recurrence within a certain time period such as within 1, 2, 3, 4, or 5 years. If the methylation levels of the biomarkers measured in a sample are sufficiently close to the reference methylation levels of the recurrent gastric cancer sample, then the sample in question can be classified as being of that characteristic.
  • the degree of closeness in methylation levels required to be classified as a match may be predetermined using a statistical analysis. In some embodiments, the predetermined amount of closeness is within one standard deviation of the mean expression level of the reference cohort.
  • the predetermined amount is within 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 10, 15, or 20% of the reference methylation level, or any range derivable therein.
  • a sample may be classified as belonging to a recurrent or nonrecurrent type despite the methylation levels of one or more biomarkers deviating from a reference expression level by a substantial amount. For instance, if a substantial number of other biomarker methylation levels sufficiently match the reference methylation, then the sample metastasis may be classified as belonging to the subtype.
  • a computer-based classifier programmed to perform a statistical analysis may be used to determine whether methylation levels of a sufficient number of biomarkers in a sample are sufficiently close to the reference methylation levels of a particular molecular subtype to classify the sample as belonging to that subtype.
  • the methods described herein may involve a comparison between methylation levels measured for a sample and reference methylation levels that are indicative of either recurrent or nonrecurrent gastric cancer.
  • the measured methylation level for a biomarker is lower than, higher than, close to, higher by a predetermined amount than, lower by a predetermined amount than, or within a predetermined amount of the methylation level of the biomarker from a cohort of recurrent gastric cancer patients or nonrecurrent gastric cancer patients.
  • a unique collection of biomarkers as a genetic classifier with respect to methylation states in a cancer tissue is provided that is useful in determining likelihood of recurrence of cancer.
  • the panel also provides relevant information about recurrence and/or treatment with other cancer treatment such as chemotherapeutic s, radiation, and/or immunotherapeutics.
  • Such a collection may be termed a“biomarker panel,”“expression classifier,” or“classifier.”
  • a score is calculated based on the methylation profile of a patient.
  • the value assigned to represent the expression of one or more genes may be adjusted.
  • a weight is attached to one or more values.
  • the term “weight” refers to the relative importance of an item in a statistical calculation. The weight of each biomarker in a methylation level classifier may be determined on a data set of patient samples using analytical methods known in the art. III. Sample Preparation
  • methods involve obtaining a sample from a subject.
  • the methods of obtaining provided herein may include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy.
  • the sample is obtained from a biopsy from esophageal tissue by any of the biopsy methods previously mentioned.
  • the sample may be obtained from any of the tissues provided herein that include but are not limited to non-cancerous or cancerous tissue and non-cancerous or cancerous tissue from the serum, gall bladder, mucosal, skin, heart, lung, breast, pancreas, blood, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue.
  • the sample may be obtained from any other source including but not limited to blood, sweat, hair follicle, buccal tissue, tears, menses, feces, or saliva.
  • any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing.
  • the biological sample can be obtained without the assistance of a medical professional.
  • a sample may include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject.
  • the biological sample may be a heterogeneous or homogeneous population of cells or tissues.
  • the biological sample may be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein.
  • the sample may be obtained by non-invasive methods including but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen.
  • the sample may be obtained by methods known in the art.
  • the samples are obtained by biopsy.
  • the sample is obtained by swabbing, endoscopy, scraping, phlebotomy, or any other methods known in the art.
  • the sample may be obtained, stored, or transported using components of a kit of the present methods.
  • multiple samples such as multiple esophageal samples may be obtained for diagnosis by the methods described herein.
  • multiple samples such as one or more samples from one tissue type (for example esophagus) and one or more samples from another specimen (for example serum) may be obtained for diagnosis by the methods.
  • multiple samples such as one or more samples from one tissue type (e.g.
  • samples from another specimen may be obtained at the same or different times.
  • Samples may be obtained at different times are stored and/or analyzed by different methods. For example, a sample may be obtained and analyzed by routine staining methods or any other cytological analysis methods.
  • the biological sample may be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or a pulmonologist.
  • the medical professional may indicate the appropriate test or assay to perform on the sample.
  • a molecular profiling business may consult on which assays or tests are most appropriately indicated.
  • the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.
  • the sample is obtained by an invasive procedure including but not limited to: biopsy, needle aspiration, endoscopy, or phlebotomy.
  • the method of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy.
  • multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.
  • the sample is a fine needle aspirate of a esophageal or a suspected esophageal tumor or neoplasm.
  • the fine needle aspirate sampling procedure may be guided by the use of an ultrasound, X-ray, or other imaging device.
  • the molecular profiling business may obtain the biological sample from a subject directly, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party.
  • the biological sample may be obtained by the molecular profiling business after the subject, a medical professional, or a third party acquires and sends the biological sample to the molecular profiling business.
  • the molecular profiling business may provide suitable containers, and excipients for storage and transport of the biological sample to the molecular profiling business.
