WO2020132494A2 - Procédés de pronostic, de diagnostic et de traitement du cancer gastrique - Google Patents

Procédés de pronostic, de diagnostic et de traitement du cancer gastrique Download PDF

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WO2020132494A2
WO2020132494A2 PCT/US2019/067942 US2019067942W WO2020132494A2 WO 2020132494 A2 WO2020132494 A2 WO 2020132494A2 US 2019067942 W US2019067942 W US 2019067942W WO 2020132494 A2 WO2020132494 A2 WO 2020132494A2
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patient
expression
mir
identified
samples
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WO2020132494A3 (fr
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Ajay Goel
Tadanobu SHIMURA
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Baylor Research Institute
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Baylor Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/158—Expression markers
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates generally to the fields of molecular biology and oncology. More particularly, it concerns methods and compositions involving cancer prognosis, diagnosis and treatment.
  • CT computed tomography
  • PET positron emission tomography
  • CT and PET are commonly used to diagnose peritoneal metastasis.
  • the sensitivity of CT and PET is inadequate for identifying peritoneal metastatic lesions, and though staging laparoscopy can be used to identify peritoneal metastasis at a much higher rate than CT or PET-CT, this radical procedure is invasive and requires general anesthesia which increases the risk of complications.
  • the current disclosure fulfills a need in the art by providing a miRNA-based signature for diagnosing gastric cancer patients with peritoneal metastasis.
  • aspects of the disclosure relate to a method for treating a patient with gastric cancer peritoneal metastasis, the method comprising treating the patient for gastric cancer after the expression level of 1, 2, 3, 4 or 5 of the biomarkers selected from miR-30a-5p, miR-134-5p, miR-337-3p, miR-659-3p, and miR-3917 has been measured or determined in a sample from the patient.
  • Further aspects relate to method for evaluating a gastric cancer patient comprising measuring the level of expression of 1, 2, 3, 4, or 5 of the biomarkers selected from miR-30a-5p, miR-134-5p, miR-337-3p, miR-659-3p, and miR-3917 in a sample from the patient.
  • Further aspects of the disclosure relate to a method of prognosing and/or diagnosing a patient with gastric cancer comprising a) measuring the level of expression of one or more of miR- 30a-5p, miR-134-5p, miR-337-3p, miR-659-3p, and miR-3917 in a sample from the patient; b) comparing the level(s) of expression to a control sample(s) or control level(s) of expression; and, c) prognosing and/or diagnosing the patient based on the levels of measured expression.
  • At least miR-30a-5p, miR-659-3p and miR-3917 were determined or measured in a sample from the patient. In some embodiments, at least miR-30a-5p and miR-659-3p were determined or measured in a sample from the patient. In some embodiments, at least miR-30a-5p was determined or measured in a sample from the patient. In some embodiments, at least miR-134-5p was determined or measured in a sample from the patient. In some embodiments, at least miR-337-3p was determined or measured in a sample from the patient. In some embodiments, at least miR-659-3p was determined or measured in a sample from the patient. In some embodiments, at least miR-3917 was determined or measured in a sample from the patient.
  • 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.
  • Further embodiments include isolating or analyzing protein expression in a biological sample for the expression of the biomarker.
  • a microarray may be used to measure or assay the level of the biomarkers in a sample.
  • the methods may further comprise recording the biomarker expression or activity level in a tangible medium or reporting the expression or activity level to the patient, a health care payer, a physician, an insurance agent, or an electronic system.
  • the method further comprises determining the macroscopic Borrmann type of the gastric tumor.
  • the patient has been diagnosed with gastric cancer.
  • the patient has not been diagnosed with distant metastasis.
  • the patient has not been diagnosed with peritoneal metastasis.
  • the patient has not been diagnosed with or has not been treated for peritoneal metastasis or Stage IV gastric cancer.
  • the method further comprises treating the patient for cancer after measuring the level of expression of one or more listed biomarkers.
  • the biomarker is measured prior to surgical resection of the tumor or prior to total or subtotal gastrectomy. In some embodiments, the biomarker is measured after surgical resection of the tumor or after total or subtotal gastrectomy. In some embodiments, the patient has undergone surgery to resect all or part of the cancer. In some embodiments, the patient has not undergone surgical resection of the tumor.
  • the level of expression of miR-30a-5p was determined or measured pre-operative and/or post-operative. In some embodiments, the level of expression of miR-134-5p was determined or measured pre-operative and/or post-operative. In some embodiments, the level of expression of miR-337-3p was determined or measured pre-operative and/or post-operative. In some embodiments, the level of expression of miR-659-3p was determined or measured pre-operative and/or post-operative. In some embodiments, the level of expression of miR-3917 was determined or measured pre-operative and/or post-operative. In some embodiments, the patient has not undergone laparoscopy of gastric cancer tissues.
  • the expression levels of the one or more biomarkers in the patient was determined to be i) increased compared to the levels of expression in samples from patients identified as not having peritoneal metastasis, identified as low risk, or in normal tissues or ii) within the range of expression levels in samples of patients identified as having peritoneal metastasis or identified as high risk.
  • the expression levels of at least one of the biomarkers in the patient was determined to be i) increased compared to the levels of expression in samples from patients identified as not having peritoneal metastasis, identified as low risk, or in normal tissues or ii) within the range of expression levels in samples of patients identified as having peritoneal metastasis or identified as high risk.
  • the expression levels of at least two of the biomarkers in the patient was determined to be i) increased compared to the levels of expression in samples from patients identified as not having peritoneal metastasis, identified as low risk, or in normal tissues or ii) within the range of expression levels in samples of patients identified as having peritoneal metastasis or identified as high risk.
  • the expression levels of at least three of the biomarkers in the patient was determined to be i) increased compared to the levels of expression in samples from patients identified as not having peritoneal metastasis, identified as low risk, or in normal tissues or ii) within the range of expression levels in samples of patients identified as having peritoneal metastasis or identified as high risk
  • the expression levels of at least four of the biomarkers in the patient was determined to be i) increased compared to the levels of expression in samples from patients identified as not having peritoneal metastasis, identified as low risk, or in normal tissues or ii) within the range of expression levels in samples of patients identified as having peritoneal metastasis or identified as high risk.
  • the expression levels of at least five of the biomarkers in the patient was determined to be i) increased compared to the levels of expression in samples from patients identified as not having peritoneal metastasis, identified as low risk, or in normal tissues or ii) within the range of expression levels in samples of patients identified as having peritoneal metastasis or identified as high risk.
  • the patient was determined to have a macroscopic Borrmann type III or IV gastric tumor.
  • the patient is treated for peritoneal metastasis.
  • the treatment comprises one or more of cytoreductive surgery, hyperthermic intraperitoneal chemotherapy (HIPEC), chemotherapy, neo-adjuvant chemotherapy, adjuvant chemotherapy, subtotal or total gastrectomy, tumor resection, and endoscopic resection.
  • the chemotherapy comprises paclitaxel.
  • the chemotherapy is administered by intraperitoneal administration.
  • the chemotherapy comprises one or more chemotherapeutic agents described herein.
  • the chemotherapy excludes one or more chemotherapeutic agents described herein.
  • the expression levels of the one or more biomarkers in the patient was determined to be i) decreased compared to the levels of expression in samples from patients identified as having peritoneal metastasis or identified as low risk or ii) within the range of expression levels in samples of patients identified as not having peritoneal metastasis or in normal tissues.
  • the expression levels of at least one of the biomarkers in the patient was determined to be i) decreased compared to the levels of expression in samples from patients identified as having peritoneal metastasis or identified as low risk or ii) within the range of expression levels in samples of patients identified as not having peritoneal metastasis or in normal tissues.
  • the expression levels of at least two of the biomarkers in the patient was determined to be i) decreased compared to the levels of expression in samples from patients identified as having peritoneal metastasis or identified as low risk or ii) within the range of expression levels in samples of patients identified as not having peritoneal metastasis or in normal tissues.
