False Biomarker Discovery due to Reactivity of a Commercial ELISA for CUZD1 with Cancer Antigen CA125 I. Prassas, D. Brinc, S. Farkona, F. Leung, A. Dimitromanolakis, C.C. Chrystoja, R. Brand, V. Kulasingam, I.M. Blasutig, and E.P. Diamandis February © Copyright 2014 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry Introduction: The Biomarker Problem  Despite the recent advances in throughput of genomic and proteomic technologies, very few (<15) novel biomarkers have been introduced in the clinic the last 2 decades  Bringing a biomarker to the clinic is a multi-step process: discovery, verification, preclinical validation, large blinded clinical validation  Every step of this process is prone to errors that can put the whole pipeline at risk

© Copyright 2009 by the American Association for Clinical Chemistry Introduction: The problem with commercial ELISAs  The most common method for biomarker validation is the development of antibody-based assays (ELISAs)  In house ELISA development is costly, labor-intensive and expensive  Commercially available kits represent the easiest option  As our case illustrates, these reagents are sometimes sold without having been rigorously validated and this can result in substantial amounts of wasted time and investments.

© Copyright 2009 by the American Association for Clinical Chemistry Question  What are some common errors (analytical, statistical, methodological) that can happen during the different steps of a biomarker discovery and validation pipeline?

© Copyright 2009 by the American Association for Clinical Chemistry Introduction: Our case  Commercial ELISA kits were used to assess the performance of our top-ranked (recently discovered) candidate pancreatic cancer (PDAC) biomarkers  Of these, CUZD1 appeared as the most promising candidate biomarker for the diagnosis of pancreatic cancer  In our early pre-clinical verification studies of CUZD1 (measured by a commercial CUZD1 ELISA kit), this biomarker showed strong diagnostic performance  Before we moved to further clinical validation studies, we rigorously characterized the specificity of this commercial kit

© Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods  We performed detailed experiments to investigate the specificity of the commercial CUZD1 ELISA assay: 1.CUZD1 protein was cloned and expressed in house in both bacteria and yeast expression vector systems 2.Recombinant pure CUZD1 antigen and several biological samples containing CUZD1, as well as commercial CUZD1 ELISA standards were analyzed by:  Western Blot  Size exclusion HPLC  Mass-Spectrometry (LC-MS Orbitrap)

© Copyright 2009 by the American Association for Clinical Chemistry Results Figure 1. (A), Assessment of CUZD1 immunoreactivity (USCN ELISA) in eluted fractions of an SEC column revealed that peak signal was always observed in the void volume (molecular weight, 500 kD). The same elution profile was obtained irrespective of the sample type used, including CUZD1 calibrators from the ELISA kit, different pancreatic ascites samples, pooled ovarian cancer serum. (B), Western blots of several CUZD1 immunoreactive sources, as shown, confirm the high molecular weight (MW) (250 kDa) of the target antigen (no bands were detected in the expected MW of CUZD1 at approximately 70 kDa).

© Copyright 2009 by the American Association for Clinical Chemistry Results Figure 2. Comparison of MS-verified CUZD1 signal with commercial “CUZD1” immunoreactivity. We used mass spectrometry (LC-MS Orbitrap) to compare CUZD1 expression (MS-verified) with the immuno-reactivity of the “CUZD1” kit against several biological fluids. As shown in the figure, all CUZD1 containing samples were negative for the CUZD1 kit and all CUZD1 non-containing samples were immunoreacting with the commercial “CUZD1” ELISA kit.

© Copyright 2009 by the American Association for Clinical Chemistry Results Figure 3. Comparison of CUZD1 immunoreactivity with immunoreactivities of other known markers (CA125 and CA19-9). (A), CUZD1 immunoreactivity (USCN) and CA125 immunoreactivity (R&D Systems) were measured in 200 sera (100 normal and 100 PDAC samples). A strong correlation (Pearson r = 0.931) was seen between these 2 assays. (B), Correlation of CUZD1 immunoreactivity (USCN ELISA) with CA19-9 immunoreactivity (Siemens automated assay) in the same dataset revealed much lower correlations (r = 0.451).

© Copyright 2009 by the American Association for Clinical Chemistry Results Figure 4A. (A), Western blot (WB) with CUZD1 antibody (from CUZD1 ELISA kit) and a CA125 antibody (from R&D Systems) against pure (MS verified) CA125 and CUZD1 recombinant proteins. Both antibodies recognize CA125 at around 170 kDa but not CUZD1. MW, molecular weight standards.

© Copyright 2009 by the American Association for Clinical Chemistry Results Figure 4B. (B), Hybrid sandwich assays with shown capture/detection antibodies. Immunoreactivities were measured in several samples (as listed in the caption) with dilutions shown in brackets. In all cases, similar signals were obtained, except for the CA125/CUZD1 assay, for which immunoreactivities followed the same trend but signals were significantly lower. None of the assays displayed any immunoreactivity with the pure CUZD1 protein (column F), whereas all assays detected pure CA125 antigen (column E).

© Copyright 2009 by the American Association for Clinical Chemistry Results Figure 4C. (C), Comparison of immunoreactivity with the CUZD1 ELISA and a clinically used CA125 ELISA (Siemens). Samples used were commercial QC sera from Bio-Rad Laboratories.

© Copyright 2009 by the American Association for Clinical Chemistry Question #2:  Mass Spectrometry can provide very definitive answers regarding the presence (and the quantity) of a protein in a sample. If this is the case, why do ELISAs remain the most common method in serum biomarker validation studies?

© Copyright 2009 by the American Association for Clinical Chemistry Conclusions  Commercial CUZD1 assay recognizes a completely different antigen (CA125) than CUZD1  This phenomenon is not a cross-reactivity issue, since the “CUZD1” kit cannot recognize any form of CUZD1 protein  All observed “CUZD1 immunoreactivity” corresponds to CA125 immunoreactivity  Rigorous validation of commercial assays can prevent substantial amounts of wasted time and investments

© Copyright 2009 by the American Association for Clinical Chemistry Question #3:  FDA poses very strict regulations when a commercial assay is intended for clinical use. However, rules are not as strict in the case of reagents designated as “for research use only.” Do you think that stricter regulations also should be imposed for the latter category of reagents?

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