  • a medical professional need not be involved in the initial diagnosis or sample acquisition.
  • An individual may alternatively obtain a sample through the use of an over the counter (OTC) kit.
  • OTC kit may contain a means for obtaining said sample as described herein, a means for storing said sample for inspection, and instructions for proper use of the kit.
  • molecular profiling services are included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately.
  • a sample suitable for use by the molecular profiling business may be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and/or adequacy are provided.
  • the subject may be referred to a specialist such as an oncologist, surgeon, or endocrinologist.
  • the specialist may likewise obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of the biological sample.
  • the medical professional may refer the subject to a testing center or laboratory for submission of the biological sample.
  • the subject may provide the sample.
  • a molecular profiling business may obtain the sample.
  • aspects of the methods include assaying nucleic acids to determine expression levels and/or methylation levels of nucleic acids.
  • Embodiments of the disclosure include the detection of one or more CpG islands, such as at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands (or any range derivable therein).
  • Each biomarker may comprise or consist of at least or at most or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands (or any range derivable therein).
  • Assays for the detection of methylated DNA are known in the art. Exemplary methods are described herein.
  • HPLC-ETV high performance liquid chromatography-ultraviolet
  • Kuo and colleagues in 1980 (described further in Kuo K.C. et ah, Nucleic Acids Res. 1980;8:4763-4776, which is herein incorporated by reference) can be used to quantify the amount of deoxycytidine (dC) and methylated cytosines (5 mC) present in a hydrolysed DNA sample.
  • the method includes hydrolyzing the DNA into its constituent nucleoside bases, the 5 mC and dC bases are separated chromatographically and, then, the fractions are measured. Then, the 5 mC/dC ratio can be calculated for each sample, and this can be compared between the experimental and control samples.
  • LC-MS/MS Liquid chromatography coupled with tandem mass spectrometry
  • HPLC-ETV high-sensitivity approach to HPLC-ETV, which requires much smaller quantities of the hydrolysed DNA sample.
  • LC-MS/MS has been validated for detecting levels of methylation levels ranging from 0.05%-l0%, and it can confidently detect differences between samples as small as -0.25% of the total cytosine residues, which corresponds to -5% differences in global DNA methylation.
  • the procedure routinely requires 50-100 ng of DNA sample, although much smaller amounts (as low as 5 ng) have been successfully profiled.
  • Another major benefit of this method is that it is not adversely affected by poor-quality DNA (e.g., DNA derived from FFPE samples).
  • ELISA enzyme-linked immunosorbent assay
  • these assays include Global DNA Methylation ELISA, available from Cell Biolabs; Imprint Methylated DNA Quantification kit (sandwich ELISA), available from Sigma- Aldrich; EpiSeeker methylated DNA Quantification Kit, available from abeam; Global DNA Methylation Assay— LINE-l, available from Active Motif; 5-mC DNA ELISA Kit, available from Zymo Research; MethylFlash Methylated DNA5-mC Quantification Kit and MethylFlash Methylated DNA5-mC Quantification Kit, available from Epigentek.
  • ELISA enzyme-linked immunosorbent assay
  • the DNA sample is captured on an ELISA plate, and the methylated cytosines are detected through sequential incubations steps with: (1) a primary antibody raised against 5 Me; (2) a labelled secondary antibody; and then (3) colorimetric/fluorometric detection reagents.
  • the Global DNA Methylation Assay LINE-l specifically determines the methylation levels of LINE-l (long interspersed nuclear elements-l) retrotransposons, of which -17% of the human genome is composed. These are well established as a surrogate for global DNA methylation. Briefly, fragmented DNA is hybridized to biotinylated LINE-l probes, which are then subsequently immobilized to a streptavidin-coated plate. Following washing and blocking steps, methylated cytosines are quantified using an anti-5 mC antibody, HRP-conjugated secondary antibody and chemiluminescent detection reagents. Samples are quantified against a standard curve generated from standards with known LINE- 1 methylation levels. The manufacturers claim the assay can detect DNA methylation levels as low as 0.5%. Thus, by analysing a fraction of the genome, it is possible to achieve better accuracy in quantification. 4. LINE-1 + Pyrosequencing
  • Levels of LINE-l methylation can alternatively be assessed by another method that involves the bisulfite conversion of DNA, followed by the PCR amplification of LINE-l conservative sequences. The methylation status of the amplified fragments is then quantified by pyrosequencing, which is able to resolve differences between DNA samples as small as -5%. Even though the technique assesses LINE-l elements and therefore relatively few CpG sites, this has been shown to reflect global DNA methylation changes very well. The method is particularly well suited for high throughput analysis of cancer samples, where hypomethylation is very often associated with poor prognosis. This method is particularly suitable for human DNA, but there are also versions adapted to rat and mouse genomes.
  • Detection of fragments that are differentially methylated could be achieved by traditional PCR-based amplification fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP) or protocols that employ a combination of both.