  • the expression levels of at least three of the biomarkers in the patient was determined to be i) decreased compared to the levels of expression in samples from patients identified as having peritoneal metastasis or identified as low risk or ii) within the range of expression levels in samples of patients identified as not having peritoneal metastasis or in normal tissues.
  • the expression levels of at least four of the biomarkers in the patient was determined to be i) decreased compared to the levels of expression in samples from patients identified as having peritoneal metastasis or identified as low risk or ii) within the range of expression levels in samples of patients identified as not having peritoneal metastasis or in normal tissues.
  • the expression levels of at least five of the biomarkers in the patient was determined to be i) decreased compared to the levels of expression in samples from patients identified as having peritoneal metastasis or identified as low risk or ii) within the range of expression levels in samples of patients identified as not having peritoneal metastasis or in normal tissues.
  • the patient was determined to have a macroscopic Borrmann type I or II gastric tumor.
  • the treatment comprises one or more of surgery with either subtotal or total gastrectomy, tumor resection, endoscopic resection.
  • the treatment excludes one or more of cytoreductive surgery, hyperthermic intraperitoneal chemotherapy (HIPEC), chemotherapy, neo-adjuvant chemotherapy, and adjuvant chemotherapy.
  • HIPEC hyperthermic intraperitoneal chemotherapy
  • chemotherapy neo-adjuvant chemotherapy
  • adjuvant chemotherapy adjuvant chemotherapy
  • low risk is indicative of a patient with a low risk for distant metastasis and/or peritoneal metastasis and good overall survival (OS) rate
  • high risk is indicative of a patient with a high risk for distant metastasis and/or peritoneal metastasis and poor overall survival (OS) rate.
  • the method further comprises comparing the level(s) of expression to a control sample(s) or control level(s) of expression.
  • the control sample(s) have expression levels that are representative of expression levels in samples from patients identified as low risk, of patients not having gastric cancer, or of patients having gastric cancer but not having peritoneal metastasis.
  • the control levels(s) comprise the levels of expression of the one or more biomarkers in non-cancerous gastric tissues.
  • the control sample(s) have expression levels that are representative of expression levels in samples from patients identified as high risk or of patients having peritoneal metastasis.
  • the expression level or activity level from a control sample may be an average value, a normalized value, a cut-off value, or an average normalized value.
  • the expression level or activity level may be an average or mean obtained from a significant proportion of patient samples.
  • the expression or activity level may also be an average or mean from one or more samples from the patient.
  • the elevated level/increased expression or reduced level/decreased expression is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 50, 100, 150, 200, 250, 500, or 1000 fold (or any derivable range therein) or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900% different than the control, or any derivable range therein.
  • a level of expression may be qualified as“low” or“high,” which indicates the patient expresses a certain gene at a level relative to a reference level or a level with a range of reference levels that are determined from multiple samples meeting particular criteria.
  • the level or range of levels in multiple control samples is an example of this.
  • that certain level or a predetermined threshold value is at, below, or above 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
  • the threshold level may be derived from a cohort of individuals meeting a particular criteria.
  • the number in the cohort may be, be at least, or be at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more (or any range derivable therein).
  • control may be the average level of expression of the miRNA in a biological sample from a subject having gastric cancer or determined to be at risk for gastric cancer.
  • the control may be the level of expression of the miRNA in a biological sample from a subject with stage I, II, III, or IV gastric cancer (or any TMN stage defined herein).
  • the decision to treat the subject for gastric cancer or diagnose or provide a prognosis that the subject has or is likely to get gastric cancer is based on the a level of expression that is similar to the control or within 1, 2, 3, 4, or 5 deviations or differs by less than 1, 3, 5, 10, 15, 20, 30, or 40% (or any derivable range therein).
  • the patient has been diagnosed with gastric cancer.
  • the method further comprises treating the patient for cancer after measuring the level of expression of one or more listed biomarkers.
  • the biomarker is measured prior to surgical resection of the tumor or prior to total or subtotal gastrectomy.
  • the biomarker is measured after surgical resection of the tumor or after total or subtotal gastrectomy.
  • the level of expression of miR-30a-5p was measured pre-operative and/or post-operative.
  • the level of expression of miR-134-5p was measured pre-operative and/or post-operative.
  • the level of expression of miR-337-3p was measured pre-operative and/or post-operative. In some embodiments, the level of expression of miR-659-3p was measured pre-operative and/or post-operative. In some embodiments, the level of expression of miR-3917 was measured pre-operative and/or post operative. [0023] In some embodiments, the method further comprises determining the macroscopic Borrmann type of the gastric tumor. In some embodiments, the patient has not been diagnosed with or has not been treated for peritoneal metastasis or Stage IV gastric cancer.
  • the patient is prognosed as high risk and/or treated when the expression levels of the one or more biomarkers in the patient was i) increased compared to the levels of expression in samples from patients identified as not having peritoneal metastasis, identified as low risk, or in normal tissues or ii) within the range of expression levels in samples of patients identified as having peritoneal metastasis or identified as high risk.
  • the patient is prognosed as high risk and/or treated when the expression levels of at least one of the biomarkers in the patient was i) increased compared to the levels of expression in samples from patients identified as not having peritoneal metastasis, identified as low risk, or in normal tissues or ii) within the range of expression levels in samples of patients identified as having peritoneal metastasis or identified as high risk.
  • the patient is prognosed as high risk and/or treated when the expression levels of at least two of the biomarkers in the patient was i) increased compared to the levels of expression in samples from patients identified as not having peritoneal metastasis, identified as low risk, or in normal tissues or ii) within the range of expression levels in samples of patients identified as having peritoneal metastasis or identified as high risk.
  • the patient is prognosed as high risk and/or treated when the expression levels of at least three of the biomarkers in the patient was i) increased compared to the levels of expression in samples from patients identified as not having peritoneal metastasis, identified as low risk, or in normal tissues or ii) within the range of expression levels in samples of patients identified as having peritoneal metastasis or identified as high risk.
  • the patient is prognosed as high risk and/or treated when the expression levels of at least four of the biomarkers in the patient was i) increased compared to the levels of expression in samples from patients identified as not having peritoneal metastasis, identified as low risk, or in normal tissues or ii) within the range of expression levels in samples of patients identified as having peritoneal metastasis or identified as high risk.
  • the patient is prognosed as high risk and/or treated when the expression levels of at least five of the biomarkers in the patient was i) increased compared to the levels of expression in samples from patients identified as not having peritoneal metastasis, identified as low risk, or in normal tissues or ii) within the range of expression levels in samples of patients identified as having peritoneal metastasis or identified as high risk.
  • the patient is prognosed as high risk and/or treated when the patient was determined to have a macroscopic Borrmann type III or IV gastric tumor.
  • the patient is diagnosed as having peritoneal metastasis.
  • the treatment comprises one or more of cytoreductive surgery, hyperthermic intraperitoneal chemotherapy (HIPEC), chemotherapy, neo-adjuvant chemotherapy, adjuvant chemotherapy, subtotal or total gastrectomy, tumor resection, and endoscopic resection.
  • the chemotherapy comprises paclitaxel.
  • the chemotherapy is administered by intraperitoneal administration.
  • the patient is prognosed as low risk and/or treated when the expression levels of the one or more biomarkers in the patient is i) decreased compared to the levels of expression in samples from patients identified as having peritoneal metastasis or identified as low risk or ii) within the range of expression levels in samples of patients identified as not having peritoneal metastasis or in normal tissues.
  • the patient is prognosed as low risk and/or treated when the expression levels of at least one of the biomarkers in the patient is i) decreased compared to the levels of expression in samples from patients identified as having peritoneal metastasis or identified as low risk or ii) within the range of expression levels in samples of patients identified as not having peritoneal metastasis or in normal tissues.
