{
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"PROJECT":{"PROJECT_TITLE":"Aspirin Metabolomics in Colorectal Cancer Chemoprevention - Blood (Part I)","PROJECT_TYPE":"NIH/NCI R01CA188038","PROJECT_SUMMARY":"Substantial evidence supports the effectiveness of aspirin for cancer chemoprevention in addition to its well established role in cardiovascular protection. In recent meta-analyses of randomized controlled trials in human, daily aspirin use reduced incidence, metastasis and mortality from several common types of cancer, especially colorectal cancer. The mechanism(s) by which aspirin exerts an anticancer benefit is uncertain; numerous effects have been described involving both cyclooxygenase-dependent and -independent pathways. The goal of this research is to elucidate the key metabolic changes that are responsible for the anticancer effects of aspirin in humans using untargeted metabolomics analysis. Metabolomics, or global metabolite profiling, is an emerging discipline that has the potential to transform the study of pharmaceutical agents. Our innovative approach will use high-resolution mass spectroscopy to detect thousands of metabolites in blood plasma and normal colon mucosa biopsies that were collected from participants in the Aspirin/Folate Polyp Prevention Study, a randomized, double-blind, placebo-controlled trial of aspirin and/or folic acid supplementation for the prevention of colorectal adenomas. Participants in the trial were assigned with equal probability to three aspirin treatment arms (placebo, 81mg, or 325mg daily). Over the three-year period, 81mg/day of aspirin reduced the risk of adenomas, whereas the 325 mg/day dose had less effect. The aims of the current proposal are to identify metabolomic signatures, including specific metabolites and metabolic pathways, that are associated with aspirin treatment in blood and normal colon mucosal tissue of participants after three years of randomized aspirin treatment; and then to assess the associations of these metabolic signatures with adenoma risk and whether they mediate the reductions in risk due to 81 mg/day aspirin treatment. We will prioritize metabolites for study by evaluating metabolite levels in patients from the placebo and treatment arms while controlling the false discovery rate, use correlation analysis to enhance identification of relevant metabolic modules associated with these prioritized metabolites, and apply pathway mapping with post-hoc application of ion dissociation spectroscopy to representative metabolites to confirm pathway identification. Because aspirin is a multifunctional drug that is thought to modify numerous pathways with potential roles in carcinogenesis, a global discovery-based metabolomics approach is the best way to identify its key activities. The public health significance of this work is substantial because understanding the mechanism of aspirin's anticancer effects is key to optimizing its use and to the development of novel drugs targeting the metabolic pathways identified.","INSTITUTE":"Emory University Dept of Medicine","DEPARTMENT":"School of Medicine","LABORATORY":"Clinical Biomarkers Laboratory","LAST_NAME":"Tran","FIRST_NAME":"ViLinh","ADDRESS":"615 Michael St, suite 225","EMAIL":"vtran6@emory.edu","PHONE":"4047275091","FUNDING_SOURCE":"NIH/NCI R01CA188038"},

"STUDY":{"STUDY_TITLE":"Aspirin Metabolomics in Colorectal Cancer Chemoprevention - blood (part-II)","STUDY_SUMMARY":"Substantial evidence supports the effectiveness of aspirin for cancer chemoprevention in addition to its well established role in cardiovascular protection. In recent meta-analyses of randomized controlled trials in human, daily aspirin use reduced incidence, metastasis and mortality from several common types of cancer, especially colorectal cancer. The mechanism(s) by which aspirin exerts an anticancer benefit is uncertain; numerous effects have been described involving both cyclooxygenase-dependent and -independent pathways. The goal of this research is to elucidate the key metabolic changes that are responsible for the anticancer effects of aspirin in humans using untargeted metabolomics analysis. Metabolomics, or global metabolite profiling, is an emerging discipline that has the potential to transform the study of pharmaceutical agents. Our innovative approach will use high-resolution mass spectroscopy