  • AFLP PCR-based amplification fragment length polymorphism
  • RFLP restriction fragment length polymorphism
  • the LUMA (luminometric methylation assay) technique utilizes a combination of two DNA restriction digest reactions performed in parallel and subsequent pyrosequencing reactions to fill-in the protruding ends of the digested DNA strands.
  • One digestion reaction is performed with the CpG methylation-sensitive enzyme Hpall; while the parallel reaction uses the methylation-insensitive enzyme Mspl, which will cut at all CCGG sites.
  • the enzyme EcoRI is included in both reactions as an internal control. Both Mspl and Hpall generate 5'-CG overhangs after DNA cleavage, whereas EcoRI produces 5'-AATT overhangs, which are then filled in with the subsequent pyrosequencing-based extension assay.
  • the measured light signal calculated as the Hpall/Mspl ratio is proportional to the amount of unmethylated DNA present in the sample.
  • the specificity of the method is very high and the variability is low, which is essential for the detection of small changes in global methylation.
  • LUMA requires only a relatively small amount of DNA (250-500 ng), demonstrates little variability and has the benefit of an internal control to account for variability in the amount of DNA input.
  • WGBS Whole genome bisulfite sequencing
  • Bisulfite sequencing methods include reduced representation bisulfite sequencing (RRBS), where only a fraction of the genome is sequenced.
  • RRBS reduced representation bisulfite sequencing
  • enrichment of CpG-rich regions is achieved by isolation of short fragments after Mspl digestion that recognizes CCGG sites (and it cut both methylated and unmethylated sites). It ensures isolation of -85% of CpG islands in the human genome.
  • the RRBS procedure normally requires -100 ng - 1 pg of DNA.
  • direct detection of modified bases without bisulfite conversion may be used to detect methylation.
  • Pacific Biosciences company has developed a way to detect methylated bases directly by monitoring the kinetics of polymerase during single molecule sequencing and offers a commercial product for such sequencing (further described in Flusberg B.A., et ah, Nat. Methods. 2010;7:461-465, which is herein incorporated by reference).
  • Other methods include nanopore-based single-molecule real-time sequencing technology (SMRT), which is able to detect modified bases directly (described in Laszlo A.H. et ah, Proc. Natl. Acad. Sci. USA. 2013 and Schreiber J., et ah, Proc. Natl. Acad. Sci. USA. 2013, which are herein incorporated by reference). ;
  • Methylated DNA fractions of the genome could be used for hybridization with microarrays.
  • arrays include: the Human CpG Island Microarray Kit (Agilent), the GeneChip Human Promoter 1.0R Array and the GeneChip Human Tiling 2. OR Array Set (Affymetrix).
  • the search for differentially-methylated regions using bisulfite-converted DNA could be done with the use of different techniques. Some of them are easier to perform and analyse than others, because only a fraction of the genome is used. The most pronounced functional effect of DNA methylation occurs within gene promoter regions, enhancer regulatory elements and 3' untranslated regions (3'UTRs).
  • Assays that focus on these specific regions can be used.
  • the arrays can be used to detect methylation status of genes, including miRNA promoters, 5' UTR, 3' UTR, coding regions (-17 CpG per gene) and island shores (regions -2 kb upstream of the CpG islands).
  • bisulfite-treated genomic DNA is mixed with assay oligos, one of which is complimentary to uracil (converted from original unmethylated cytosine), and another is complimentary to the cytosine of the methylated (and therefore protected from conversion) site.
  • primers are extended and ligated to locus-specific oligos to create a template for universal PCR.
  • labelled PCR primers are used to create detectable products that are immobilized to bar-coded beads, and the signal is measured. The ratio between two types of beads for each locus (individual CpG) is an indicator of its methylation level.
  • VeraCode Methylation assay from Illumina, 96 or 384 user- specified CpG loci are analysed with the GoldenGate Assay for Methylation. Differently from the BeadChip assay, the VeraCode assay requires the BeadXpress Reader for scanning.
  • methylation- sensitive endonuclease(s) e.g., Hpall is used for initial digestion of genomic DNA in unmethylated sites followed by adaptor ligation that contains the site for another digestion enzyme that is cut outside of its recognized site, e.g., EcoPl5I or Mmel.
  • Hpall methylation- sensitive endonuclease
  • adaptor ligation that contains the site for another digestion enzyme that is cut outside of its recognized site, e.g., EcoPl5I or Mmel.
  • small fragments are generated that are located in close proximity to the original Hpall site.
  • NGS and mapping to the genome are performed. The number of reads for each Hpall site correlates with its methylation level.
  • FspEI, MspJI and LpnPI Three methylation-dependent endonucleases that are available from New England Biolabs (FspEI, MspJI and LpnPI) are type IIS enzymes that cut outside of the recognition site and, therefore, are able to generate snippets of 32bp around the fully-methylated recognition site that contains CpG. These short fragments could be sequences and aligned to the reference genome. The number of reads obtained for each specific 32-bp fragment could be an indicator of its methylation level.