  • the patient is prognosed as low risk and/or treated when the expression levels of at least two of the biomarkers in the patient is i) decreased compared to the levels of expression in samples from patients identified as having peritoneal metastasis or identified as low risk or ii) within the range of expression levels in samples of patients identified as not having peritoneal metastasis or in normal tissues.
  • the patient is prognosed as low risk and/or treated when the expression levels of at least three of the biomarkers in the patient is i) decreased compared to the levels of expression in samples from patients identified as having peritoneal metastasis or identified as low risk or ii) within the range of expression levels in samples of patients identified as not having peritoneal metastasis or in normal tissues.
  • the patient is prognosed as low risk and/or treated when the expression levels of at least four of the biomarkers in the patient is i) decreased compared to the levels of expression in samples from patients identified as having peritoneal metastasis or identified as low risk or ii) within the range of expression levels in samples of patients identified as not having peritoneal metastasis or in normal tissues.
  • the patient is prognosed as low risk and/or treated when the expression levels of at least five of the biomarkers in the patient is i) decreased compared to the levels of expression in samples from patients identified as having peritoneal metastasis or identified as low risk or ii) within the range of expression levels in samples of patients identified as not having peritoneal metastasis or in normal tissues.
  • the patient is prognosed as low risk and/or treated when the patient was determined to have a macroscopic Borrmann type I or II gastric tumor.
  • the treatment comprises one or more of surgery with either subtotal or total gastrectomy, tumor resection, endoscopic resection.
  • the treatment excludes one or more of cytoreductive surgery, hyperthermic intraperitoneal chemotherapy (HIPEC), chemotherapy, neo-adjuvant chemotherapy, and adjuvant chemotherapy.
  • HIPEC hyperthermic intraperitoneal chemotherapy
  • methods will involve determining or calculating a prognosis score based on data concerning the expression or activity level of one or more of the biomarkers, meaning that the expression or activity level of one or more of the biomarkers is at least one of the factors on which the score is based.
  • a prognosis score will provide information about the patient, such as the general probability whether the patient is sensitive to a particular therapy or has poor survival or high chances of recurrence.
  • a prognosis value is expressed as a numerical integer or number that represents a probability of 0% likelihood to 100% likelihood that a patient has a chance of poor survival or cancer recurrence or poor response to a particular treatment.
  • the prognosis score is expressed as a number that represents a probability of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
  • a difference between or among weighted coefficients or expression or activity levels or between or among the weighted comparisons may be, be at least or be at most about 0.1, 0.2,
  • 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,
  • the prognosis score is calculated using one or more statistically significantly differentially expressed biomarkers (either individually or as difference pairs), including expression or activity levels in a biomarker, gene, or protein.
  • the sample from the patient comprises gastric cancer tissue.
  • the normal tissues comprises non-cancerous gastric tissues.
  • the sample from the patient comprises a serum sample.
  • the sample from the patient comprises nucleic acids. In some embodiments, the sample from the patient comprises a fractionated serum sample comprising nucleic acids. In some embodiments, the samples from patients identified as not having peritoneal metastasis or identified as low risk comprises the level of expression of the one or more biomarkers in a serum sample or samples from patients without peritoneal metastasis. In some embodiments, the expression level of no other biomarker in the biological sample was determined or measured.
  • the biological sample from the patient is a sample from a primary gastric cancer tumor.
  • the biological sample is from a tissue or organ as described herein.
  • the method may comprise obtaining a sample of the subject or patient.
  • the sample include 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 a serum sample, a plasma sample or a whole blood sample.
  • the methods of obtaining a sample of the subject or patient 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 intestinal, stomach, or other associated gastric tissues.
  • 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, esophagus, or thyroid tissue.
  • 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.
  • FFPE formalin-fixed, paraffin embedded
  • 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.
  • kits comprising 1, 2, 3, 4, or 5 detection agents for determining expression levels of biomarkers for gastric cancer, wherein the biomarkers comprise one or more of miR-30a-5p, miR-134-5p, miR-337-3p, miR-659-3p, and miR-3917.
  • the kit further comprises one or more negative or positive control samples and/or control detection agents.
  • the detection agents compnse nucleic acids.
  • the detection agents comprise nucleic acid probes that hybridize to a biomarker gene or fragment thereof.
  • the detection agents comprise a pair of nucleic acid primers that are capable of amplifying a biomarker gene or a fragment thereof.
  • the detection agent comprises an antibody that specifically binds to a biomarker protein.
  • the probes are labeled.
  • the kit further comprises nucleic acid probes for detecting a control.
  • the control comprises a RNA, miRNA, or a biomarker protein or gene not differentially expressed in liver cancer or in fast or slow DT liver cancer or HCC.
  • the probe comprises nucleic acid primers that are capable of amplifying the RNA or a cDNA made from the RNA by PCR.
  • the kit further comprises reagents for performing one or more of reverse transcriptase PCR, DNA amplification by PCR, and real time PCR. In some embodiments, the kit further comprises instructions for use.
  • 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 or activity level of a gene, biomarker or protein in a sample from a patient; and b) determining a difference value in the expression or activity levels using the information corresponding to the expression or activity levels in the sample compared to a control or reference expression or activity 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 gastric cancer, administering the same treatment as the first treatment to the patient if the patient does not have increased expression or activity level; administering a different treatment from the first treatment to the patient if the patient has increased expression or activity level.
  • receiving information comprises receiving from a tangible data storage device information corresponding to the expression or activity 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 gastric therapy if the patient does not have expression or activity levels, and/or or treating the patient with an alternative gastric therapy if the patient has increased expression or activity 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 comprising a thymidylate synthase inhibitor to a patient that is determined to have a decreased expression or activity level.
  • the terms“subject,”“mammal,” and“patient” are used interchangeably.
  • the subject is a mammal.
  • the subject is a human.
  • the subject is a mouse, rat, rabbit, dog, donkey, or a laboratory test animal such as fruit fly, zebrafish, etc.
  • the biomarkers described herein, such as the gene or RNA biomarkers may correspond to the human gene or RNA In some embodiments, the biomarkers corresponds to a human, mammalian, mouse, dog, cat, or a homolog of a human gene or RNA.
  • 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.
  • compositions and methods for their use can“comprise,”“consist essentially of,” or“consist of’ any of the ingredients or steps disclosed throughout the specification.
  • 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-C Identification of candidate miRNAs in primary tumors from gastric cancer patients with peritoneal metastasis.
  • A Schematic of the study design for biomarker discovery and validation in various patient cohorts.
  • B Heatmap illustrating differentially expressed miRNAs between peritoneal metastasis positive and negative samples identified from the miRNA- microarray dataset.
  • C Differentially expressed candidate miRNAs between stage IV and stage IB-III GC patients in the TCGA dataset. Abbreviations; PM, peritoneal metastasis. *p ⁇ 0.05, by Mann- Whitney U test.
  • FIG. 2A-C Candidate three miRNAs were differentially expressed in peritoneal metastasis positive patients in the testing cohort.
  • A The expression of candidate five miRNAs between PM positive and negative samples.
  • B Differential expression of candidate miRNAs in the sub-group analysis of stage IV patients between PM positive and negative patients.
  • C Kaplan meier analysis for overall survival (OS) between two groups dichotomized by Youden’s index for PM in combined miRNA signature. Abbreviations; PM, peritoneal metastasis. *p ⁇ 0.05, **p ⁇ 0.01, by Mann- Whitney U test.
  • FIG. 3A-D The expression of candidate five miRNAs between PM positive and negative samples.
  • B Differential expression of candidate miRNAs in the sub-group analysis of stage IV patients between PM positive and negative patients.
  • C Kaplan meier analysis for overall survival (OS) between two groups dichotomized by Youden’s index for PM in combined miRNA signature. Abbreviations; PM, peritoneal metasta
  • Candidate miRNAs were differentially expressed in peritoneal metastasis positive patients in the testing cohort.
