#METABOLOMICS WORKBENCH vykoukal_20200519_113122 DATATRACK_ID:2017 STUDY_ID:ST001408 ANALYSIS_ID:AN002353 PROJECT_ID:PR000964 VERSION 1 CREATED_ON June 26, 2020, 2:56 pm #PROJECT PR:PROJECT_TITLE AS Biomarker Project PR:PROJECT_TYPE Biomarker PR:PROJECT_SUMMARY Metabolomic profiling of baseline plasmas from a longitudinal prospective cohort PR:PROJECT_SUMMARY of 491 active surveillance (AS) participants with the objective of identifying PR:PROJECT_SUMMARY metabolic fingerprints that predict Gleason grade progression. PR:INSTITUTE University of Texas MD Anderson Cancer Center PR:DEPARTMENT Department of Clinical Cancer Prevention PR:LAST_NAME Vykoukal PR:FIRST_NAME Jody PR:ADDRESS 1515 Holcombe Boulevard, Houston, Texas, 77030, USA PR:EMAIL jody@mdanderson.org PR:PHONE 8327243044 #STUDY ST:STUDY_TITLE Metabolomic profiling of baseline plasmas from a longitudinal prospective cohort ST:STUDY_TITLE of 491 active surveillance (AS) participants ST:STUDY_TYPE Biomarker study ST:STUDY_SUMMARY Untargeted metabolomics analyses were performed on clinically matched baseline ST:STUDY_SUMMARY plasma samples (n = 16 per group) prospectively collected from patients with ST:STUDY_SUMMARY clinically low-risk early stage prostate cancer undergoing AS who exhibited ST:STUDY_SUMMARY early disease progression (DP) (defined as upgrading of Gleason score (GS) ST:STUDY_SUMMARY and/or increased tumor volume on surveillance biopsy within 18 months after ST:STUDY_SUMMARY start of AS) or indolent disease (no progression for five or more years after ST:STUDY_SUMMARY start of AS) as well as 459 baseline plasma samples prospectively collected from ST:STUDY_SUMMARY patients with early-stage prostate cancer undergoing AS. ST:INSTITUTE University of Texas MD Anderson Cancer Center ST:DEPARTMENT Department of Clinical Cancer Prevention ST:LAST_NAME Vykoukal ST:FIRST_NAME Jody ST:ADDRESS 6767 Bertner Ave, Houston, TX 77030 ST:EMAIL jvykouka@mdanderson.org ST:PHONE 713-834-6095 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - PL3B1_Lipids_39106 AS participant:AS participant RAW_FILE_NAME=P_PL3B1_004_39106.raw SUBJECT_SAMPLE_FACTORS - PL3B1_Lipids_39122 AS participant:AS participant RAW_FILE_NAME=P_PL3B1_005_39122.raw SUBJECT_SAMPLE_FACTORS - PL3B1_Lipids_909977 AS participant:AS participant RAW_FILE_NAME=P_PL3B1_006_909977.raw SUBJECT_SAMPLE_FACTORS - PL3B1_Lipids_43883 AS participant:AS participant RAW_FILE_NAME=P_PL3B1_007_43883.raw SUBJECT_SAMPLE_FACTORS - PL3B1_Lipids_45269 AS participant:AS participant RAW_FILE_NAME=P_PL3B1_009_45269.raw SUBJECT_SAMPLE_FACTORS - 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PL26_HA_39767 AS participant:AS participant RAW_FILE_NAME=P_HA_PL26_B1_057_39767.raw SUBJECT_SAMPLE_FACTORS - PL26_HA_45814 AS participant:AS participant RAW_FILE_NAME=P_HA_PL26_B1_059_45814.raw SUBJECT_SAMPLE_FACTORS - PL26_HA_48762 AS participant:AS participant RAW_FILE_NAME=P_HA_PL26_B1_061_48762.raw SUBJECT_SAMPLE_FACTORS - PL26_HA_53570 AS participant:AS participant RAW_FILE_NAME=P_HA_PL26_B1_063_53570.raw SUBJECT_SAMPLE_FACTORS - PL26_HA_54885 AS participant:AS participant RAW_FILE_NAME=P_HA_PL26_B1_065_54885.raw SUBJECT_SAMPLE_FACTORS - PL26_HA_46568 AS participant:AS participant RAW_FILE_NAME=P_HA_PL26_B1_067_46568.raw SUBJECT_SAMPLE_FACTORS - PL26_HA_52086R AS participant:AS participant RAW_FILE_NAME=P_HA_PL26_B1_073_52086R.raw SUBJECT_SAMPLE_FACTORS - PL26_HA_46328R AS participant:AS participant RAW_FILE_NAME=P_HA_PL26_B1_075_46328R.raw SUBJECT_SAMPLE_FACTORS - PL26_HA_51148R AS participant:AS participant RAW_FILE_NAME=P_HA_PL26_B1_077_51148R.raw #COLLECTION CO:COLLECTION_SUMMARY Patients in this prospective clinical cohort included men diagnosed with CO:COLLECTION_SUMMARY localized prostate cancer and enrolled on an AS trial protocol between February CO:COLLECTION_SUMMARY 2006 and February 2014 (n=825). Of these, 616 patients had at least 1 year CO:COLLECTION_SUMMARY follow-up and 491 patients had baseline plasma samples, enabling inclusion in CO:COLLECTION_SUMMARY the study. The surveillance protocol was conducted by a multidisciplinary team CO:COLLECTION_SUMMARY of urologists, radiation oncologists and medical oncologists, was approved by CO:COLLECTION_SUMMARY the Institutional Review Board, and is registered on clinicaltrials.gov CO:COLLECTION_SUMMARY (NCT00490763). EDTA was used in all plasma collections and