  • short fragments could be generated from methylated CpG islands with Escherichia coli’s methyl-specific endonuclease McrBC, which cuts DNA between two half-sites of (G/A) mC that are lying within 50 bp-3000 bp from each other.
  • McrBC methyl- specific endonuclease
  • DNA including bisulfite-converted DNA could be used for the amplification of the region of interest followed by sequencing.
  • Primers are designed around the CpG island and used for PCR amplification of bisulfite-converted DNA.
  • the resulting PCR products could be cloned and sequenced.
  • aspects of the disclosure may include sequencing nucleic acids to detect methylation of nucleic acids and/or biomarkers.
  • the methods of the disclosure include a sequencing method. Exemplary sequencing methods include those described below.
  • MPSS Massively parallel signature sequencing
  • MPSS massively parallel signature sequencing
  • MPSS MPSS
  • the powerful Illumina HiSeq2000, HiSeq2500 and MiSeq systems are based on MPSS.
  • the Polony sequencing method developed in the laboratory of George M. Church at Harvard, was among the first next-generation sequencing systems and was used to sequence a full genome in 2005. It combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of >99.9999% and a cost approximately 1/9 that of Sanger sequencing.
  • the technology was licensed to Agencourt Biosciences, subsequently spun out into Agencourt Personal Genomics, and eventually incorporated into the Applied Biosystems SOLiD platform, which is now owned by Life Technologies.
  • a parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.
  • the method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.
  • the sequencing machine contains many picoliter-volume wells each containing a single bead and sequencing enzymes.
  • Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs. This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.
  • Solexa now part of Illumina, developed a sequencing method based on reversible dye-terminators technology, and engineered polymerases, that it developed internally.
  • the terminated chemistry was developed internally at Solexa and the concept of the Solexa system was invented by Balasubramanian and Klennerman from Cambridge University's chemistry department.
  • Solexa acquired the company Manteia Predictive Medicine in order to gain a massivelly parallel sequencing technology based on "DNA Clusters", which involves the clonal amplification of DNA on a surface.
  • the cluster technology was co-acquired with Lynx Therapeutics of California. Solexa Ltd. later merged with Lynx to form Solexa Inc.
  • DNA molecules and primers are first attached on a slide and amplified with polymerase so that local clonal DNA colonies, later coined "DNA clusters", are formed.
  • RT-bases reversible terminator bases
  • 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 for the next cycle to begin.
  • the DNA chains are extended one nucleotide at a time and image acquisition can be performed at a delayed moment, allowing for very large arrays of DNA colonies to be captured by sequential images taken from a single camera.
  • Applied Biosystems' (now a Thermo Fisher Scientific brand) SOLiD technology employs sequencing by ligation.
  • a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position.
  • Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position.
  • the DNA is amplified by emulsion PCR.
  • the resulting beads, each containing single copies of the same DNA molecule, are deposited on a glass slide. The result is sequences of quantities and lengths comparable to Illumina sequencing. This sequencing by ligation method has been reported to have some issue sequencing palindromic sequences. 6.
  • Ion Torrent Systems Inc. (now owned by Thermo Fisher Scientific) developed a system based on using standard sequencing chemistry, but with a novel, semiconductor based detection system. This method of sequencing is based on the detection of hydrogen ions that are released during the polymerization of DNA, as opposed to the optical methods used in other sequencing systems.
  • a microwell containing a template DNA strand to be sequenced is flooded with a single type of nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. 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.
  • DNA nanoball sequencing is a type of high throughput sequencing technology used to determine the entire genomic sequence of an organism.
  • the company Complete Genomics uses this technology to sequence samples submitted by independent researchers.
  • the method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Unchained sequencing by ligation is then used to determine the nucleotide sequence.
  • This method of DNA sequencing allows large numbers of DNA nanoballs to be sequenced per run and at low reagent costs compared to other next generation sequencing platforms. However, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult. This technology has been used for multiple genome sequencing projects.
  • Heliscope sequencing is a method of single-molecule sequencing developed by Helicos Biosciences. It uses DNA fragments with added poly-A tail adapters which are attached to the flow cell surface. The next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides (one nucleotide type at a time, as with the Sanger method). The reads are performed by the Heliscope sequencer. The reads are short, up to 55 bases per run, but recent improvements allow for more accurate reads of stretches of one type of nucleotides. This sequencing method and equipment were used to sequence the genome of the M13 bacteriophage. 9. Single molecule real time (SMRT) sequencing.
  • SMRT Single molecule real time
  • 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 labelled nucleotides flowing freely in the solution.
  • the wells are constructed in a way that only the fluorescence occurring by 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.
  • this methodology allows detection of nucleotide modifications (such as cytosine methylation). This happens through the observation of polymerase kinetics. This approach allows reads of 20,000 nucleotides or more, with average read lengths of 5 kilobases.
  • methods involve amplifying and/or sequencing one or more target genomic regions using at least one pair of primers specific to the target genomic regions.