  • A Diagnostic robustness of miRNA candidates represented by receiver operating characteristic (ROC) curves.
  • B The waterfall plot representing risk score of PM positive and negative patients based on the combined miRNAs signature.
  • C ROC curve of the combined miRNA signature for the detection of PM. Abbreviations; Sen, sensitivity; Spe, specificity.
  • D Kaplan-Meier analysis for overall survival (OS) between two groups dichotomized by Youden’s index for PM in individual overexpressing miRNAs.
  • FIG. 4A-D Candidate three miRNAs were differentially expressed in peritoneal metastasis positive patients in the validation cohort.
  • A The expression of candidate three miRNAs between PM positive and negative samples.
  • B Diagnostic robustness of miRNA candidates represented by ROC curve.
  • C Differential expression of candidate three miRNAs in the sub-group analysis of stage IV patients between PM positive and negative patients.
  • D Kaplan meier analysis for OS in individual three miRNAs. Abbreviations; PM, peritoneal metastasis; Sen, sensitivity; Spe, specificity. *p ⁇ 0.05, by Mann- Whitney U test.
  • FIG. 5A-F Diagnostic accuracy of peritoneal metastasis detection and prognostic significance of combined miRNA signature in validation cohort and performance evaluation cohort.
  • FIG. 6A-D Candidate three miRNAs were differentially expressed in peritoneal metastasis positive patients in the performance evaluation cohort.
  • A The expression of candidate three miRNAs between PM positive and negative samples.
  • B Diagnostic robustness of miRNA candidates represented by ROC curve.
  • C Differential expression of candidate three miRNAs in the sub-group analysis of stage IV patients between PM positive and negative patients.
  • D Kaplan meier analysis for OS in individual three miRNAs. Abbreviations; PM, peritoneal metastasis; Sen, sensitivity; Spe, specificity. **p ⁇ 0.01, ***p ⁇ 0.001, by Mann-Whitney U test.
  • FIG. 7A-D Diagnostic robustness of peritoneal metastasis detection of combined miRNA signature and tumor macroscopic type in testing cohort
  • A ROC curve of the combined miRNA signature, tumor macroscopic type, and their combination for detection of PM.
  • B Nomogram
  • C Predicted probability plot
  • D Cost-benefit curve for PM detection.
  • FIG. 8A-H Diagnostic accuracy of peritoneal metastasis detection of combined miRNA signature and tumor macroscopic type in validation cohort and performance evaluation cohort. ROC curves derived from the combination miRNA signature, tumor macroscopic type, and their combination for detection of PM in the validation (A), and performance evaluation cohort
  • FIG. 9A-C Identification of candidate miRNAs in primary tumors from peritoneal metastasis positive patients.
  • A Schematics of target miRNA identification using miRNA- microarray and TCGA datasets.
  • B Heatmap of differentially expressed miRNAs between peritoneal metastasis positive and negative samples identified from miRNA-microarray dataset.
  • FIG. 10A-D Candidate miRNAs were differentially expressed in peritoneal metastasis positive patients in cohort 1.
  • A The expression of candidate miRNAs between peritoneal metastasis positive and negative samples.
  • B Diagnostic robustness of miRNA candidates represented by receiver operating characteristic (ROC) curve.
  • C ROC curve of the combined miRNAs signature for detect peritoneal metastasis (left). The waterfall plot representing risk score of peritoneal metastasis positive and negative patients based on the combined miRNAs signature.
  • ROC receiver operating characteristic
  • FIG. 11A-C Differential expression of candidate miRNAs between peritoneal metastasis positive and negative patients in cohorts (A) 1, (B) 2, and (C) 3.
  • FIG. 12A-D Candidate miRNAs were differentially expressed in peritoneal metastasis positive patients in cohort 2.
  • A The expression of candidate three miRNAs between peritoneal metastasis positive and negative samples.
  • B Diagnostic robustness of miRNA candidates represented by receiver operating characteristic (ROC) curve.
  • C ROC curve of the combined miRNAs signature for detect peritoneal metastasis (left). The waterfall plot representing risk score of peritoneal metastasis positive and negative patients based on the combined miRNAs signature.
  • D Kaplan meier analysis for overall survival in individual three miRNAs and combined miRNA signature. * p ⁇ 0.05, ** p ⁇ 0.01, by Mann-Whitney U test.
  • FIG. 13A-D Differential expression of candidate miRNAs between peritoneal metastasis positive and negative samples in fresh frozen cohort.
  • A The expression of candidate three miRNAs between peritoneal metastasis positive and negative samples.
  • B Diagnostic robustness of miRNA candidates represented by receiver operating characteristic (ROC) curve (right).
  • C ROC curve of the combined miRNAs signature for detect peritoneal metastasis (left). The waterfall plot representing risk score of peritoneal metastasis positive and negative patients based on the combined miRNAs signature.
  • D Kaplan meier analysis for overall survival in individual three miRNAs and combined miRNA signature. * p ⁇ 0.05, ** p ⁇ 0.01, by Mann- Whitney U test.
  • FIG. 14A-C Diagnostic capacity of the miRNA signature upon combination of tumor macroscopic type with the miRNA signature in patient cohorts (A) 1, (B) 2, and (C) 3.
  • Described herein are improved methods for treating gastric cancer patients.
  • the inventors aimed to do a genomewide transcriptomic profiling to develop a miRNA-based signature for the identification of peritoneal metastasis (PM) in patients with gastric cancer (GC).
  • PM peritoneal metastasis
  • GC gastric cancer
  • Systematic biomarker discovery was performed by analyzing miRNA expression profiles in primary tumors from GC patients with and without PM, and subsequently validated the expression of candidate miRNA biomarkers in three independent clinical cohorts of 354 patients with advanced GC.
  • the novel miRNA-based signature is a robust diagnostic tool for identifying peritoneal metastasis in GC patients, which could lead to improved survival outcomes in gastric cancer and more efficient and effective treatment of gastric cancer patients..
  • antibody encompasses antibodies and antibody fragments thereof, derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate including human), that specifically bind to an antigenic polypeptide.
  • exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; multispecific antibodies (e.g., bispecific antibodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotype antibodies, and may be any intact molecule or fragment thereof.
  • “Prognosis” refers to as a prediction of how a patient will progress, and whether there is a chance of recovery.“Cancer prognosis” generally refers to a forecast or prediction of the probable course or outcome of the cancer, with or without a treatment.
  • cancer prognosis includes the forecast or prediction of any one or more of the following: duration of survival of a patient susceptible to or diagnosed with a cancer, duration of recurrence-free survival, duration of progression free survival of a patient susceptible to or diagnosed with a cancer, response rate in a group of patients susceptible to or diagnosed with a cancer, duration of response in a patient or a group of patients susceptible to or diagnosed with a cancer, and/or likelihood of metastasis in a patient susceptible to or diagnosed with a cancer.
  • Prognosis also includes prediction of favorable responses to cancer treatments, such as a conventional cancer therapy.
  • a response may be either a therapeutic response (sensitivity or recurrence-free survival) or a lack of therapeutic response (residual disease, which may indicate resistance or recurrence).
  • the terms“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. Alternatively, or in conjunction, the terms refer to a level of expression that is less than 2, 1.5, or 1.25 fold different or less than 2, 1, or 0.5 standard deviations than the expression or activity level it is compared to.
  • 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 expression” or “elevated expression” or “decreased expression” refers to an expression 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 fast doubling time HCC, or with slow doubling time HCC, 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. In certain embodiments, a reference level is a normalized level.
  • determining or“evaluating” as used herein may refer to measuring, quantitating, or quantifying (either qualitatively or quantitatively).
  • gastric cancer comprises a cancer stage, TNM, and/or is further characterized as having features described below.
  • 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.
