Elucidating Novel Serum Biomarkers Associated with Pulmonary Tuberculosis Treatment

et al. (2013) Elucidating Novel Serum Biomarkers Associated with Pulmonary Tuberculosis Treatment. PLoS ONE 8(4): e61002. doi:10.1371/journal.pone.0061002 Elucidating Novel Serum Biomarkers Associated with Pulmonary Tuberculosis Treatment Mary A. De Groote 0 Payam Nahid 0 Leah Jarlsberg 0 John L. Johnson 0 Marc Weiner 0 Grace Muzanyi 0 Nebojsa Janjic 0 David G. Sterling 0 Urs A. Ochsner 0 Robert B. Sim, Oxford University, United Kingdom 0 1 SomaLogic, Inc., Boulder, Colorado, United States of America, 2 Department of Microbiology, Immunology and Pathology, Colorado State University Campus, Fort Collins, Colorado, United States of America, 3 Pulmonary and Critical Care Medicine, University of California San Francisco , San Francisco , California, United States of America, 4 Tuberculosis Research Unit, Division of Infectious Diseases, Case Western Reserve University , Cleveland , Ohio, United States of America, 5 Division of Infectious Diseases, University of Texas Health Science Center , San Antonio, Texas , United States of America, 6 Uganda-Case Western Reserve University Research Collaboration , Kampala , Uganda In an unbiased approach to biomarker discovery, we applied a highly multiplexed proteomic technology (SOMAscan, SomaLogic, Inc, Boulder, CO) to understand changes in proteins from paired serum samples at enrollment and after 8 weeks of TB treatment from 39 patients with pulmonary TB from Kampala, Uganda enrolled in the Center for Disease Control and Prevention's Tuberculosis Trials Consortium (TBTC) Study 29. This work represents the first large-scale proteomic analysis employing modified DNA aptamers in a study of active tuberculosis (TB). We identified multiple proteins that exhibit significant expression differences during the intensive phase of TB therapy. There was enrichment for proteins in conserved networks of biological processes and function including antimicrobial defense, tissue healing and remodeling, acute phase response, pattern recognition, protease/anti-proteases, complement and coagulation cascade, apoptosis, immunity and inflammation pathways. Members of cytokine pathways such as interferon-gamma, while present, were not as highly represented as might have been predicted. The top proteins that changed between baseline and 8 weeks of therapy were TSP4, TIMP-2, SEPR, MRC-2, Antithrombin III, SAA, CRP, NPS-PLA2, LEAP-1, and LBP. The novel proteins elucidated in this work may provide new insights for understanding TB disease, its treatment and subsequent healing processes that occur in response to effective therapy. - Using an unbiased approach with a highly multiplexed comprehensive platform, our goal in this study was to identify and quantify protein markers that are present in patients diagnosed with culture-confirmed active TB and that change in response to highly efficacious drug therapy. In our previous work in lung cancer we performed a large scale application of our platform technology to identify markers capable of diagnosing early onset lung cancer [1]. In this study we used the same modified aptamer array to quantitate 1,030 proteins in serum of patients diagnosed with active TB disease and monitor changes in all markers to better understand the evolution of protein markers as the disease improves on anti-mycobacterial therapy. SOMAscan proteomic technology is based on slow offrate modified aptamers (SOMAmers), with improved binding properties due to long dissociation rates (generally .30 min) and the incorporation of modified nucleotides that lead to higher affinity of these reagents as compared to standard RNA or DNA aptamers. SOMAmers are made from single-stranded DNA (ssDNA) that contain pyrimidine residues modified at their 5prime position to introduce functional groups not present in natural nucleic acids, such as mimics of amino acid side-chains. SOMAmers have several advantages over antibodies, including lower molecular weight, higher multiplexing capabilities (low cross-reactivity, universally-applicable assay conditions), chemical stability (to heat, drying, and solvents, reversible renaturation), ease of reagent manufacturing, consistent lot-to-lot performance and lower cost (fully synthetic). The Version 2 SOMAscan assay generates simultaneous quantitative measurements of 1,030 human proteins in serum, plasma, CSF or tissue lysate [2] in a small (,100 ml) sample volume. Across all 1,030 proteins, the median lower limit of quantitation is 0.3 picomolar (pM), with a dynamic range of .5 logs, and a median coefficient of variation (%CV) of 5% [3]. This proof-of-principle study is the first large-scale unbiased targeted proteomic analysis employing modified DNA aptamers and we sought to identify and quantify protein markers that are associated with active TB and that changed in response to fourdrug treatment. This pilot study was made possible due to an archived collection of specimens nested into Tuberculosis Trials Consortium (TBTC) Study 29, a phase 2B clinical trial which evaluated rifapentine in place of rifampin in combination with isoniazid, ethambutol and pyrazinamide for the treatment of drugsusceptible TB [4]. Participant Characteristics Clinical characteristics of the 39 participants included in this study are listed in Table 1. Participants completed between 6 and 24 months of anti-TB treatment. Follow-up through the end of treatment did not reveal any treatment failures. One participant had INH and RIF resistant tuberculosis; one participant had mono-drug resistance to INH and one had dual resistance (to streptomycin and rifampin), all were detected after the participants completed intensive phase treatment. Four participants received between 35 days of 4 drug, standard chemotherapy prior to enrollment. Sample Handling and Analysis A small systematic difference (4%) in the overall protein concentrations between the baseline and 8 week sample sets was removed during normalization. Three samples had elevated hemoglobin levels and correspondingly low haptoglobin levels (data not shown) when compared both to other subjects and internal assay calibrators suggesting some degree of hemolysis. No other evidence of sample processing errors [3] was observed so all samples were considered fit for inclusion in the subsequent data analysis. Nonspecific Markers of Active TB Serum protein concentrations in TB patients at baseline were compared to those measured in the same patients after 8 weeks of therapy. The acute phase reactants C-reactive protein (CRP) and serum amyloid A protein (SAA) decreased from baseline to week 8 in all but one subject (Figure 1). Serum albumin increased between baseline and week 8 in all but one subject. Other known important acute phase reactants including haptoglobin, alpha-1 antitrypsin (AAT) and serum amyloid A protein declined from baseline to week 8 consistent with a reduction in the disease burden. Correlations with Severity of Disease Microbiological and radiographic parameter (...truncated)