to detect thousands of metabolites in blood plasma that were collected from participants in the Aspirin/Folate Polyp Prevention Study, a randomized, double-blind, placebo-controlled trial of aspirin for the prevention of colorectal adenomas. Participants in the trial were assigned with equal probability to three aspirin treatment arms (placebo, 81mg, or 325mg daily). Over the three-year period, 81mg/day of aspirin reduced the risk of adenomas, whereas the 325 mg/day dose had less effect. The aims of the current proposal are to identify metabolomic signatures, including specific metabolites and metabolic pathways, that are associated with aspirin treatment in blood of participants after three years of randomized aspirin treatment; and then to assess the associations of these metabolic signatures with adenoma risk and whether they mediate the reductions in risk due to 81 mg/day aspirin treatment. We will prioritize metabolites for study by evaluating metabolite levels in patients from the placebo and treatment arms while controlling the false discovery rate, use correlation analysis to enhance identification of relevant metabolic modules associated with these prioritized metabolites, and apply pathway mapping with post-hoc application of ion dissociation spectroscopy to representative metabolites to confirm pathway identification. Because aspirin is a multifunctional drug that is thought to modify numerous pathways with potential roles in carcinogenesis, a global discovery-based metabolomics approach is the best way to identify its key activities. The public health significance of this work is substantial because understanding the mechanism of aspirin's anticancer effects is key to optimizing its use and to the development of novel drugs targeting the metabolic pathways identified.","INSTITUTE":"Emory University","DEPARTMENT":"School of Medicine","LABORATORY":"Clincal Biomarkers Laboratory","LAST_NAME":"Uppal","FIRST_NAME":"Karan","ADDRESS":"615 Michael St, Suite 225","EMAIL":"kuppal2@emory.edu","PHONE":"(404) 727 5027","TOTAL_SUBJECTS":"600","STUDY_COMMENTS":"Aspirin Metabolomics Priority 1"},

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"Sample ID":"40001440_1",
"Factors":{"Sex":"1","Treatment":"2"},
"Additional sample data":{"SubjectID":"40250","RAW_FILE_NAME":"VT_160226_079"}
},
{
"Subject ID":"Plasma Study Sample",
"Sample ID":"40022363_1",
"Factors":{"Sex":"2","Treatment":"-"},
"Additional sample data":{"SubjectID":"20270","RAW_FILE_NAME":"VT_160226_085"}
},
{
"Subject ID":"Plasma Study Sample",
"Sample ID":"40022384_1",
"Factors":{"Sex":"2","Treatment":"1"},
"Additional sample data":{"SubjectID":"30350","RAW_FILE_NAME":"VT_160226_091"}
},
{
"Subject ID":"Plasma Study Sample",
"Sample ID":"400106024_1",
"Factors":{"Sex":"1","Treatment":"-"},
"Additional sample data":{"SubjectID":"10090","RAW_FILE_NAME":"VT_160226_097"}
},
{
"Subject ID":"Plasma Study Sample",
"Sample ID":"40022734_1",
"Factors":{"Sex":"1","Treatment":"2"},
"Additional sample data":{"SubjectID":"70321","RAW_FILE_NAME":"VT_160226_103"}
},
{
"Subject ID":"Plasma Study Sample",
"Sample ID":"40022462_1",
"Factors":{"Sex":"1","Treatment":"-"},
"Additional sample data":{"SubjectID":"60118","RAW_FILE_NAME":"VT_160226_109"}
},
{
"Subject ID":"Plasma Study Sample",
"Sample ID":"40005890_1",
"Factors":{"Sex":"2","Treatment":"1"},
"Additional sample data":{"SubjectID":"30350","RAW_FILE_NAME":"VT_160226_115"}
},
{
"Subject ID":"Plasma Study Sample",
"Sample ID":"400105771_1",
"Factors":{"Sex":"1","Treatment":"2"},
"Additional sample data":{"SubjectID":"60432","RAW_FILE_NAME":"VT_160226_121"}
},
{
"Subject ID":"Plasma Study Sample",
"Sample ID":"40000892_1",
"Factors":{"Sex":"2","Treatment":"-"},
"Additional sample data":{"SubjectID":"20270","RAW_FILE_NAME":"VT_160226_127"}
},
{
"Subject ID":"Quality Control",
"Sample ID":"q3June2014_30c_1",
"Factors":{"Sex":"NA","Treatment":"NA"},
"Additional sample data":{"SubjectID":"NA","RAW_FILE_NAME":"VT_160226_133"}
},
{
"Subject ID":"Quality Control",
"Sample ID":"nist2_1",
"Factors":{"Sex":"NA","Treatment":"NA"},