  • GIST gastrointestinal stromal 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.
  • 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:
  • Another commonly used classification method for gastric cancer is the Borrmann classification system.
  • Borchard FJH Classification of gastric carcinoma, Hepato- Gastroenterology, 1990;37(2):223-232.
  • the Borrmann system stratifies gastric cancer patients based on tumor location and macroscopic appearance and growth state of the tumor.
  • gastric cancers are divided into four Types: Borrmann Type I, primarily exogenous growth, usually broad-based polypoid carcinomas with protrusions; Borrmann Type II, with a central, bowl- shaped ulcer and elevated margins and a relatively distinct boundary between the cancer and surrounding tissue; Borrmann Type III, centrally ulcerating carcinoma without a ridge, elevated margins and indistinct borders; Borrmann Type IV, diffuse tumor with indistinct borders and infiltration of the gastric wall.
  • Type I and II are localized types of tumors, whereas Types III and IV and infiltrative type tumors.
  • The“cancer” referred to in the methods described herein may include or exclude any of the above stages or TNM categories.
  • 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 IV cancer.
  • 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 MO; Tl, Nl, and/or MO; T2, NO, and/or MO; Tl, N2, and/or MO; T2, Nl, and/or MO; T3, NO, and/or MO; 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, N2, and/or MO; T4b and/or NO; Nl and/or MO; T4a, N3, and/or MO; T4b and/or N2; N3 and/or MO; Any T, any N, and/or Ml.
  • Methods of the disclosure relate to treating subjects and patients with a cancer therapy.
  • the cancer therapy may be one described below and may be given with respect to a patient having been determined to have a certain biomarker profile.
  • the therapy described below is given to a patient with a poor prognosis, unfavorable prognosis, or to a patient determined to be high risk.
  • the therapy described below is given to a patient with a favorable prognosis, or to a patient determined to be low risk. Also contemplated are combinations of the therapies described below.
  • endoscopic mucosal resection For a very early stage (Tla) cancer, some doctors may recommend a non- surgical treatment called endoscopic mucosal resection. This is the removal of the tumor with an endoscope.
  • stages 0 or I when the cancer is still only in the stomach, surgery is used to remove the part of the stomach with cancer and nearby lymph nodes. This is called a subtotal or partial gastrectomy.
  • a partial gastrectomy the surgeon connects the remaining part of the stomach to the esophagus or small intestine.
  • the cancer has spread to the outer stomach wall with or without having spread to the lymph nodes, surgery plus chemotherapy or chemotherapy and radiation therapy may be used.
  • the surgeon can perform 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 are often removed during surgery because the cancer may have spread to those lymph nodes. This is called a lymphadenectomy.
  • Cytoreductive surgery with hyperthermic intraperitoneal chemotherapy is also used to treat cancers that have originated in or spread to the abdominal cavity, such as gastric cancer.
  • hyperthermic intraperitoneal chemotherapy HIPEC
  • a high dose chemotherapy solution heated to between 107.6°F - 109.4°F (42-43°C) is delivered directly into the abdominal cavity through catheters for approximately 90 minutes to eliminate remaining cancer cells while preserving healthy cells.
  • 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.
  • Patients with stomach cancer usually receive 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 the use of drugs to destroy cancer cells, usually by stopping the cancer cells’ ability to grow and divide. Chemotherapy is given by a medical oncologist. Systemic chemotherapy gets into the bloodstream to reach cancer cells throughout the body. Common ways to give chemotherapy include an intravenous (IV) tube placed into a vein using a needle or in a pill or capsule that is swallowed (orally). A chemotherapy regimen usually comprises 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.
  • IV intravenous
  • a chemotherapy regimen usually comprises 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.
  • chemotherapeutic regimens include, for example, the combination of fluorouracil (5-FU, Adrucil) and cisplatin (Platinol).
  • Other drugs commonly used include 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.
  • 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.
  • prodmgs 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 (VP16), 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
  • 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 a 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 a gastroesophageal cancer
  • 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, and a doctor will give other options for treating the cancer.
  • ramucirumab is a type of targeted therapy called an anti- angiogenic. It is focused on stopping angiogenesis
  • 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.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting.
  • the treatment is a gene therapy.
  • the therapeutic gene is a tumor suppressor gene.
  • a tumor suppressor gene is a gene that, when present in a cell, reduces the tumorigenicity, malignancy, or hyperproliferative phenotype of the cell. This definition includes both the full length nucleic acid sequence of the tumor suppressor gene, as well as non-full length sequences of any length derived from the full length sequences. It being further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.
  • tumor suppressor nucleic acids within this definition include, but are not limited to APC, CYLD, HIN-I, KRAS 2b, plo, pl9, p21, p27, p27mt, p53, p57, p73, PTEN, Rb, Uteroglobin, Skp2, BRCA-I, BRCA-2, CHK2, CDKN2A, DCC, DPC4, MADR2/JV18, MEN1, MEN2, MTS1, NF1, NF2, VHL, WRN, WT1, CFTR, C-CAM, CTS-I, zacl, scFV, 5 MMAC1, FCC, MCC, Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), 101F6, Gene 21 (NPRL2), or a gene encoding a SEM A3 polypeptide and FUS1.
  • tumor suppressor genes are described in a database of tumor suppressor genes at www.cise.ufl.edu/ ⁇ yyl/HTML-TSGDB/Homepage.litml. This database is herein specifically incorporated by reference into this and all other sections of the present application.
  • Nucleic acids encoding tumor suppressor genes include tumor suppressor genes, or nucleic acids derived therefrom (e.g., cDNAs, cRNAs, mRNAs, and subsequences thereof encoding active fragments of the respective tumor suppressor amino acid sequences), as well as vectors comprising these sequences.
  • cDNAs, cRNAs, mRNAs, and subsequences thereof encoding active fragments of the respective tumor suppressor amino acid sequences
  • the methods described herein may include or exclude any of the cancer therapies described in the disclosure.
  • the biomarker-based 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 miRNA described above.
  • the methods of the disclosure further include one or more monitoring tests.
  • 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.
  • the monitoring test comprises radiographic imaging. Examples of radiographic imaging this is useful in the methods of the disclosure includes hepatic ultrasound, computed tomographic (CT) abdominal scan, liver magnetic resonance imaging (MRI), body CT scan, and body MRI.
  • 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.
  • Methods of the disclosure relate to the detection of one or more of miR-30a-5p, miR- 134-5p, miR-337-3p, miR-659-3p, and miR-3917 for treating gastric cancer.
  • the known sequences of the micro RNAs are exemplified by the following:
  • hsa-miR-30a-5p uguaaacauccucgacuggaag (SEQ ID NO: l); hsa-miR-134-5p: ugugacugguugaccagagggg (SEQ ID NO:2); hsa-miR-337-3p : cuccuauaugaugccuuucuuc (SEQ ID NOG); hsa-miR-659-3p: cuugguucagggagggucccca (SEQ ID NO:4); and hsa-miR-3917: gcucggacugagcagguggg (SEQ ID NOG).
  • 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 intestinal or mucosal 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.
  • the sample is obtained from cystic fluid or fluid derived from a tumor or neoplasm.
  • the cyst, tumor, or neoplasm is gastric or in the digestive system.
  • 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 gastric 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 gastric) 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 e.g. serum
  • samples from another specimen e.g. serum
  • 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.
  • 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 or activity levels.
  • Arrays can be used to detect differences between two samples.
  • Specifically contemplated applications include identifying and/or quantifying differences between RNA from a sample that is normal and from a sample that is not normal, between a cancerous condition and a non-cancerous condition, between one cancerous condition, such as fast doubling time cells and another cancer condition, such as slow doubling time cells, or between two differently treated samples.
  • RNA may be compared between a sample believed to be susceptible to a particular disease or condition and one believed to be not susceptible or resistant to that disease or condition.
