#METABOLOMICS WORKBENCH hormel101_20170711_140549 DATATRACK_ID:1117 STUDY_ID:ST000789 ANALYSIS_ID:AN001257 PROJECT_ID:PR000574 VERSION 1 CREATED_ON July 12, 2017, 11:23 am #PROJECT PR:PROJECT_TITLE Mayo Pilot and Feasibility: Identifying metabolic adaptations characteristic of PR:PROJECT_TITLE multiple myeloma cells via mass spectrometry-based metabolite profiling PR:PROJECT_SUMMARY Multiple myeloma (MM) is a clonal plasma cell malignancy that remains incurable PR:PROJECT_SUMMARY in most afflicted patients. It can be preceded by an asymptomatic, premalignant PR:PROJECT_SUMMARY stage known as smoldering multiple myeloma (SMM) that does not require therapy PR:PROJECT_SUMMARY but has an increased life-long risk of progression to MM. However, one-third of PR:PROJECT_SUMMARY SMM patients are “high risk” for imminent progression to MM within two years PR:PROJECT_SUMMARY of diagnosis compared to the remainder of SMM patients who continue on an PR:PROJECT_SUMMARY indolent asymptomatic course for several years. The diagnosis of MM cannot be PR:PROJECT_SUMMARY made until they experience overt end-organ damage such as renal failure, lytic PR:PROJECT_SUMMARY bone destruction, anemia and hypercalcemia. Currently, we lack sensitive PR:PROJECT_SUMMARY biomarkers that can identify all SMM patients at high risk of progression to MM. PR:PROJECT_SUMMARY Being able to identify high risk SMM patients could allow us to initiate PR:PROJECT_SUMMARY systemic chemotherapy before they progress to MM. Cancer cells undergo distinct PR:PROJECT_SUMMARY metabolic adaptations to meet the augmented cellular demand for energy and PR:PROJECT_SUMMARY nutrients created by their increased rates of cellular proliferation. The PR:PROJECT_SUMMARY presence of an altered cellular metabolism in clonal PCs from MM patients and PR:PROJECT_SUMMARY its role as an essential factor in the progression of SMM to MM is unknown. We PR:PROJECT_SUMMARY hypothesize that the clonal PCs in high risk SMM patients likely have an altered PR:PROJECT_SUMMARY metabolic phenotype similar to those present in MM patients but different when PR:PROJECT_SUMMARY compared to clonal PCs in the remainder of the SMM patients whose clinical PR:PROJECT_SUMMARY course remains indolent. Thus, two specific aims are proposed in this study: Aim PR:PROJECT_SUMMARY 1 will verify if there are differences in the regulation of the metabolic PR:PROJECT_SUMMARY pathways in clonal PCs from MM patients compared to normal PCs from healthy PR:PROJECT_SUMMARY patients; Aim 2 will assess whether the clonal PCs from high risk SMM patients PR:PROJECT_SUMMARY bear a distinct metabolic phenotype compared to clonal PCs from standard risk PR:PROJECT_SUMMARY SMM patients. PR:INSTITUTE Mayo Clinic PR:LAST_NAME Gonsalves PR:FIRST_NAME Wilson PR:ADDRESS 200 First St. SW, Rochester, Minnesota, 55905, USA PR:EMAIL gonsalves.wilson@mayo.edu PR:PHONE 507-266-0792 #STUDY ST:STUDY_TITLE Identifying metabolic adaptations characteristic of multiple myeloma cells via ST:STUDY_TITLE mass spectrometry-based metabolite profiling from (CD138+ Plasma Cells) ST:STUDY_SUMMARY Will be assessing the untargeted metabolomic profiles of high risk versus low ST:STUDY_SUMMARY risk smoldering myeloma patients based on peripheral blood plasma, bone marrow ST:STUDY_SUMMARY plasma and Cd138+ plasma cells. Cd138+ plasma cells ms5974 ST:INSTITUTE Mayo Clinic ST:LAST_NAME Gonsalves ST:FIRST_NAME Wilson ST:ADDRESS 200 First St. SW, Rochester, Minnesota, 55905, USA ST:EMAIL gonsalves.wilson@mayo.edu ST:PHONE 507-266-0792 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - nC18-16mar16-001-r001 groups:Quick Progress | rep:1 type=NC18 SUBJECT_SAMPLE_FACTORS - nC18-16mar16-002-r001 groups:Slow Progress | rep:1 type=NC18 SUBJECT_SAMPLE_FACTORS - nC18-16mar16-004-r001 groups:Quick Progress | rep:1 type=NC18 SUBJECT_SAMPLE_FACTORS - nC18-16mar16-005-r001 groups:Slow Progress | rep:1 type=NC18 SUBJECT_SAMPLE_FACTORS - nC18-16mar16-006 groups:Quick Progress | rep:1 type=NC18 SUBJECT_SAMPLE_FACTORS - nC18-16mar16-007 groups:Slow Progress | rep:1 type=NC18 SUBJECT_SAMPLE_FACTORS - nC18-16mar16-008 groups:Slow Progress | rep:1 type=NC18 SUBJECT_SAMPLE_FACTORS - 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philic_16mar16-017-r002 groups:Quick Progress | rep:2 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-018-r001 groups:Slow Progress | rep:1 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-018-r002 groups:Slow Progress | rep:2 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-019-r001 