Dom Zichi

Unlocking Biomarker Discovery: Large Scale Application of Aptamer Proteomic Technology for Early Detection of Lung Cancer

Background Lung cancer is the leading cause of cancer deaths worldwide. New diagnostics are needed to detect early stage lung cancer because it may be cured with surgery. However, most cases are diagnosed too late for curative surgery. Here we present a comprehensive clinical biomarker study of lung cancer and the first large-scale clinical application of a new aptamer-based proteomic technology to discover blood protein biomarkers in disease. Methodology/Principal Findings We conducted a multi-center case-control study in archived serum samples from 1,326 subjects from four independent studies of non-small cell lung cancer (NSCLC) in long-term tobacco-exposed populations. Sera were collected and processed under uniform protocols. Case sera were collected from 291 patients within 8 weeks of the first biopsy-proven lung cancer and prior to tumor removal by surgery. Control sera were collected from 1,035 asymptomatic study participants with ≥10 pack-years of cigarette smoking. We measured 813 proteins in each sample with a new aptamer-based proteomic technology, identified 44 candidate biomarkers, and developed a 12-protein panel (cadherin-1, CD30 ligand, endostatin, HSP90α, LRIG3, MIP-4, pleiotrophin, PRKCI, RGM-C, SCF-sR, sL-selectin, and YES) that discriminates NSCLC from controls with 91% sensitivity and 84% specificity in cross-validated training and 89% sensitivity and 83% specificity in a separate verification set, with similar performance for early and late stage NSCLC. Conclusions/Significance This study is a significant advance in clinical proteomics in an area of high unmet clinical need. Our analysis exceeds the breadth and dynamic range of proteome interrogated of previously published clinical studies of broad serum proteome profiling platforms including mass spectrometry, antibody arrays, and autoantibody arrays. The sensitivity and specificity of our 12-biomarker panel improves upon published protein and gene expression panels. Separate verification of classifier performance provides evidence against over-fitting and is encouraging for the next development phase, independent validation. This careful study provides a solid foundation to develop tests sorely needed to identify early stage lung cancer.

From SOMAmer-Based Biomarker Discovery to Diagnostic and Clinical Applications: A SOMAmer-Based, Streamlined Multiplex Proteomic Assay

Recently, we reported a SOMAmer-based, highly multiplexed assay for the purpose of biomarker identification. To enable seamless transition from highly multiplexed biomarker discovery assays to a format suitable and convenient for diagnostic and life-science applications, we developed a streamlined, plate-based version of the assay. The plate-based version of the assay is robust, sensitive (sub-picomolar), rapid, can be highly multiplexed (upwards of 60 analytes), and fully automated. We demonstrate that quantification by microarray-based hybridization, Luminex bead-based methods, and qPCR are each compatible with our platform, further expanding the breadth of proteomic applications for a wide user community.

The stability of the circulating human proteome to variations in sample collection and handling procedures measured with an aptamer-based proteomics array

Blood-based protein biomarkers hold great promise to advance medicine with applications that detect and diagnose diseases and aid in their treatment. We are developing such applications with our proteomics technology that combines high-content with low limits of detection. Biomarker discovery relies heavily on archived blood sample collections. Blood is dynamic and changes with different sampling procedures potentially confounding biomarker studies. In order to better understand the effects of sampling procedures on the circulating proteome, we studied three sample collection variables commonly encountered in archived sample sets. These variables included (1) three different sample tube types, PPT plasma, SST serum, and Red Top serum, (2) the time from venipuncture to centrifugation, and (3) the time from centrifugation to freezing. We profiled 498 proteins for each of 240 samples and compared the results by ANOVA. The results found no significant variation in the measurements for most proteins (approximately 99%) when the two sample processing times tested were 2h or less, regardless of sample tube type. Even at the longest timepoints, 20 h, approximately 82% of the proteins, on average for the three collection tube types, showed no significant change. These results are encouraging for proteomic biomarker discovery.