  • a sample that is not normal is one exhibiting phenotypic trait(s) of a disease or condition or one believed to be not normal with respect to that disease or condition. It may be compared to a cell that is normal with respect to that disease or condition.
  • Phenotypic traits include symptoms of, or susceptibility to, a disease or condition of which a component is or may or may not be genetic or caused by a hyperproliferative or neoplastic cell or cells.
  • an array may be used.
  • 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 ah, 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.
  • biomarker expression examples include, but are not limited to, nucleic amplification, polymerase chain reaction, quantitative PCR, RT-PCR, in situ hybridization, Northern hybridization, hybridization protection assay (HP A)( GenProbe ), branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (US Genomics), Invader assay (ThirdWave Technologies), and/or Bridge Litigation Assay (Genaco).
  • RNA sequencing also called whole transcriptome shotgun sequencing, uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment in time.
  • NGS next-generation sequencing
  • RNA-Seq is used to analyze the continually changing cellular transcriptome. Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression.
  • RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling. RNA-Seq can also be used to determine exon/intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries.
  • 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.
  • 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 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, 500, 1000 pg/kg, mg/kg, pg/day, or mg/day or any range derivable therein.
  • 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 pM to 150 pM.
  • the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (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 mM to 100 pM. It is also understood that 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.
  • compositions or agents for use in the methods are suitably contained in a pharmaceutically acceptable carrier.
  • the carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent.
  • the agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e. to a specific location of the body, such as skeletal muscle or other tissue) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. Certain aspects of the disclosure also contemplate local administration of the compositions by coating medical devices and the like.
  • Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol.
  • sterile, fixed oils may be employed as a solvent or suspending medium.
  • any biocompatible oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.
  • the carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s).
  • a delivery vehicle may include, by way of non limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.
  • the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • compositions may comprise, for example, at least about 0.1 % of an active agent, such as an isolated exosome, a related lipid nanovesicle, or an exosome or nanovesicle loaded with therapeutic agents or diagnostic agents.
  • an active agent such as an isolated exosome, a related lipid nanovesicle, or an exosome or nanovesicle loaded with therapeutic agents or diagnostic agents.
  • the active agent may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a range of about 5 microgram/kg/body weight to about 100 mg/kg/body weight, about 5 micro gram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered.
  • Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified.
  • a typical composition for such purpose comprises a pharmaceutically acceptable carrier.
  • the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline.
  • Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial agents, antgifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.
  • Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
  • the compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • the pharmaceutical compositions may include classic pharmaceutical preparations.
  • Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Topical administration may be particularly advantageous for the treatment of skin cancers, to prevent chemotherapy-induced alopecia or other dermal hyperproliferative disorder.
  • administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
  • aerosol delivery can be used for treatment of conditions of the lungs. Volume of the aerosol is between about 0.01 ml and 0.5 ml.
  • An effective amount of the pharmaceutical composition is determined based on the intended goal.
  • unit dose or“dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen.
  • Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.
  • 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, 16, 17, 18, 19, 20, 21, 22,
  • kits for evaluating biomarker activity in a cell are provided.
  • 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, primers, synthetic nucleic acids, nonsynthetic nucleic acids, biomarker binding polypeptides, antibodies, 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, and any such molecules that hybridize to a biomarker nucleic acid.
  • control nucleic acids, probes, and inhibitors are included in some kit embodiments.
  • the control molecules can be used to verify efficiency and/or control for sample quality or to normalize expression.
  • a kit 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.
  • 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 and/or probes and detecting agents.
  • 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 Genomewide expression profiling identifies a novel miRNA-based signature for the detection of peritoneal metastasis in patients with gastric cancer
  • the novel miRNA-based signature is a robust diagnostic tool for identifying peritoneal metastasis in GC patients, which could lead to improved survival outcomes in gastric cancer.
  • GC Gastric cancer
  • PM peritoneal metastasis
  • CT computed tomography
  • PET positron emission tomography
  • CT computed tomography
  • PET positron emission tomography
  • the current study consisted of a systematic and comprehensive biomarker discovery and a validation phases.
  • the inventors generated microarray-based miRNA expression profiling results, followed by additional comparison in The Cancer Genome Atlas (TCGA) dataset, for the identification of candidate miRNAs that can detect PM in GC patients.
  • TCGA Cancer Genome Atlas
  • the inventors identified differentially expressed miRNAs in the primary tumors from PM positive vs. negative patients, with a p value of ⁇ 0.05 as an initial cut-off criteria in the miRNA- profiling data.
  • the inventors analyzed TCGA dataset to identify miRNAs that were differentially expressed in the stage IV vs. other advanced GC patients.
  • the inventors selected miRNAs that were commonly dysregulated in both datasets of GC patients.
  • the inventors examined their expression by TaqMan-based qRT-PCR assays in three independent cohorts of GC patients.
  • the three clinical validation cohorts enrolled 354 gastric cancer patients, comprising of a testing cohort of 65 patients enrolled at the Mie University, a validation cohort of 85 patients from the Kumamoto University, and a performance evaluation cohort of 204 patients from the Nagoya University, Japan.
  • the testing and validation cohorts included paraffin embedded tissues, while the performance evaluation cohort comprised of frozen tissues. Further information on patient demographics and clinicopathological characteristics are provided in the Table 1. A written informed consent was obtained from all patients, and the Institutional Review Boards of all participating institutions approved the study. 2.
  • the raw data of each spot was normalized by substitution with a mean intensity of the background signal determined by signal intensities of all blank spots with 95% confidence intervals. Measurements of spots with the signal intensities greater than 2 standard deviations (SD) of the background signal intensity were considered to be valid. The relative expression level of a given miRNA was calculated by comparing the signal intensities of the valid spots throughout the microarray experiments.
  • “LIMMA” package R studio, Boston, MA
  • quantile normalization of the array results was used for background corrections, followed by quantile normalization of the array results.
  • the inventors performed miRNA expression profiling in six patients each, with and without PM. The inventors used a p-value of ⁇ 0.05 as the initial criteria to identify differentially expressed miRNAs (Figure 1A). Subsequently, the inventors identified 513 candidate miRNAs, of which 364 were upregulated in the primary tumors of GC patients with PM ( Figure IB). To further narrow down the list of miRNA candidates for developing a clinically relevant diagnostic signature, the inventors next analyzed the TCGA dataset to identify miRNAs that were specifically differentially expressed in stage IV vs. other stages of GC patients.
  • stage IV vs. stage IB-III tumors identified 104 differentially expressed miRNAs, of which 46 were upregulated and 58 were downregulated.
  • the inventors thereafter overlapped miRNAs that were dysregulated both in PM and stage IV GC patients, and identified eight miRNAs, of which five were upregulated (miR-30a-5p, miR-134-5p, miR-337-3p, miR-659-3p, and miR-3917) and three were downregulated (miR-718, miR-1281, and miR-3162). Since the read count of the three downregulated miRNAs were extremely low, the inventors excluded these and were able to successfully validate the remaining five miRNAs in the TCGA dataset ( Figure 1C).
  • the candidate miRNA biomarkers were overexpressed gastric cancer patients with peritoneal metastasis
  • Tumor macroscopic type together with the miRNA signature further improved the diagnostic accuracy for identifying peritoneal metastasis in gastric cancer patients.
  • the inventors assessed the performance of various clinicopathological factors associated with PM, in conjunction with the miRNA signature, to determine whether the miRNA signature might serve as an independent factor for diagnosing PM in GC patients.
  • the inventors also aimed to identify if any of the clinical factor(s) could be used in conjunction with the miRNA signature in further improving its diagnostic accuracy.
  • the inventors thereafter assessed the efficacy of a five miRNA panel in the two independent clinical cohorts, which led to the identification of three miRNAs which were consistently overexpressed in both cohorts (testing and validation), and the combination miRNA signature was significantly more robust in discriminating GC patients with vs. without PM.