  • the primers are heptamers.
  • enzymes are added such as primases or primase/polymerase combination enzyme to the amplification step to synthesize primers.
  • arrays can be used to detect nucleic acids of the disclosure.
  • An array comprises a solid support with nucleic acid probes attached to the support.
  • Arrays typically comprise a plurality of different nucleic acid probes that are coupled to a surface of a substrate in different, known locations.
  • These arrays also described as “microarrays” or colloquially “chips” have been generally described in the art, for example, U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186 and Fodor et al cache 1991), each of which is incorporated by reference in its entirety for all purposes.
  • arrays may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces.
  • Arrays may be nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, which are hereby incorporated in their entirety for all purposes.
  • nucleic acids include, but are not limited to, nucleic amplification, polymerase chain reaction, quantitative PCR, RT-PCR, in situ hybridization, digital PCR, dd PCR (digital droplet PCR), nCounter (nanoString), BEAMing (Beads, Emulsions, Amplifications, and Magnetics) (Inostics), ARMS (Amplification Refractory Mutation Systems), RNA-Seq, TAm-Seg (Tagged- Amplicon deep sequencing), PAP (Pyrophosphorolysis-activation polymerization), next generation RNA sequencing, northern hybridization, hybridization protection assay (HPA)(GenProbe), branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (US Genomics), Invader assay (Thir
  • Amplification primers or hybridization probes can be prepared to be complementary to a genomic region, biomarker, probe, or oligo described herein.
  • the term "primer” or“probe” as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process and/or pairing with a single strand of an oligo of the disclosure, or portion thereof.
  • primers are oligonucleotides from ten to twenty and/or thirty nucleic acids in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded and/or single- stranded form, although the single- stranded form is preferred.
  • a probe or primer of between 13 and 100 nucleotides particularly between 17 and 100 nucleotides in length, or in some aspects up to 1-2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective.
  • Molecules having complementary sequences over contiguous stretches greater than 20 bases in length may be used to increase stability and/or selectivity of the hybrid molecules obtained.
  • One may design nucleic acid molecules for hybridization having one or more complementary sequences of 20 to 30 nucleotides, or even longer where desired.
  • Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.
  • each probe/primer comprises at least 15 nucleotides.
  • each probe can comprise at least or at most 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400 or more nucleotides (or any range derivable therein). They may have these lengths and have a sequence that is identical or complementary to a gene described herein.
  • each probe/primer has relatively high sequence complexity and does not have any ambiguous residue (undetermined "n" residues).
  • the probes/primers can hybridize to the target gene, including its RNA transcripts, under stringent or highly stringent conditions. It is contemplated that probes or primers may have inosine or other design implementations that accommodate recognition of more than one human sequence for a particular biomarker.
  • relatively high stringency conditions For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids.
  • relatively low salt and/or high temperature conditions such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C.
  • Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.
  • quantitative RT-PCR (such as TaqMan, ABI) is used for detecting and comparing the levels or abundance of nucleic acids in samples.
  • concentration of the target DNA in the linear portion of the PCR process is proportional to the starting concentration of the target before the PCR was begun.
  • concentration of the PCR products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. This direct proportionality between the concentration of the PCR products and the relative abundances in the starting material is true in the linear range portion of the PCR reaction.
  • the final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. Therefore, the sampling and quantifying of the amplified PCR products may be carried out when the PCR reactions are in the linear portion of their curves.
  • relative concentrations of the amplifiable DNAs may be normalized to some independent standard/control, which may be based on either internally existing DNA species or externally introduced DNA species. The abundance of a particular DNA species may also be determined relative to the average abundance of all DNA species in the sample.
  • the PCR amplification utilizes one or more internal PCR standards.
  • the internal standard may be an abundant housekeeping gene in the cell or it can specifically be GAPDH, GUSB and b-2 microglobulin. These standards may be used to normalize expression levels so that the expression levels of different gene products can be compared directly. A person of ordinary skill in the art would know how to use an internal standard to normalize expression levels.
  • a problem inherent in some samples is that they are of variable quantity and/or quality. This problem can be overcome if the RT-PCR is performed as a relative quantitative RT-PCR with an internal standard in which the internal standard is an amplifiable DNA fragment that is similar or larger than the target DNA fragment and in which the abundance of the DNA representing the internal standard is roughly 5-100 fold higher than the DNA representing the target nucleic acid region.
  • the relative quantitative RT-PCR uses an external standard protocol. Under this protocol, the PCR products are sampled in the linear portion of their amplification curves. The number of PCR cycles that are optimal for sampling can be empirically determined for each target DNA fragment. In addition, the nucleic acids isolated from the various samples can be normalized for equal concentrations of amplifiable DNAs.
  • a nucleic acid array can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more different polynucleotide probes, which may hybridize to different and/or the same biomarkers. Multiple probes for the same gene can be used on a single nucleic acid array. Probes for other disease genes can also be included in the nucleic acid array.
