Sarcoma is general term for a group of over 70 cancers that begin in bones or connective/soft tissue. Sarcomas are primarily classified using genomic techniques to identify characteristic gene fusions in tumor tissues. However, this technique requires relatively invasive biopsies of tumor tissues and is not conducive to monitoring disease progression or response to treatment. To overcome these limitations, we sought to develop a method capable of quantitative measurements of relevant proteins in plasma, including the proteins coinciding to the aforementioned gene fusions.
First, discovery experiments for peptide selection were performed on EWSR1 and FLI1 recombinant proteins purchased from Origene and cells purchased from American Type Culture Collection derived from Ewing’s Sarcoma tissues and known to express fusion proteins. Recombinant proteins were diluted to a concentration of 10 pmol/mL in 0.1% BSA in water. Cell lines were lysed by sonication in 20 mM Tris-HCl pH 8, 100 mM NaCl, and 1% NP-40. Approximately 10 million cells were used for each discovery experiment. EWSR1, FLI1, and fusion proteins were isolated using antibody-based immunopurification. Anti-EWSR1 (NB200-182, Novus Biologicals) and anti-FLI1 (MA5-38312, ThermoFisher Scientific) were immobilized on magnetic beads (14204, ThermoFisher Scientific) and 2.5 μg of each antibody was added to each sample prior to a 2 hour incubation. After incubation, the beads were washed twice with PBS, twice with water, and eluted with 0.2% trifluoroacetic acid, 0.002% Z316. The eluates were neutralized with 1 M Tris-HCl pH 8, reduced, alklylated, and digested with chymotrypsin for 15 hours at 37°C. The resulting solution was then loaded onto EvoTips and LC separation was performed using the extended method on an EvoSep One. Data-dependent analysis was performed using an Exploris 480 (ThermoFisher Scientific) mass spectrometer. After these peptide selection experiments, target peptide precursor ion masses were used for subsequent LC-MS/MS analysis. This sample preparation technique and LC-MS/MS method was then used to analyze nine samples from patients diagnosed with Ewing’s Sarcoma and 40 clinical residual samples representing the normal population.
Discovery experiments yielded approximately 50% sequence coverage for EWSR1, FLI1, and the fusion proteins. Based on these experiments, 14 peptides spanning the EWSR1, FLI1, and common fusion proteins were selected for targeted LC-MS/MS measurements with the intention of being able to derive the concentration of the respective proteins from which the measured peptides were produced. Quality controls samples derived from Ewing’s Sarcoma cells produced %CVs of less than 25% for intra and interday replicate measurements. Measurements of clinical residual plasma samples were used to calculate a “reference range” for each peptide, which was applied to plasma samples from patients diagnosed with Ewing’s Sarcoma to identify signals that were above normal. The Ewing’s Sarcoma patients exhibited several peptide concentrations above the normal patient population confidence interval on average. Additionally, the concentrations showed a general downward trend with treatment. However, the small sample size (n=9) and the imprecision of replicate measurements make it impossible to make definitive conclusions on the clinical utility of this technique.
We present a novel method for measuring protein biomarkers of Ewing’s sarcomas. When utilized for measurement of plasma samples, the results were very intriguing. However, the method would require analytical and throughput improvements as well as additional clinical validation before clinical implementation can occur.