  • the inventors evaluated the robustness of the miRNA biomarkers in a performance evaluation cohort of fresh frozen specimens that would mimic biopsies in pre-surgical endoscopy settings, and the miRNA signature performed equally robustly even in this cohort. Furthermore, high expression of all three miRNAs individually and their combination was associated with inferior overall survival.
  • the inventors established a diagnostic probability nomogram by combining risk scores from the miRNA signature together with the macroscopic Borrmann’s type, which were significantly superior and offered a positive coskbenefit for their clinical application in diagnosing peritoneal metastasis in gastric cancer patients.
  • MiRNAs are short, single-stranded, noncoding RNAs which are frequently dysregulated in cancers. Previous studies have demonstrated that several miRNAs appear to have functional associations with PM in GC patients [23-25]. Using a peritoneal metastatic GC derived cell line, miR-136 was identified as a tumor- suppressive miRNA which attenuated metastatic potential of GC cells [23]. Similarly, a series of in vitro and in vivo experiments demonstrated miR-3978 as a potential tumor suppressor-miRNA, which inhibited legumain, a lysosomal cysteine endopeptidase [24, 25]. Collectively, these studies supported functional relevance of miRNAs in GC and suggested their potential clinical significance for the diagnosis of patients with PM.
  • miR-30a Among the initially identified panel of miRNAs overexpressed in primary tumors of PM-positive patients, miR-30a, miR-659 and miR-3917 were consistently overexpressed and successfully validated in three independent patient cohorts.
  • high expression of miR-30a was shown to promote migratory and invasive potential through upregulation of epithelial-to-mesenchymal-transition related genes [27].
  • overexpression of miR-30, including miR-30a resulted in suppression of SOCS3, a key regulator of Jak/STAT3 pathway, and subsequently enhanced glioma stem cell growth [28].
  • miR-30 is overexpressed in both GC tissues and its overexpression enhanced cellular proliferation and suppressed apoptosis through inhibition of p53 [29].
  • miR-30a acts as an oncogene.
  • functional role of miR-659 and miR-3917 is unclear and remains to be elucidated.
  • the current study demonstrates that a combination of the miRNA signature together with the tumor macroscopic type (Borrmann’s type III and IV) was significantly superior in identifying GC patients with PM.
  • the Borrmann’s classification was developed in 1926, and is widely used to classify GCs based on endoscopic characteristics.
  • Several studies have shown that Borrmann’s type correlates with other clinicopathological factors including depth of invasion, tumor stage, lymph node metastasis, distant metastasis and PM [30-32].
  • GCs with macroscopic Borrmann’s type III and IV are consistently associated with PM, it was not surprising that macroscopic type was identified as one of the key factors for PM diagnosis. Since, macroscopic stages for GC can be determined during endoscopy prior to the surgery, the risk probability nomogram, which combines macroscopic type with the miRNA signature offers a potentially attractive choice for the early diagnosis of PM in gastric cancer patients.
  • the inventors have developed a novel miRNA-based signature for the detection of PM in GC patients, and validated its robustness in multiple independent clinical cohorts.
  • the inventors have established a risk-diagnosis nomogram that offers a potentially attractive approach for the diagnosis of PM patients in gastric cancer patients, providing a more personalized approach for improving the overall survival and mortality in this patient population.
  • Table 2 Multivariate logistic regression analysis for the diagnosis of peritoneal metastasis in gastric cancer patients in each cohort
  • Example 2 A miRNA-based signature for detection of gastric cancer peritoneal metastasis
  • microRNA microRNA
  • the inventors have comprehensively characterized microRNA (miRNA) profile of primary tumors from patients with peritoneal metastasis and compared to those without by microarray.
  • the inventors examined biomarker potential of candidate miRNAs found to be dysregulated in tumors from patients with peritoneal metastasis in three independent cohorts, 354 in total, comprised of advanced gastric cancers (tumors with T stage greater than T2) by qRT-PCR.
  • CT computed tomography
  • PET positron emission tomography
  • CA125 and CA72-4 are tumor markers typically upregulated in advanced gastric cancers and also shown to be overexpressed in serum of patients with gastric cancer peritoneal metastasis [8-10]. However, individually or even as a combination the sensitivity of these tumor markers for detecting peritoneal metastasis is poor [11]. Furthermore, if peritoneal metastasis can be detected prior to the surgery, it is possible to perform alternative treatments such as hyperthermic intraperitoneal chemotherapy (HIPEC) with cytoreductive surgery.
  • HIPEC hyperthermic intraperitoneal chemotherapy
  • HIPEC has been shown to be an effective treatment for gastric cancer patients with peritoneal metastasis [12-15]. Therefore, if there is a robust molecular marker which could identify patients with gastric cancer peritoneal metastasis, alternative treatment strategies can be implemented for patients with peritoneal metastasis.
  • the inventors conducted a comprehensive miRNA profiling of primary tumors derived from peritoneal metastasis patients and compared to that of those without peritoneal metastasis using miRNA-microarray. Subsequently clinical significance of key miRNAs dysregulated in the primary tumors from gastric cancer patients with peritoneal metastasis identified from microarray profiling were evaluated in multiple independent clinical patient cohorts comprised of advanced gastric cancers. The data indicates that miRNA-based signature could be used to diagnose gastric cancer patients with peritoneal metastasis.
  • This study analyzed 354 tissue specimens, which comprised of 150 formalin-fixed paraffin-embedded (FFPE) primary gastric cancer tissues and 204 fresh primary gastric cancer tissues. These tissues were collected from patients enrolled at Mie University Hospital, Kumamoto University Hospital, and Nagoya University Hospital in Japan. Further information on patient demographics and clinicopathological characteristics are provided in the Supplementary Table 1.
  • the current study consists of discovery phase and validation phase. In the discovery phase, the inventors used miRNA-microarray data that the inventors have generated as well as the Cancer Genome Atlas (TCGA) dataset to identify miRNA candidates for further validation.
  • TCGA Cancer Genome Atlas
  • the inventors identified differentially expressed miRNAs in the primary tumors of peritoneal metastasis positive patients compared to those without peritoneal metastasis using miRNA-microarray data with p ⁇ 0.05 as the initial cutoff criteria. Furthermore, the inventors used TCGA dataset to identify miRNAs differentially expressed in the stage IV patients compared to other gastric cancer stages. Subsequently, the inventors have selected miRNAs identified in both datasets as candidate miRNAs. To evaluate the efficacy of the miRNA candidates identified in the discovery phase, the inventors examined the expression of the candidate miRNAs by TaqMan- based qRT-PCR in three independent tissue validation cohorts. Written informed consent was obtained from all patients, and the Institutional Review Boards of all participating institutions approved the study.
  • RNA was extracted according to the manufacturer’s protocols. Quantitative evaluation of total RNA was performed by Nanodrop. Extracted total RNA were labeled using the miRNA Complete Labeling Kit, Labeled RNAs were hybridized onto Human miRNA Microarray Kit Release, 8x60K. Arrays were scanned and images analyzed by the Feature Extraction Software from Agilent Technologies.
  • RNA extraction from FFPE tissue samples and fresh frozen tissue samples stored in RNA later were carried out with miRNeasy FFPE Kit (Qiagen, Valencia, CA) and miRNeasy Mini Kit (Qiagen) respectively according to the manufacturer's instructions.
  • miRNAs was analyzed by TaqMan miRNA real-time qRT-PCR assays (Applied Biosystems, Foster City, CA) using QuantStudioTM 7 Flex Real-Time PCR System (Applied Biosystems).
  • miR-30a-5p, miR-134-5p, miR-337-3p, miR-669-3p (assay no: 000417, 001186, 002157, 001514 Catalog no: 4427975, Thermo Fisher Scientific) and miR-3917 (Assay no: 464692_mat, Cat no: 440886, Thermo Fisher Scientific).