  • the probe density on the array can be in any range. In some embodiments, the density may be or may be at least 50, 100, 200, 300, 400, 500 or more probes/cm2 (or any range derivable therein).
  • chip-based nucleic acid technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization (see also, Pease et al., 1994; and Fodor et al, 1991). It is contemplated that this technology may be used in conjunction with evaluating the expression level of one or more cancer biomarkers with respect to diagnostic, prognostic, and treatment methods.
  • Certain embodiments may involve the use of arrays or data generated from an array. Data may be readily available. Moreover, an array may be prepared in order to generate data that may then be used in correlation studies. V. Administration of Therapeutic Compositions
  • the therapy provided herein may comprise administration of a combination of therapeutic agents, such as a first cancer therapy and a second cancer therapy.
  • the therapies may be administered in any suitable manner known in the art.
  • the first and second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time).
  • the first and second cancer treatments are administered in a separate composition.
  • the first and second cancer treatments are in the same composition.
  • Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions.
  • the different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions.
  • Various combinations of the agents may be employed, for example, a first cancer treatment is “A” and a second cancer treatment is“B”:
  • the therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration.
  • the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
  • the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
  • the appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.
  • the treatments may include various“unit doses.”
  • Unit dose is defined as containing a predetermined-quantity of the therapeutic composition.
  • the quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts.
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
  • a unit dose comprises a single administrable dose.
  • the quantity to be administered, both according to number of treatments and unit dose depends on the treatment effect desired.
  • An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these agents.
  • doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400,
  • Such doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.
  • the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 mM to 150 mM.
  • the effective dose provides a blood level of about 4 mM to 100 mM.; or about 1 mM to 100 mM; or about 1 mM to 50 mM; or about 1 mM to 40 mM; or about 1 mM to 30 mM; or about 1 mM to 20 mM; or about 1 mM to 10 mM; or about 10 mM to 150 mM; or about 10 mM to 100 mM; or about 10 mM to 50 mM; or about 25 mM to 150 mM; or about 25 mM to 100 mM; or about 25 mM to 50 mM; or about 50 mM to 150 mM; or about 50 mM to 100 mM (or any range derivable therein).
  • the dose can provide the following blood level of the agent
  • the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent.
  • the blood levels discussed herein may refer to the unmetabolized therapeutic agent.
  • Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.
  • dosage units of pg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of pg/ml or mM (blood levels), such as 4 pM to 100 pM.
  • uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.
  • kits containing compositions of the invention or compositions to implement methods of the invention.
  • kits can be used to evaluate one or more biomarkers.
  • a kit contains, contains at least or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • kits for evaluating biomarker activity in a cell there are kits for evaluating biomarker activity in a cell.
  • Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.
  • Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more.
  • Kits for using probes, synthetic nucleic acids, nonsynthetic nucleic acids, and/or inhibitors of the disclosure for prognostic or diagnostic applications are included as part of the disclosure.
  • any such molecules corresponding to any biomarker identified herein which includes nucleic acid primers/primer sets and probes that are identical to or complementary to all or part of a biomarker, which may include noncoding sequences of the biomarker, as well as coding sequences of the biomarker.
  • kits may include a sample that is a negative or positive control for methylation of one or more biomarkers.
  • a control includes a nucleic acid that contains at least one CpG or is capable of identifying a CpG methylation site.
  • any embodiment of the disclosure involving specific biomarker by name is contemplated also to cover embodiments involving biomarkers whose sequences are at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the mature sequence of the specified nucleic acid.
  • kits for analysis of a pathological sample by assessing biomarker profile for a sample comprising, in suitable container means, two or more biomarker probes, wherein the biomarker probes detect one or more of the biomarkers identified herein.
  • the kit can further comprise reagents for labeling nucleic acids in the sample.
  • the kit may also include labeling reagents, including at least one of amine-modified nucleotide, poly(A) polymerase, and poly(A) polymerase buffer. Labeling reagents can include an amine- reactive dye.
  • Example 1- CpG methylation-based recurrence prediction nomogram in gastric adenocarcinoma
  • CRPN CpG methylation-based recurrence prediction nomogram
  • the inventors next examined the performance of these 14 methylated probes in a clinical training cohort using quantitative pyrosequencing.
  • a stepwise logistic regression analysis identified 6 probes that efficiently discriminated GC patients with recurrence.
  • the inventors further examined these probes in an independent validation cohort, and discovered that methylation levels of ONECUT2 significantly discriminated patients with and without recurrence - an observation that was also confirmed in the training cohort patients.
  • the inventors thereafter compared the predictive power of ONECUT2 with currently used clinicopathological risk-factors, and established a CMRPN, which achieved an impressive AUROC of 0.774 (95% Cl 0.697-0.839, P ⁇ 0.000l) and 0.753 (95% Cl 0.674-0.822, P ⁇ 0.000l), in both the clinical training and validation cohorts.
