Impact of Kidney Function on the Blood Proteome and on Protein Cardiovascular Risk Biomarkers in Patients With Stable Coronary Heart Disease.

Background Chronic kidney disease (CKD) confers increased cardiovascular risk, not fully explained by traditional factors. Proteins regulate biological processes and inform the risk of diseases. Thus, in 938 patients with stable coronary heart disease from the Heart and Soul cohort, we quantified 1054 plasma proteins using modified aptamers (SOMAscan) to: (1) discern how reduced glomerular filtration influences the circulating proteome, (2) learn of the importance of kidney function to the prognostic information contained in recently identified protein cardiovascular risk biomarkers, and (3) identify novel and even unique cardiovascular risk biomarkers among individuals with CKD. Methods and Results Plasma protein levels were correlated to estimated glomerular filtration rate (eGFR) using Spearman-rank correlation coefficients. Cox proportional hazard models were used to estimate the association between individual protein levels and the risk of the cardiovascular outcome (first among myocardial infarction, stroke, heart failure hospitalization, or mortality). Seven hundred and nine (67.3%) plasma proteins correlated with eGFR at P<0.05 (ρ 0.06-0.74); 218 (20.7%) proteins correlated with eGFR moderately or strongly (ρ 0.2-0.74). Among the previously identified 196 protein cardiovascular biomarkers, just 87 remained prognostic after correction for eGFR. Among patients with CKD (eGFR <60 mL/min per 1.73 m2), we identified 21 protein cardiovascular risk biomarkers of which 8 are unique to CKD. Conclusions CKD broadly alters the composition of the circulating proteome. We describe protein biomarkers capable of predicting cardiovascular risk independently of glomerular filtration, and those that are prognostic of cardiovascular risk specifically in patients with CKD and even unique to patients with CKD.

Modified nucleotides may have enhanced early RNA catalysis.

The modern version of the RNA World Hypothesis begins with activated ribonucleotides condensing (nonenzymatically) to make RNA molecules, some of which possess (perhaps slight) catalytic activity. We propose that noncanonical ribonucleotides, which would have been inevitable under prebiotic conditions, might decrease the RNA length required to have useful catalytic function by allowing short RNAs to possess a more versatile collection of folded motifs. We argue that modified versions of the standard bases, some with features that resemble cofactors, could have facilitated that first moment in which early RNA molecules with catalytic capability began their evolutionary path toward self-replication.

Stress Hyperglycemia in Critically Ill Patients: Insight Into Possible Molecular Pathways.

Severe sepsis, systemic inflammatory response syndrome (SIRS), and traumatic brain injury are frequently associated with hyperglycemia in non-diabetic patients. In patients suffering from any of these conditions, hyperglycemia at admission to an intensive care unit (ICU) is directly correlated with increased mortality or morbidity. Although there was initial enthusiasm for insulin treatment to blood glucose levels below 110 mg/dL in these patients, recent understanding suggests that the potential for hypoglycemic complications make this approach potentially dangerous. More moderate glucose control seems to be more beneficial than the aggressive glucose lowering initially suggested. An important publication has shown that hyperlactatemia accompanying hyperglycemia could be the real culprit in bad outcomes. This suggests that coupling moderate glucose lowering with therapeutic agents which might treat the underlying metabolic disturbances in these conditions may be a better strategy. The key metabolic disturbance in these three conditions seems to be persistent glycolysis as an energy source even in the presence of adequate tissue oxygenation (the Warburg Effect). We look at recent advances in understanding aerobic glycolysis and possibly the action of DPP4 on incretins resulting in insulin dysregulation and suggest key metabolic pathways involved in hyperglycemia regulation.

On the Mechanisms of Action of the Low Molecular Weight Fraction of Commercial Human Serum Albumin in Osteoarthritis.