groups:Quick Progress | rep:1 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-019-r002 groups:Quick Progress | rep:2 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-020-r001 groups:Quick Progress | rep:1 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-020-r002 groups:Quick Progress | rep:2 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-021-r001 groups:Slow Progress | rep:1 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-021-r002 groups:Slow Progress | rep:2 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-022-r001 groups:Quick Progress | rep:1 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-022-r002 groups:Quick Progress | rep:2 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-023-r001 groups:Quick Progress | rep:1 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-023-r002 groups:Quick Progress | rep:2 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-024-r001 groups:Slow Progress | rep:1 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-024-r002 groups:Slow Progress | rep:2 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-025-r001 groups:Quick Progress | rep:1 type=PHILIC SUBJECT_SAMPLE_FACTORS - philic_16mar16-025-r002 groups:Quick Progress | rep:2 type=PHILIC #COLLECTION CO:COLLECTION_SUMMARY "In order to analyze the metabolites of clonal PCs (intracellular) and BM plasma CO:COLLECTION_SUMMARY (extracellular) separately, they have to be separated out from the BM samples. CO:COLLECTION_SUMMARY Thus, upon acquiring the BM samples from patients they will be placed in CO:COLLECTION_SUMMARY centrifuged at 2500 rpm for 10 minutes to separate out the plasma from the CO:COLLECTION_SUMMARY cellular fraction. The separated BM plasma is then stored in separate vials and CO:COLLECTION_SUMMARY snap frozen under liquid nitrogen for 20 seconds before storing at -80οC for CO:COLLECTION_SUMMARY further analysis. The leftover cellular component present as a pellet will be CO:COLLECTION_SUMMARY washed and reconstituted with an equal volume of RPMI 1640 medium. Erythrocytes CO:COLLECTION_SUMMARY are lysed using ammonium chloride lysing solution. After incubation on ice for 5 CO:COLLECTION_SUMMARY min, the cell suspension is diluted with RPMI medium. The cells are again CO:COLLECTION_SUMMARY pelleted by centrifugation and then suspended in RoboSep buffer (250ml PBS, 2% CO:COLLECTION_SUMMARY BSA, 1mM EDTA). The clonal CD138 positive PCs are purified using positive CO:COLLECTION_SUMMARY selection by mixing the cells with a CD138 positive selection cocktail and CO:COLLECTION_SUMMARY anti-CD138 magnetic-activated cell separation microbeads (ROBOSEP™ cell CO:COLLECTION_SUMMARY separation system, StemCell Technologies Inc) in the automated RoboSep cell CO:COLLECTION_SUMMARY separation system. The purified samples containing only CD138 positive cells are CO:COLLECTION_SUMMARY re‐suspended and then centrifuged to form a cell pellet. The goal will be to CO:COLLECTION_SUMMARY obtain at least 1-2 x 107 clonal PCs per sample. The cell pellet will be snap CO:COLLECTION_SUMMARY frozen under liquid nitrogen for 20 seconds before storing at -80οC for further CO:COLLECTION_SUMMARY analysis. Both the BM plasma samples as well as the clonal PCs pellet will be CO:COLLECTION_SUMMARY provided to the metabolomics core for sample preparation for LC-MS analysis. For CO:COLLECTION_SUMMARY Aim 2, we will be using stored BM samples from SMM patients that have already CO:COLLECTION_SUMMARY had their BM plasma and clonal PCs separated from each other and stored at CO:COLLECTION_SUMMARY -80οC. It is important to note that all these samples were collected and frozen CO:COLLECTION_SUMMARY in a timely manner. Furthermore consistency in sample collection, storage and CO:COLLECTION_SUMMARY processing is imperative for the optimal conduct of metabolomics-based CO:COLLECTION_SUMMARY experiments. This ensures that all samples being analyzed and compared with each CO:COLLECTION_SUMMARY other would have been manipulated similarly, limiting any handling biases. All CO:COLLECTION_SUMMARY patient samples in the Predolin Foundation Biobank were collected and stored by CO:COLLECTION_SUMMARY following a standardized operating procedure. BM samples were processed for BM CO:COLLECTION_SUMMARY plasma separation and clonal PCs enrichment but were then immediately snap CO:COLLECTION_SUMMARY frozen for storage at -80οC within approximately 3 hours of collection from the CO:COLLECTION_SUMMARY patient. All samples were collected in patients who have been fasting for at