  • GC Gastric cancer
  • the inventors analyzed in total 399 GC cases from TCGA 450K methylation dataset and two clinical validation cohorts.
  • the methylation and clinical data was available for 399 GC patients from which information on 114 stage II and III cases which have 2 years follow-up were extracted from the data.
  • the TCGA cohort comprised of 50 samples from patients with recurrence within 2 years after surgery (Rec) and 64 samples from the ones without recurrence for more than 2 years (Non-rec).
  • the inventors selected 266,201 CpG probes by filtering out the probes that did not pass quality control (“NA” for all samples), are on X and Y chromosomes, are cross-reactive and mapped to single nucleotide polymorphisms.
  • the inventors fit the following linear model to each CpG probe; methylation M value ⁇ group (Rec or Non-rec) + age + gender, and 17754 probes were found to be significant between Rec and Non-rec patients with the P value ⁇ 0.05.
  • the inventors selected probes that belonged to CpG islands (CGIs) or differentially methylated regions (DMRs) with more than 2 probes. Further using stringent criterion of at least 10% hyper or hypo methylation in Rec vs. Non-rec patients, the inventors identified 7 hyper methylated and 46 hypo-methylated probes. [00209] For validation of the discovered methylation candidates, the inventors examined 285 GC specimens from two independent stage II and III GC patient cohorts, training cohort and validation cohort, totaling 88 Rec and 197 Non-rec, respectively. The detailed patient demographics and clinicopathological characteristics are provided in Table 1.
  • a training cohort included 144 GC frozen samples (43 Rec, 101 Non-rec) from stage II and III patients who underwent curative resection without preoperative treatment at Nagoya University between 1997 and 2013.
  • a validation cohort included 141 FFPE samples (45 LNP, 96 LNN) from stage II and III patients who underwent curative resection without preoperative treatment at Kumamoto University between 2007 and 2015.
  • DNA was extracted from fresh frozen primary tissues using AllPrep DNA/RNA/miRNA Universal (Qiagen, Hilden, Germany) as per manufacturer’s instructions. Following DNA quantification using Nanodrop system (ThermoFisher Scientific, Massachusetts, USA), 500 ng of genomic DNA was bisulfite converted with EZ-DNA methylation Gold-Kit (Zymo, Irvine, CA, USA).
  • BSPCR bisulfite-specific PCR
  • Primers for bisulfite-specific PCR were designed using the PyroMark Assay Design Software 2.0 (Qiagen) and with amplicon size ranging from 120- 250 base pairs (supplementary table 1.
  • Bisulfite converted DNA was amplified by BSPCR using PyroMark PCR Mastermix (Qiagen). Briefly, lOng of bisulfite converted DNA was mixed with the HotStarTaq Master Mix (Qiagen) and bisulfite specific primers under the following conditions: 95 °C for 10 min, 45 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s, and an elongation step of 72 °C for 10 min.
  • the amplified products were run on 2% agarose gels to check the specificity of primers.
  • Pyrosequencing was performed on Pyromark Q48 Autoprep using Q48 advanced CpG Reagents (Qiagen). Concisely, 10 pl of BSPCR product was added to 3 m ⁇ of magnetic beads and 2 m ⁇ of 4 mM sequencing primer on the Pyromark Q48 discs as per manufacturer’s instructions.
  • Output data were analyzed using PyroMark Q48 Autoprep Software (Qiagen), which calculates the CpG methylation value as the percentage (methylated cytosine/ [methylated cytosine+ unmethylated cytosine]) for each CpG site, allowing quantitative comparisons. Controls to assess proper bisulfite conversion of the DNA were included in each assay to ensure the fidelity of the measurements. Analysis was performed on the average methylation of all CpGs of a particular gene taken together.
  • LASSO Cox regression analysis was used to select the recurrence prediction markers of the candidate CpG sites and to construct a multi-CpG-based classifier for predicting the recurrence free survival of patients with GC in TCGA cohort.
  • LASSO regression shrinks the coefficient estimates towards zero, with the degree of shrinkage depending on an additional parameter, l. In this way, coefficient estimates can be forced to be exactly zero, thereby effectively eliminating a number of variables.
  • the inventors adopted the LASSO regression model to achieve shrinkage and variable selection simultaneously. Ten-time cross-validations were used to determine the optimal values of l. The inventors chose l via l-s.e.
  • the optimal l is the largest value for which the partial likelihood deviance is within 1 s.e. of the smallest value of partial likelihood deviance24.
  • the inventors used R software version 3.3.1 (R Foundation for Statistical Computing, Vienna, Austria) and the‘glmnet’ package to perform LASSO regression analysis.
  • Confidence intervals for the ROC curves were calculated using the method of DeLong.
  • the Kaplan-Meier method was used to analyze the correlation between methylation-based classifier and patient survival, and the log-rank test for comparing survival differences between groups using Medcalc.
  • the hazard ratio of the signature as well as the other clinical variables was calculated with Cox proportionality hazard model.