The low molecular weight fraction of commercial human serum albumin (LMWF5A) has been shown to successfully relieve pain and inflammation in severe osteoarthritis of the knee (OAK). LMWF5A contains at least three active components that could account for these antiinflammatory and analgesic effects. We summarize in vitro experiments in bone marrow-derived mesenchymal stem cells, monocytic cell lines, chondrocytes, peripheral blood mononuclear cells, fibroblast-like synoviocytes, and endothelial cells on the biochemistry of anti-inflammatory changes induced by LMWF5A. We then look at four of the major pathways that cut across cell-type considerations to examine which biochemical reactions are affected by mTOR, COX-2, CD36, and AhR pathways. All three components show anti-inflammatory activities in at least some of the cell types. The in vitro experiments show that the effects of LMWF5A in chondrocytes and bone marrow- derived stem cells in particular, coupled with recent data from previous clinical trials of single and multiple injections of LMWF5A into OAK patients demonstrated improvements in pain, function, and Patient Global Assessment (PGA), as well as high responder rates that could be attributed to the multiple mechanism of action (MOA) pathways are summarized here. In vitro and in vivo data are highly suggestive of LMWF5A being a disease-modifying drug for OAK.

Overcoming the Warburg Effect: Is it the key to survival in sepsis?

Sepsis is a leading cause of mortality in the U.S. and Europe. Sepsis and septic shock are the results of severe metabolic abnormalities following infection. Aerobic glycolysis (the Warburg Effect) is as much a hallmark of sepsis as it is of cancer. Warburg observed that cancer cells generated energy through glycolysis (generation of ATP through degradation of glucose, usually associated with anaerobic conditions) rather than through oxidative phosphorylation (generation of ATP through the mitochondrial inner membrane via the tricarboxylic acid cycle, usually associated with aerobic conditions). Although the initial pathways of cancer and sepsis may be different, the mechanisms which allow aerobic glycolysis to occur, even in the presence of oxygen, are similar. This review provides some evidence that reversing these steps reverses the Warburg Effect in model systems and some pathological consequences of this effect. Therefore, this implies that these steps might be modifiable in sepsis to reverse the Warburg Effect and possibly lead to better outcomes.

Use of Saline as a Placebo in Intra-articular Injections in Osteoarthritis: Potential Contributions to Nociceptive Pain Relief.

BACKGROUND: Osteoarthritis of the knee (OAK) is a severe debilitating condition characterized by joint pain, stiffness, and resultant limited mobility. In recent years, intra-articular (IA) injections have been used to relieve symptoms and have succeeded to varying degrees either with sodium hyaluronate preparations or with a biologic. OBJECTIVE: The objective of this review is to evaluate multiple studies that demonstrate some relief from the symptoms of OAK in the saline arm of various clinical trials. METHOD: A thorough literature search (PubMed) was performed assessing the pain efficacy of various compounds compared to saline injections in clinical trials. A total of 73 studies were identified in the literature search including a total of 5,816 patients. These clinical trials all involved the IA injection of a viscosupplement (hyaluronate, platelet rich plasma (PRP), etc.) or a biologic (the low molecular weight fraction (< 5kDa) of human serum albumin (LMWF-5A)). For all of these studies, the control arm was injection of sterile physiological saline that approximates the salt concentration and total solute concentration of blood and most tissues. RESULTS: Based on our review of the current literature, the tested compounds performed with mixed results when compared to saline injections. Moreover, OAK is a variable disease, with severity measured on the Kellgren and Lawrence (KL) scale where various hyaluronate preparations have a therapeutic effect mostly on KL 2-3 patients while a biologic works best on KL 3-4 patients. CONCLUSION: Since the effect of saline injection is always greater than no treatment, the evaluations of these treatments can be confounded in clinical trials. Therefore, the question of whether there are known therapeutic effects of saline injections might explain these results.

The low molecular weight fraction of commercial human serum albumin induces acetylation of α-tubulin and reduces transcytosis in retinal endothelial cells.

It has long been appreciated that the microtubule network plays a critical role in endothelial cell function. Chemical inhibition of tubulin polymerization has been shown to drastically increases endothelial permeability via interactions with the actin cytoskeleton. Conversely, stabilization of microtubules significantly decreases vascular permeability. The purpose of this investigation was to determine if the low molecular weight fraction of commercial 5% human serum albumin (LMWF5A) alters endothelial cell cytoskeletal dynamics and function. To investigate this, human retinal endothelial cells (HREC) were treated with LMWF5A and the acetylation of α-tubulin was determined by immunofluorescent staining and immunoblotting. In addition, permeability assays were performed to evaluate functional changes. We found that HREC treated with LMWF5A exhibit a rapid increase in the amount and distribution of acetylated α-tubulin. This was accompanied by a reduction in macromolecular permeability. Calcium depletion and inhibition of PI3-kinase reduced LMWF5A-induced acetylation while p38 MAPK inhibition potentiated this effect. These findings suggest that LMWF5A mediates changes in the microtubule network and reduces transcytosis in HREC.