Aptamers and the RNA World, Past and Present

Aptamers and the SELEX process were discovered over two decades ago. These discoveries have spawned a productive academic and commercial industry. The collective results provide insights into biology, past and present, through an in vitro evolutionary exploration of the nature of nucleic acids and their potential roles in ancient life. Aptamers have helped usher in an RNA renaissance. Here we explore some of the evolution of the aptamer field and the insights it has provided for conceptualizing an RNA world, from its nascence to our current endeavor employing aptamers in human proteomics to discover biomarkers of health and disease.

High-content affinity-based proteomics: unlocking protein biomarker discovery

Single protein biomarkers measured with antibody-based affinity assays are the basis of molecular diagnostics in clinical practice today. There is great hope in discovering new protein biomarkers and combinations of protein biomarkers for advancing medicine through monitoring health, diagnosing disease, guiding treatment, and developing new therapeutics. The goal of high-content proteomics is to unlock protein biomarker discovery by measuring many (thousands) or all (∼23,000) proteins in the human proteome in an unbiased, data-driven approach. High-content proteomics has proven technically difficult due to the diversity of proteins, the complexity of relevant biological samples, such as blood and tissue, and large concentration ranges (in the order of 1012 in blood). Mass spectrometry and affinity methods based on antibodies have dominated approaches to high-content proteomics. For technical reasons, neither has achieved adequate simultaneous performance and high-content. Here we review antibody-based protein measurement, multiplexed antibody-based protein measurement, and limitations of antibodies for high-content proteomics due to their inherent cross-reactivity. Finally, we review a new affinity-based proteomic technology developed from the ground up to solve the problem of high content with high sensitivity and specificity. Based on a new generation of slow off-rate modified aptamers (SOMAmers), this technology is unlocking biomarker discovery.

Life's simple measures: unlocking the proteome.

Using modified nucleotides and selecting for slow off-rates in the SELEX procedure, we have evolved a special class of aptamers, called SOMAmers (slow off-rate modified aptamers), which bind tightly and specifically to proteins in body fluids. We use these in a novel assay that yields 1:1 complexes of the SOMAmers with their cognate proteins in body fluids. Measuring the SOMAmer concentrations of the resultant complexes reflects the concentration of the proteins in the fluids. This is simply done by hybridization to complementary sequences on solid supports, but it can also be done by any other DNA quantification technology (including NexGen sequencing). We use measurements of over 1000 proteins in under 100 μL of serum or plasma to answer important medical questions, two of which are reviewed here. A number of bioinformatics methods have guided our discoveries, including principal component analysis. We use various methods to evaluate sample handling procedures in our clinical samples and can identify many parameters that corrupt proteomics analysis.

A streamlined, aptamer-based multiplex proteomic assay for diagnostic applications

To enable the application of protein signatures identified in our highly multiplexed biomarker discovery platform to real-world diagnostics, we developed a streamlined, plate-based assay that employs the same principles as the larger SOMAScan assay. The plate-based assay is sensitive (sub-picomolar), robust, rapid, automatable, and can multiplex upwards of 60 analytes. We have used this platform to seamlessly translate sets of biomarkers identified in our SOMAScan assay to a practical non-small cell lung cancer diagnostic.

Highly multiplexed SOMAmer assays as a flexible platform for protein biomarker discovery research

SomaLogic presents a transformative proteomic biomarker discovery technology that measures ~1000 human proteins in low volumes (~15 of uL biological sample) with a high-performance, high-throughput, and economical assay. Limits of detection average 1 pM and the overall dynamic range spans 7 logs with ~5% average coefficient of variation. This technology is enabled by a new class of DNA aptamers — “SOMAmers” — that contain novel chemicallymodified nucleotides, which greatly expand the physicochemical diversity of the large combinatorial SELEX libraries from which they are selected. Proteins are measured with a process that transforms a signature of protein concentrations into a representative DNA concentration signature, which is quantified with a DNA microarray. We demonstrate the utility of this technology in a large clinical biomarker discovery study of non-small cell lung cancer, as well as in alternate biological matrices.