  • the regression model and median cut- off derived from the methylation-based nomogram in the training cohort was applied to the independent validation cohort for calculating the AUC for recurrence prediction.
  • the 450K methylation array data of 114 GC patients from in-silico TCGA cohort was analyzed in the discovery step for identifying the most differentially methylated CpG probes between Rec and Non-rec patients. From the 450K methylation array data, the inventors identified 7 hyper methylated and 46 hypo-methylated probes (details in method part). The heatmap shows the methylation values of these 53 probes in the Rec and Non-rec groups (FIG. 1A).
  • the inventors performed Lasso-Cox regression analysis on all 53 probes, which lead to the identification of 14 CGIs/DMRs which resulted in an AUC of 0.908 to identify a clinically feasible number of DMPs for recurrence prediction (FIG. 1B).
  • the inventors identified 14 CGIs/DMRs that were differentially methylated in GC patients with recurrence.
  • the details of the 14 CGIs/DMRs are summarized in supplementary table 1. To evaluate the recurrence predictive power of these 14 CGIs/DMRs, logistic regression analysis was performed.
  • the AUC values and hazard ratio (HR) of each 6 CGIs for predicting recurrence were analyzed using univariate logistic regression and Cox regression in both cohorts (FIG. 2A).
  • the methylation level of ONECUT2 was discovered to be able to significantly discriminate the GC patients with and without recurrence by itself throughout both the training and validation cohort (AUC 0.769, 0.713, respectively) (FIG. 2A).
  • the inventors stratified Stage II and III GC patients by the methylation level using a median value as a cut-off, and compared relapse free survival and overall survival in the training cohort and the validation cohort.
  • ONECUT2-hypomethylated patients had significantly poorer relapse free survival than hypermethylated patients in both cohorts (training cohort, P ⁇ 0.0001; validation cohort, P ⁇ 0.0001) (FIG. 2B).
  • CRPN CpG methylation-based recurrence prediction nomogram
  • ONECUT2 as an independent prognostic factor, the inventors performed multivariate Cox regression analysis based on relapse free survival using ONECUT2 and clinicopathological factors that are routinely evaluated in these patients, including age, gender, conventional tumor markers, tumor size and lymph node metastasis status (table 2).
  • the inventors next asked whether they can further improve the predictive potential of ONECUT2 by combining it with the clinicopathological factors.
  • the inventors analyzed the training cohort and applied backward step-wise elimination approach using logistic regression analysis for predicting recurrence.
  • tumor size and lymph node metastasis status were identified as co-factors, and CMRPN was developed in the training cohort (FIG. 3A).
  • This nomogram was even more superior in predicting Rec patients with the AUC of 0.774 (95%CI 0.697 - 0.839) (FIG. 3B), while the range of AUCs for each clinicopathological factors ranged from 0.528 to 0.641 in the training cohort (table 3).
  • the inventors applied the methylation data of the validation cohort to the nomogram.
  • the robustness of the nomogram in predicting recurrence was successfully validated with the AUC of 0.753 (95%CI 0.674 - 0.822), which is consistent result of the one in the training cohort (FIG. 3B).
  • the inventors stratified Stage II and III GC patients based on the risk score for the recurrence calculated by the nomogram using a median cut-off value, and compared relapse free survival and overall survival in the training cohort and the validation cohort.
  • the inventors established CMRPN using ONECUT2 and clinical factors in the training cohort, which could stratify GC patients into two distinct subgroups with high or low risks of recurrence in the validation cohort.
  • the nomogram may have clinical implications for individualized follow-up and therapeutic strategies including adjuvant chemotherapy regimens for the patients with gastric cancer.
  • Torre LA Bray F
  • Siegel RL Ferlay J
  • Lortet-Tieulent J Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 20l5;65(2):87-l08.
  • MicroRNA499 rs3746444 A/G polymorphism functions as a biomarker to predict recurrence following endoscopic submucosal dissection in primary early gastric cancer. Mol Med Rep. 2017;15(5):3245-51.

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Abstract

L'invention concerne des procédés et des compositions utilisant la détermination des niveaux de méthylation d'un ou de plusieurs biomarqueurs chez des patients atteints d'un cancer. Dans certains modes de réalisation, les niveaux de méthylation sont utilisés pour évaluer des patients atteints d'un cancer gastrique et le risque de récurrence.
PCT/US2019/022031 2018-03-13 2019-03-13 Procédés et compositions liés à la méthylation et à la récurrence chez des patients atteints d'un cancer gastrique WO2019178214A1 (fr)

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WO2022181496A1 (fr) * 2021-02-25 2022-09-01 富士フイルム株式会社 Procédé d'évaluation de réactif bisulfite et procédé de test génétique

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WO2017142484A1 (fr) * 2016-02-16 2017-08-24 Agency For Science, Technology And Research Profilage épigénomique révélant le paysage de promoteur somatique d'adénocarcinome gastrique primaire

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