CO:COLLECTION_SUMMARY least 6 to 8 hours prior. To further ensure consistency in our analysis of this CO:COLLECTION_SUMMARY study, we will only use samples that have never been thawed since initial CO:COLLECTION_SUMMARY storage to preserve the stability of the metabolites originally present in the CO:COLLECTION_SUMMARY samples." #TREATMENT TR:TREATMENT_SUMMARY We will use matched BM plasma and purified clonal marrow PCs from the BM samples TR:TREATMENT_SUMMARY of SMM patients collected at the time of their diagnosis and stored within the TR:TREATMENT_SUMMARY Mayo Clinic Predolin Foundation Biobank. We will select BM samples from patients TR:TREATMENT_SUMMARY with SMM who progressed to MM within 2 years of their samples being collected TR:TREATMENT_SUMMARY and stored; this will constitute the high risk SMM group. We will also select BM TR:TREATMENT_SUMMARY samples from SMM patients who have not progressed to MM within at least 2 years TR:TREATMENT_SUMMARY of follow up of their samples being collected and stored; this will constitute TR:TREATMENT_SUMMARY the standard risk SMM group. The strength of this approach is that we will TR:TREATMENT_SUMMARY utilize stored SMM samples from one of the most comprehensively characterized TR:TREATMENT_SUMMARY monoclonal gammopathy biobanks available. Secondly, all samples will be obtained TR:TREATMENT_SUMMARY from collection dates at least 2 years prior in order to ensure adequate TR:TREATMENT_SUMMARY follow-up time to assess their current clinical status. This would avoid the TR:TREATMENT_SUMMARY need to prospectively collect samples from SMM patients and clinically follow TR:TREATMENT_SUMMARY them for a number of years before we are able to gauge who were clinically high TR:TREATMENT_SUMMARY or standard risk for progression. There are over 700 SMM patients who have had TR:TREATMENT_SUMMARY their BM samples collected and stored in the biobank from 1996 to 2013; more TR:TREATMENT_SUMMARY than half these patients have progressed to MM. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The metabolite profiling as well as sample preparations will be undertaken at SP:SAMPLEPREP_SUMMARY the Mayo Clinic Metabolomics Resource Core. The matched BM plasma and clonal PCs SP:SAMPLEPREP_SUMMARY pellets from the stored patient samples will undergo metabolite extraction and SP:SAMPLEPREP_SUMMARY analysis by LC-MS. They will be deproteinized with cold methanol (1:4 ratio) and SP:SAMPLEPREP_SUMMARY centrifuged at 15,000 g for 20 minutes at 4οC. The supernatants will be divided SP:SAMPLEPREP_SUMMARY into 2 aliquots and dried down under a stream of nitrogen for 90 minutes and SP:SAMPLEPREP_SUMMARY stored at -20C until ready for analysis. These samples will undergo preparation SP:SAMPLEPREP_SUMMARY for metabolite separation and analysis on a Time-of-Flight (TOF) Mass SP:SAMPLEPREP_SUMMARY Spectrometer (Agilent Technologies 6220 TOF) coupled with an Ultra High Pressure SP:SAMPLEPREP_SUMMARY Liquid Chromatograph (Waters Acquity). The dried metabolites will be SP:SAMPLEPREP_SUMMARY re‐suspended in H2O/acetonitrile solution containing injection standards. SP:SAMPLEPREP_SUMMARY Profiling data will be acquired under both positive and negative electrospray SP:SAMPLEPREP_SUMMARY ionization modes separately over a scan range of 50 - 1200 m/z at a resolution SP:SAMPLEPREP_SUMMARY of 10,000. Metabolite separation will be achieved using two columns of differing SP:SAMPLEPREP_SUMMARY polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x SP:SAMPLEPREP_SUMMARY 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica SP:SAMPLEPREP_SUMMARY 2.1 x 150 mm, 1.8 mm; Waters). For positive ion detection, mobile phase A will SP:SAMPLEPREP_SUMMARY contain 100% water with 0.1% formic acid and mobile phase B will contain 100% SP:SAMPLEPREP_SUMMARY methanol with 0.1% formic acid. For negative ion detection, formic acid will be SP:SAMPLEPREP_SUMMARY replaced with 0.1% ammonium bicarbonate. For each column, the run time will be SP:SAMPLEPREP_SUMMARY 20 min using a flow rate of 400 microL/min. A total of four runs per sample will SP:SAMPLEPREP_SUMMARY be performed to give maximum coverage of metabolites. Samples will be injected SP:SAMPLEPREP_SUMMARY in triplicate with blank injections between samples. Quality control samples, SP:SAMPLEPREP_SUMMARY made up of a subset of samples from the study will be injected several times SP:SAMPLEPREP_SUMMARY during a run. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_RESULTS_FILE ST000789_AN001257_Results.txt UNITS:intensity #END