all specimens CO:COLLECTION_SUMMARY underwent a similar number of freeze/thaw cycles prior to obtaining metabolomics CO:COLLECTION_SUMMARY data. Sample ages varied, as the study began accrual in 2006 and continued over CO:COLLECTION_SUMMARY a ten year time period. Plasma was not obtained from fasted individuals. CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Plasma metabolites were extracted from pre-aliquoted EDTA plasma (10 µL) with SP:SAMPLEPREP_SUMMARY 30µL of LCMS grade methanol (ThermoFisher) in a 96-well microplate (Eppendorf). SP:SAMPLEPREP_SUMMARY Plates were heat sealed, vortexed for 5 min at 750 rpm, and centrifuged at 2000 SP:SAMPLEPREP_SUMMARY × g for 10 minutes at room temperature. The supernatant (10 µL) was carefully SP:SAMPLEPREP_SUMMARY transferred to a 96-well plate, leaving behind the precipitated protein. The SP:SAMPLEPREP_SUMMARY supernatant was further diluted with 10 µL of 100 mM ammonium formate, pH3. For SP:SAMPLEPREP_SUMMARY Hydrophilic Interaction Liquid Chromatography (HILIC) analysis, the samples were SP:SAMPLEPREP_SUMMARY diluted with 60 µL LCMS grade acetonitrile (ThermoFisher), whereas samples for SP:SAMPLEPREP_SUMMARY C18 analysis were diluted with 60 µL water (GenPure ultrapure water system, SP:SAMPLEPREP_SUMMARY Thermofisher). Each sample solution was transferred to 384-well microplate SP:SAMPLEPREP_SUMMARY (Eppendorf) for LCMS analysis. For lipidomics, Pre-aliquoted EDTA plasma samples SP:SAMPLEPREP_SUMMARY (10 µL) were extracted with 30µL of LCMS grade 2-propanol (ThermoFisher) in a SP:SAMPLEPREP_SUMMARY 96-well microplate (Eppendorf). Plates were heat sealed, vortexed for 5min at SP:SAMPLEPREP_SUMMARY 750 rpm, and centrifuged at 2000 x g for 10 minutes at room temperature. The SP:SAMPLEPREP_SUMMARY supernatant (10µL) was carefully transferred to a 96-well plate, leaving behind SP:SAMPLEPREP_SUMMARY the precipitated protein. The supernatant was further diluted with 90µL of SP:SAMPLEPREP_SUMMARY 1:3:2 100mM ammonium formate, pH3 (Fischer Scientific): acetonitrile: 2-propanol SP:SAMPLEPREP_SUMMARY and transferred to a 384-well microplate (Eppendorf) for lipids analysis using SP:SAMPLEPREP_SUMMARY LCMS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separation was performed using C18 (Acquity™ UPLC HSS T3, 100 CH:CHROMATOGRAPHY_SUMMARY Å, 1.8 µm,, 2.1×100mm, Water Corporation, Milford, U.S.A) columns at 45°C. CH:CHROMATOGRAPHY_SUMMARY Quaternary solvent system mobile phases were (A) 0.1% formic acid in water, (B) CH:CHROMATOGRAPHY_SUMMARY 0.1% formic acid in acetonitrile and (D) 100mM ammonium formate, pH 3. For C18 CH:CHROMATOGRAPHY_SUMMARY separation, a chromatography gradient of was as follows: starting conditions, CH:CHROMATOGRAPHY_SUMMARY 100% A, with linear increase to final conditions of 5% A, 95% B followed by CH:CHROMATOGRAPHY_SUMMARY isocratic gradient at 95% B, 5% D for 1 min.Waters Acquity™ UPLC system with CH:CHROMATOGRAPHY_SUMMARY 2D column regeneration configuration CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity UPLC CH:COLUMN_NAME Waters Acquity UPLC HSS T3 (100 Ã…, 1.8 µm, 2.1×100mm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo G2-XS MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Mass spectrometry data was acquired using ‘sensitivity’ mode in positive and MS:MS_COMMENTS negative electrospray ionization mode within 50-1200 Da range. For the MS:MS_COMMENTS electrospray acquisition, the capillary voltage was set at 1.5 kV (positive), MS:MS_COMMENTS 3.0kV (negative), sample cone voltage 30V, source temperature at 120° C, cone MS:MS_COMMENTS gas flow 50 L/h and desolvation gas flow rate of 800 L/h with scan time of 0.5 MS:MS_COMMENTS sec in continuum mode. Leucine Enkephalin; 556.2771 Da (positive) and 554.2615 MS:MS_COMMENTS Da (negative) was used for lockspray correction and scans were performed at 0.5 MS:MS_COMMENTS min. The injection volume for each sample was 3µL, unless otherwise specified. MS:MS_COMMENTS The acquisition was carried out with instrument auto gain control to optimize MS:MS_COMMENTS instrument sensitivity over the samples acquisition time. LC-MS and LC-MSe data MS:MS_COMMENTS were processed using Progenesis QI (Nonlinear, Waters) and values were reported MS:MS_COMMENTS as area units. MS:MS_RESULTS_FILE ST001408_AN002353_Results.txt UNITS:normalized ion abundance Has m/z:Yes Has RT:Yes RT units:Minutes #END