"Additional sample data":{"SubjectID":"NA","RAW_FILE_NAME":"VT_160226_139"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Blood plasma samples were collected from non-fasting participants in the Aspirin/Folate Polyp Prevention Study at enrollment (baseline) and after three years of study treatment. Blood samples were collected in tubes containing EDTA and after centrifugation aliquots of plasma were transferred to 1.8ml freezer tubes and stored frozen at -20C or lower. The samples were shipped on dry ice from the clinical centers to the Dartmouth biorepository storage facility (for storage at -70C) and subsequently to the metabolimics analysis lab at Emory University.","SAMPLE_TYPE":"Blood (plasma)","STORAGE_CONDITIONS":"Described in summary"},

"TREATMENT":{"TREATMENT_SUMMARY":"Samples were received frozen in aliquouts of <250uL. Prior to analysis, samples were thawed and prepared for HRM analysis using the standard protocols described in the Sample Preparation section."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Samples were prepared for metabolomics analysis using established methods(Johnson et al. (2010). Analyst; Go et al. (2015). Tox Sci). Prior to analysis, plasma aliquots were removed from storage at -80 degrees C and thawed on ice. Each cryotube was then vortexed briefly to ensure homogeneity, and 50 microliters was transferred to a clean microfuge tube. Immediately after, the plasma was treated with 100 microliters of ice-cold LC-MS grade acetonitrile (Sigma Aldrich) containing 2.5 microliters of internal standard solution with eight stable isotopic chemicals selected to cover a range of chemical properties. Following addition of acetonitrile, urine was equilibrated for 30min on ice, upon which precipitated proteins were removed by centrifuge (14,000rpm at 4 degrees C for 10 min). The resulting supernatant (100 microliters) was removed, added to a low volume autosampler vial and maintained at 4 degrees C until analysis (<22 h).","SAMPLEPREP_PROTOCOL_ID":"HRM_SP_082016_01","SAMPLEPREP_PROTOCOL_FILENAME":"EmoryUniversity_HRM_SP_082016_01.pdf","SAMPLEPREP_PROTOCOL_COMMENTS":"Date effective: 30 July 2016","EXTRACTION_METHOD":"2:1 acetonitrile: sample followed by vortexing and centrifugation"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"The C18 column is operated parallel to the HILIC column for simultaneous analytical separation and column flushing through the use of a dual head HPLC pump equipped with 10-port and 6- port switching valves. During operation of the C18 method, the MS is operated in negative ion mode and 10 uL of sample is injected onto the C18 column while the HILIC column is flushing with wash solution. Flow rate is maintained at 0.35 mL/min until 2 min, increased to 0.4 mL/min at 6 min and held for 4 min. Solvent A is 100% LC-MS grade water, solvent B is 100% LC-MS grade acetonitrile and solvent C is 2% formic acid (v/v) in LC-MS grade water. Initial mobile phase conditions are 80% A, 15% B, 5% C hold for 0.5 min, with linear gradient to 0% A, 95% B, 5% C at 2 min, hold for 4min, resulting in a total analytical run time of 10 min. During the flushing phase (HILIC analytical separation), the C18 column is equilibrated with a wash solution of 0% A, 95% B, 5% C until 7.5 min, followed by an equilibration solution of 80% A, 15% B, 5% C for 2.5 min.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Dionex UltiMate 3000","COLUMN_NAME":"Thermo Fisher Accucore C18 50x2.1mm 2.6u with Thermo accucore","FLOW_RATE":"0.35 mL/min for 2 min; linear increase to 0.4 mL/min at 6 min held for 4 min","COLUMN_TEMPERATURE":"60C","METHODS_FILENAME":"20160120_posC18120kres10mim_ESI_hilicposwash_acore.meth","SOLVENT_A":"LC-MS grade water","SOLVENT_B":"LC-MS grade acetonitrile","SAMPLE_INJECTION":"10 uL","ANALYTICAL_TIME":"10 min","SAMPLE_LOOP_SIZE":"15 uL","SAMPLE_SYRINGE_SIZE":"100 uL"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"Clinical Biomarkers Laboratory","OPERATOR_NAME":"Vilinh Tran","DETECTOR_TYPE":"Orbitrap","SOFTWARE_VERSION":"Xcalibur 3.0.63","ACQUISITION_DATE":"1/20/16-2/26/16","ANALYSIS_PROTOCOL_FILE":"EmoryUniversity_HRM_QEHF-MS_092017_v1.pdf","DATA_FORMAT":"Profile"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive HF hybrid Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"None","CAPILLARY_TEMPERATURE":"250C","COLLISION_GAS":"N2","DRY_GAS_FLOW":"45","DRY_GAS_TEMP":"150C","MASS_ACCURACY":"< 3ppm","SPRAY_VOLTAGE":"-4000","ACTIVATION_PARAMETER":"5.00E+05","ACTIVATION_TIME":"118ms","INTERFACE_VOLTAGE":"S-Lens RF level= 55","MS_RESULTS_FILE":"ST001422_AN002379_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Seconds"}

}