The low molecular weight fraction of human serum albumin upregulates COX2, prostaglandin E2, and prostaglandin D2 under inflammatory conditions in osteoarthritic knee synovial fibroblasts.

BACKGROUND: The ability to decrease inflammation and promote healing is important in the intervention and management of a variety of disease states, including osteoarthritis of the knee (OAK). Even though cyclooxygenase 2 (COX2) has an established pro-inflammatory role, evidence suggests it is also critical to the resolution that occurs after the initial activation phase of the immune response. In this study, we investigated the effects of the low molecular weight fraction of 5% human serum albumin (LMWF-5A), an agent that has proven to decrease pain and improve function in OAK patients after intra-articular injection, on the expression of COX2 and its downstream products, prostaglandins (PGs). METHODS: Fibroblast-like synoviocytes from the synovial membrane of OAK patients were treated with LMWF-5A or saline as a control with or without the addition of interleukin-1ß (IL-1ß) or tumor necrosis factor α (TNFα) to elicit an inflammatory response. Cells were harvested for RNA and protein at 2, 4, 8, 12, and 24 h, and media was collected at 24 h for analysis of secreted products. COX2 mRNA expression was determined by qPCR, and COX2 protein expression was determined by western blot analysis. Levels of prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2) in the media were quantified by competitive ELISA. RESULTS: In the presence of either IL-1ß or TNFα, LMWF-5A increased the expression of both COX2 mRNA and protein, and this increase was significant compared to that observed with IL-1ß- or TNFα-stimulated, saline-treated cells. Downstream of COX2, the levels of PGE2 were increased only in TNFα-stimulated, LMWF-5A-treated cells; however, in both IL-1ß- and TNFα-stimulated cells, LMWF-5A increased the release of the anti-inflammatory prostaglandin PGD2. CONCLUSION: LMWF-5A appears to trigger increased anti-inflammatory PG signaling, and this may be a primary component of its therapeutic mode of action in the treatment of OAK.

Association of growth differentiation factor 11/8, putative anti-ageing factor, with cardiovascular outcomes and overall mortality in humans: analysis of the Heart and Soul and HUNT3 cohorts.

AIMS: Growth differentiation factor 11 and/or its homologue growth differentiation factor 8 (GDF11/8) reverses age-related cardiac hypertrophy and vascular ageing in mice. We investigated whether GDF11/8 associates with cardiovascular outcomes, left ventricular hypertrophy (LVH), or age in humans. METHODS AND RESULTS: We measured plasma GDF11/8 levels in 928 participants with stable ischaemic heart disease in the Heart and Soul study. We adjudicated heart failure hospitalization, stroke, myocardial infarction, death, and their composite endpoint. Left ventricular hypertrophy was evaluated by echocardiography. We used multivariable Cox proportional hazards models to compare rates of cardiovascular events and death across GDF11/8 quartiles and logistic regression models to evaluate the association between GDF11/8 and LVH. Four hundred and fifty participants (48.5%) experienced a cardiovascular event or death during 8.9 years of follow-up. The adjusted risk of the composite endpoint was lower in the highest compared with the lowest GDF11/8 quartile [hazard ratio (HR), 0.45; 95% confidence interval (CI), 0.33-0.60; P < 0.001]. We replicated this relationship of GDF11/8 to adverse events in 971 participants in the HUNT3 cohort (adjusted HR, 0.34; 95% CI, 0.23-0.51; P < 0.001). Left ventricular hypertrophy was present in 368 participants (39.7%) at baseline. Participants in the highest quartile of GDF11/8 were less likely to have LVH than those in the lowest quartile (adjusted OR, 0.55; 95% CI, 0.35-0.86; P = 0.009). GDF11/8 levels were lower in older individuals (P < 0.001). CONCLUSION: In patients with stable ischaemic heart disease, higher GDF11/8 levels are associated with lower risk of cardiovascular events and death. Our findings suggest that GDF11/8 has similar cardioprotective properties in humans to those demonstrated in mice.