  • U6 (Assay no: 001973, Cat no: 4427975Thermo Fisher Scientific) was used as an endogenous control for data normalization.
  • the expression levels of miRNAs were calculated using the 2-ACT method.
  • ROC curve for peritoneal metastasis using combined miRNA signature and clinical risk factors which remained in multivariate analysis are also generated by logistic regression analysis.
  • OS Overall survival
  • the inventors utilized miRNA microarray to profile primary tumors obtained from six patients with peritoneal metastasis and compare to that of six without peritoneal metastasis.
  • the inventors used p-value ⁇ 0.05 as the initial criteria to identify differentially expressed miRNAs (Figure 9A).
  • the inventors identified 513 differentially expressed miRNAs, of which 364 were upregulated in the primary tumor of peritoneal metastasis positive patients ( Figure 9B).
  • the inventors next interrogated TCGA dataset to determine miRNAs that are specifically differentially expressed in stage IV gastric cancer patients.
  • TCGA does not contain specific information on the type of gastric cancer metastasis
  • the inventors wanted to ensure that miRNAs the inventors identified in miRNA-microarray dataset are specific to metastatic gastric cancers and not dysregulated in other gastric cancer stages. Accordingly, the inventors compared miRNA expression between Stage IV against Stage IB to III tumors using the following criteria of p-value ⁇ 0.05 and identified 104 differentially expressed miRNAs, of which 46 were upregulated and 58 were downregulated.
  • the inventors then overlapped miRNAs which the inventors identified in the microarray dataset and miRNAs overexpressed specifically in stage IV tumors in the TCGA dataset and identified eight miRNAs, of which five were upregulated (miR-30a-5p, miR-134-5p, miR-337-3p, miR-659-3p, and miR-3917) and three were downregulated (miR-718, miR-1281, and miR-3162). Considering that the read count of downregulated miRNAs were extremely low, the inventors have decided to use five miRNAs upregulated miRNAs as the initial candidate (Figure 9C). 2.
  • the candidate miRNAs were overexpressed in the primary tumors of gastric cancer peritoneal metastasis patients in the clinical cohort 1
  • the expression of these miRNAs should be assessed as biopsy samples harvested at the time of diagnostic upper-gastro- endoscopy. Typically these biopsy samples are stored as fresh frozen samples. Therefore, the inventors examined whether the miRNAs that the inventors have identified can be used to identify peritoneal metastatic patients in fresh frozen samples. Correlation between clinicopathological factors and relative expression of three miRNAs are summarized in Supplementary Table 4.
  • Tumor macroscopic type with combined miRNAs signature robustly identifies peritoneal metastasis patients
  • the inventors next assessed clinicopathological factors associated with peritoneal metastasis in all three cohorts to determine whether the miRNA signature is an independent factor for peritoneal metastasis diagnosis using logistic regression model. Furthermore, the inventors aimed to identify whether any other clinical factor could be combined with the miRNA signature to improve the detection of peritoneal metastasis in gastric cancer patients. The inventors specifically focused on the clinical factors which can be identified prior to the surgery, so that the clinical factors can be combined with miRNA signature to identify patients with peritoneal metastasis prior to surgery.
  • tumor macroscopic Borrmann type III or IV 0.003
  • tumor macroscopic type III or IV p ⁇ 0.0001
  • miRNA combined signature p ⁇ 0.0001
  • the inventors identified that three miRNAs that were consistently overexpressed in both cohorts and the combined miRNA signature robustly discriminated peritoneal metastasis patients from those without peritoneal metastasis and high expression of these miRNAs were associated with poor overall survival.
  • An addition of macroscopic type signature improved the diagnostic robustness of the miRNA signature in both cohorts.
  • the inventors further evaluated the robustness of these miRNAs in a clinical cohort with fresh frozen samples to mimic typical biopsy samples and showed that the miRNA signature identified patients with peritoneal metastasis effectively.
  • MiRNAs are a short single- stranded non-coding RNAs which are dysregulated in cancers.
  • the inventors identified five miRNAs overexpressed in primary tumors of patients with gastric cancer peritoneal metastasis from comprehensive miRNA profiling using two high throughput datasets in the discovery phase and validated these candidate miRNAs in multiple clinical cohorts. The data indicates that miRNAs could be used to identify patients with peritoneal metastasis. Previous studies have demonstrated that several miRNAs appear to have functional association with gastric cancer peritoneal metastasis [23-25].
  • miR-136 was identified as a tumor-suppressive miRNA which attenuated metastatic potential of gastric cancer cells when overexpressed [23].
  • miR-3978 was identified as a potential tumor suppressor-miR which inhibits legumain, a lysosomal cysteine endopeptidase of the asparaginyl endopeptidase family [24, 25].
  • miRNAs overexpressed in primary tumors of patients with gastric cancer peritoneal metastasis were consistently overexpressed in peritoneal metastasis positive patients in all three cohorts.
  • high expression of miR-30a was shown to promote migratory and invasive capability through enhancement of epithelial-to-mesenchymal-transition related genes including fibronectin, vimentin, and N-cadherin [27].
  • miR-30 overexpression of miR-30, including miR-30a, resulted in suppression of SOCS3, a key regulator of Jak/STAT3 pathway, and subsequently enhanced glioma stem cell growth [28].
  • miR-30 was shown to be overexpressed in both gastric cancer tissues and overexpression of miR-30 enhanced cellular proliferation and suppressed apoptosis through inhibition of p53 [29].
  • paclitaxel is well used conventional drug for ovarian, breast and lung cancers and acts by interfering with the function of microtubules during cell division [34-36].
  • intraperitoneal administration instead of conventional systemic administration of paclitaxel was effective for treatment of gastric cancer peritoneal metastasis [37-40].
  • cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) has been shown to be effective for gastric cancer patients with peritoneal metastasis [13-15, 41].
  • miRNAs could be utilized to identify peritoneal metastasis patients
  • the miRNA signature must be tested in a larger cohort to evaluate their true potential as a diagnostic marker.
  • the number of patients with peritoneal metastasis is relatively scarce, it would require several institutions over an extended period of time to collect samples prospectively and evaluate the efficacy of the signature.
  • the inventors were unable to evaluate whether this miRNA signature could identify metachronous peritoneal metastasis.
  • the inventors have developed miRNA-based signature for detection of gastric cancer peritoneal metastasis, and validated the robustness of the signature in multiple independent clinical cohorts.
  • Table 3 Multivariate logistic regression analysis for peritoneal metastasis diagnosis in fresh frozen cohort
  • European journal of surgical oncology the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 2016; 42: 1123-1131.

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Abstract

La présente invention concerne une signature de miARN pour la détection du cancer gastrique. Des aspects de l'invention concernent un procédé de traitement, d'évaluation, de pronostic et/ou de diagnostic d'un patient pour traiter un patient atteint d'un cancer gastrique, le procédé comprenant le traitement du patient atteint d'un cancer gastrique après que le niveau d'expression de miR-30a-5p, miR-134-5p, miR-337-3p, miR-659-3p, et miR-3917 a été déterminé dans un échantillon provenant du patient.
PCT/US2019/067942 2018-12-21 2019-12-20 Procédés de pronostic, de diagnostic et de traitement du cancer gastrique Ceased WO2020132494A2 (fr)

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CN116179691A (zh) * 2022-09-07 2023-05-30 苏州大学附属第一医院 一种胃癌组合标志物及标志物模型的建立方法

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EP3177739B1 (fr) * 2014-08-07 2019-10-23 Agency For Science, Technology And Research Biomarqueur à base de microarn utilisable en vue du diagnostic du cancer gastrique
US11746381B2 (en) * 2017-03-10 2023-09-05 Cancer Diagnostics Research Innvovations, LLC Methods for diagnosing and treating gastric cancer using miRNA expression

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