Evaluation of two high-throughput proteomic technologies for plasma biomarker discovery in immunotherapy-treated melanoma patients

Abstract

BackgroundSelective kinase and immune checkpoint inhibitors, and their combinations, have significantly improved the survival of patients with advanced metastatic melanoma. Not all patients will respond to treatment however, and some patients will present with significant toxicities. Hence, the identification of biomarkers is critical for the selection and management of patients receiving treatment. Biomarker discovery often involves proteomic techniques that simultaneously profile multiple proteins but few studies have compared these platforms.MethodsIn this study, we used the multiplex bead-based Eve Technologies Discovery assay and the aptamer-based SomaLogic SOMAscan assay to identify circulating proteins predictive of response to immunotherapy in melanoma patients treated with combination immune checkpoint inhibitors. Expression of four plasma proteins were further validated using the bead-based Millipore Milliplex assay.ResultsBoth the Discovery and the SOMAscan assays detected circulating plasma proteins in immunotherapy-treated melanoma patients. However, these widely used assays showed limited correlation in relative protein quantification, due to differences in specificity and the dynamic range of protein detection. Protein data derived from the Discovery and Milliplex bead-based assays were highly correlated.ConclusionsOur study highlights significant limitations imposed by inconsistent sensitivity and specificity due to differences in the detection antibodies or aptamers of these widespread biomarker discovery approaches. Our findings emphasize the need to improve these technologies for the accurate identification of biomarkers.Electronic supplementary materialThe online version of this article (10.1186/s40364-017-0112-9) contains supplementary material, which is available to authorized users.

Circulating Cytokines Predict Immune-Related Toxicity in Melanoma Patients Receiving Anti-PD-1–Based Immunotherapy

“…The low levels of cytokine expression in plasma meant that RFU signals were often below the standard protein curves used in the detection assays and this prevented absolute cytokine concentration calculations. However, we have previously shown that RFU are accurate indicators of relative cytokine levels (23), and RFU analysis provided more statistical power for testing differences in cytokine expression levels (24). Expression of the 65 cytokines in the cohorts was also measured across different assay plates and batches, which may have contributed to some discrepancies in RFU due to variations in assay lot (i.e., changes in assay reagents and antibodies) and machine calibration.…”

“…The 65-plex discovery assay utilized 150 mL of undiluted plasma per run and each run was performed in duplicate; duplicates did not vary by more than 4% (data not shown). Fluorescence intensity values were derived from the discovery assay and are in direct proportion to reflect the amount of proteins in the samples (23). Fluorescence values were…”

Finding a Needle in a Haystack

“…Although aptamer and PEA platforms are commercially available, both are expensive and not without their shortcomings. For example, commercially available aptamers have limited dynamic range and ability to detect proteins generated from single nucleotide polymorphisms ( 33 , 34 ). Current PEA assays have inconsistent relative error in measurement that depends on the analyte, rather than protein abundance ( 35 ).…”

Liquid biomarkers in melanoma: detection and discovery

“…Method testing and validation of these multiplex immunoassays are critical but few studies have examined performance of these assays in detail [ 41 ]. Indeed, we recently compared a bead-based and an aptamer-based affinity assay and found poor correlation in relative plasma protein quantification between the two assays [ 45 ]. This highlights the discrepancies introduced when using different assays, which will limit the comparison and validation of potential biomarkers in independent studies.…”