Investigating a novel multiplex proteomics technology for detection of changes in serum protein concentrations that may correlate to tumor burden

Abstract

Background: To account for cancer heterogeneity, we previously introduced the concept of “personalized” tumor markers, which are biomarkers that are informative in subsets of patients or even a single patient. Recent developments in various multiplex protein technologies create excitement for the discovery of markers of tumor burden in individual patients, but the reliability of the technologies remains to be tested for this purpose. Here, we sought to explore the potential of a novel proteomics platform, which utilizes a multiplexed antibody microarray, to detect changes in serum protein concentration that may correlate to tumor burden in pancreatic cancer. Methods: We applied the Quantibody® Human Kiloplex Array to simultaneously measure 1,000 proteins in sera obtained pre- and post-surgically from five pancreatic cancer patients. We expected that proteins which decreased post-surgery may correlate to tumor burden. Sera from two healthy individuals, split into two aliquots each, were used as controls. To validate the multiplexed results, we used single-target ELISA assays to measure the proteins with the largest serum concentration changes after surgery in sera collected pre- and post-surgically from the previous five patients and 10 additional patients. Results: The multiplexed array revealed nine proteins with more than two-fold post-surgical decrease in at least two of five patients. However, validation using single ELISAs showed that only two proteins tested displayed more than two-fold post-surgical decrease in one of the five original patients. In the independent cohort, six of the proteins tested showed at least a two-fold decrease post-surgery in at least one patient. Conclusions: Our study found that the Quantibody® Human Kiloplex Array results could not be reliably replicated with individual ELISA assays and most hits would likely represent false positives if applied to biomarker discovery. These findings suggest that data from novel, high-throughput proteomic platforms need stringent validation to avoid false discoveries.

Comparison of two multiplexed technologies for profiling >1,000 serum proteins that may associate with tumor burden

“…10 Our previous evaluation of QKA demonstrated lower precision and a weaker correlation between the technical duplicates (r = 0.853 to 0.862). 12 A closer look at intra-assay variation observed that 97.6% of the 1073 proteins analyzed by PEA showed a favorable coefficient of variation (CV) below 20%, 89.3% had a CV below 10%, and 0.3% showed a CV over 100% (data not shown). In contrast, 67.9% of the 1000 proteins assayed by QKA showed a CV below 20%, 47.6% had a CV below 10%, while 20.5% exhibited high variation with a CV over 100% (data not shown).…”

“…The workflow of the array was previously described. 12 In brief, each array included protein standards to generate a standard curve for absolute protein quantitation. The samples were analyzed in technical quadruplicates, and the technical personnel were blinded to the sample status.…”

“…67 However, we previously reported discrepancies between QKA and ELISA results for validating nine candidate proteins that may correlate to tumor burden in pancreatic cancer patients. 12 The analytical precision of multiplexed platforms may be highly analyte-dependent, and multiplexed data may benefit from independent validation to verify the protein quantitation.…”

Uncovering the Depths of the Human Proteome: Antibody-based Technologies for Ultrasensitive Multiplexed Protein Detection and Quantification

“…The micro-ELISA array technology from RayBiotech combines 25 nonoverlapping glass-slide arrays to perform up to 1000 miniature sandwich ELISAs simultaneously, targeting mostly inflammation-related proteins, including cytokines, chemokines, growth factors, tumor markers, and transcription factors ( 37 , 38 , 39 , 40 , 41 , 42 , 43 ). Each 75 mm × 25 mm glass slide contains 16 identical antibody subarrays, where each subarray multiplexes 40 different proteins arranged in technical quadruplicates.…”

“…However, Ren et al. ( 41 ) reported incongruities between the micro-ELISA array and ELISA data for nine protein candidates in pancreatic cancer. The overall findings illustrated that inconsistencies in protein quantification between different ultrasensitive multiplex immunoassays are highly analyte-dependent.…”