Low Molecular Weight Fraction of Commercial Human Serum Albumin Induces Morphologic and Transcriptional Changes of Bone Marrow-Derived Mesenchymal Stem Cells.

Osteoarthritis (OA) is the most common chronic disease of the joint; however, the therapeutic options for severe OA are limited. The low molecular weight fraction of commercial 5% human serum albumin (LMWF5A) has been shown to have anti-inflammatory properties that are mediated, in part, by a diketopiperazine that is present in the albumin preparation and that was demonstrated to be safe and effective in reducing pain and improving function when administered intra-articularly in a phase III clinical trial. In the present study, bone marrow-derived mesenchymal stem cells (BMMSCs) exposed to LMWF5A exhibited an elongated phenotype with diffuse intracellular F-actin, pronounced migratory leading edges, and filopodia-like projections. In addition, LMWF5A promoted chondrogenic condensation in "micromass" culture, concurrent with the upregulation of collagen 2α1 mRNA. Furthermore, the transcription of the CXCR4-CXCL12 axis was significantly regulated in a manner conducive to migration and homing. Several transcription factors involved in stem cell differentiation were also found to bind oligonucleotide response element probes following exposure to LMWF5A. Finally, a rapid increase in PRAS40 phosphorylation was observed following treatment, potentially resulting in the activation mTORC1. Proteomic analysis of synovial fluid taken from a preliminary set of patients indicated that at 12 weeks following administration of LMWF5A, a microenvironment exists in the knee conducive to stem cell infiltration, self-renewal, and differentiation, in addition to indications of remodeling with a reduction in inflammation. Taken together, these findings imply that LMWF5A treatment may prime stem cells for both mobilization and chondrogenic differentiation, potentially explaining some of the beneficial effects achieved in clinical trials.

Large-scale serum protein biomarker discovery in Duchenne muscular dystrophy.

Serum biomarkers in Duchenne muscular dystrophy (DMD) may provide deeper insights into disease pathogenesis, suggest new therapeutic approaches, serve as acute read-outs of drug effects, and be useful as surrogate outcome measures to predict later clinical benefit. In this study a large-scale biomarker discovery was performed on serum samples from patients with DMD and age-matched healthy volunteers using a modified aptamer-based proteomics technology. Levels of 1,125 proteins were quantified in serum samples from two independent DMD cohorts: cohort 1 (The Parent Project Muscular Dystrophy-Cincinnati Children's Hospital Medical Center), 42 patients with DMD and 28 age-matched normal volunteers; and cohort 2 (The Cooperative International Neuromuscular Research Group, Duchenne Natural History Study), 51 patients with DMD and 17 age-matched normal volunteers. Forty-four proteins showed significant differences that were consistent in both cohorts when comparing DMD patients and healthy volunteers at a 1% false-discovery rate, a large number of significant protein changes for such a small study. These biomarkers can be classified by known cellular processes and by age-dependent changes in protein concentration. Our findings demonstrate both the utility of this unbiased biomarker discovery approach and suggest potential new diagnostic and therapeutic avenues for ameliorating the burden of DMD and, we hope, other rare and devastating diseases.

Inflammatory Pathways in Knee Osteoarthritis: Potential Targets for Treatment.

Osteoarthritis (OA) of the knee is a wide-spread, debilitating disease that is prominent in Western countries. It is associated with old age, obesity, and mechanical stress on the knee joint. By examining the recent literature on the effect of the anti-inflammatory prostaglandins 15d-PGJ2 and Δ12-PGJ2, we propose that new therapeutic agents for this disease could facilitate the transition from the COX-2-dependent pro-inflammatory synthesis of the prostaglandin PGE2 (catalyzed by mPGES-1), to the equally COX-2-dependent synthesis of the aforementioned anti-inflammatory prostaglandins. This transition could be instrumental in halting the breakdown of cartilage via matrix metalloproteinases (MMPs) and aggrecanases, as well as promoting the matrix regeneration and synthesis of cartilage by chondrocytes. Another desirable property of new OA therapeutics could involve the recruitment of mesenchymal stem cells to the damaged cartilage and bone, possibly resulting in the generation of chondrocytes, synoviocytes, and, in the case of bone, osteoblasts. Moreover, we propose that research promoting this transition from pro-inflammatory to anti-inflammatory prostaglandins could aid in the identification of new OA therapeutics.

Sepsis, oxidative stress, and hypoxia: Are there clues to better treatment?

Sepsis is a clinical syndrome characterized by systemic inflammation, usually in response to infection. The signs and symptoms are very similar to Systemic Inflammatory Response Syndrome (SIRS), which typically occur consequent to trauma and auto-immune diseases. Common treatments of sepsis include administration of antibiotics and oxygen. Oxygen is administered due to ischemia in tissues, which results in the production of free radicals. Poor utilization of oxygen by the mitochondrial electron transport chain can increase oxidative stress during ischemia and exacerbate the severity and outcome in septic patients. This course of treatment virtually mimics the conditions seen in ischemia-reperfusion disorders. Therefore, this review proposes that the mechanism of free radical production seen in sepsis and SIRS is identical to the oxidative stress seen in ischemia-reperfusion injury. Specifically, this is due to a biochemical mechanism within the mitochondria where the oxidation of succinate to fumarate by succinate dehydrogenase (complex II) is reversed in sepsis (hypoxia), leading to succinate accumulation. Oxygen administration (equivalent to reperfusion) rapidly oxidizes the accumulated succinate, leading to the generation of large amounts of superoxide radical and other free radical species. Organ damage possibly leading to multi-organ failure could result from this oxidative burst seen in sepsis and SIRS. Accordingly, we postulate that temporal administration with anti-oxidants targeting the mitochondria and/or succinate dehydrogenase inhibitors could be beneficial in sepsis and SIRS patients.

Oxidative stress in severe acute illness.

The overall redox potential of a cell is primarily determined by oxidizable/reducible chemical pairs, including glutathione-glutathione disulfide, reduced thioredoxin-oxidized thioredoxin, and NAD(+)-NADH (and NADP-NADPH). Current methods for evaluating oxidative stress rely on detecting levels of individual byproducts of oxidative damage or by determining the total levels or activity of individual antioxidant enzymes. Oxidation-reduction potential (ORP), on the other hand, is an integrated, comprehensive measure of the balance between total (known and unknown) pro-oxidant and antioxidant components in a biological system. Much emphasis has been placed on the role of oxidative stress in chronic diseases, such as Alzheimer's disease and atherosclerosis. The role of oxidative stress in acute diseases often seen in the emergency room and intensive care unit is considerable. New tools for the rapid, inexpensive measurement of both redox potential and total redox capacity should aid in introducing a new body of literature on the role of oxidative stress in acute illness and how to screen and monitor for potentially beneficial pharmacologic agents.

Validation of a blood protein signature for non-small cell lung cancer.

BACKGROUND: CT screening for lung cancer is effective in reducing mortality, but there are areas of concern, including a positive predictive value of 4% and development of interval cancers. A blood test that could manage these limitations would be useful, but development of such tests has been impaired by variations in blood collection that may lead to poor reproducibility across populations. RESULTS: Blood-based proteomic profiles were generated with SOMAscan technology, which measured 1033 proteins. First, preanalytic variability was evaluated with Sample Mapping Vectors (SMV), which are panels of proteins that detect confounders in protein levels related to sample collection. A subset of well collected serum samples not influenced by preanalytic variability was selected for discovery of lung cancer biomarkers. The impact of sample collection variation on these candidate markers was tested in the subset of samples with higher SMV scores so that the most robust markers could be used to create disease classifiers. The discovery sample set (n = 363) was from a multi-center study of 94 non-small cell lung cancer (NSCLC) cases and 269 long-term smokers and benign pulmonary nodule controls. The analysis resulted in a 7-marker panel with an AUC of 0.85 for all cases (68% adenocarcinoma, 32% squamous) and an AUC of 0.93 for squamous cell carcinoma in particular. This panel was validated by making blinded predictions in two independent cohorts (n = 138 in the first validation and n = 135 in the second). The model was recalibrated for a panel format prior to unblinding the second cohort. The AUCs overall were 0.81 and 0.77, and for squamous cell tumors alone were 0.89 and 0.87. The estimated negative predictive value for a 15% disease prevalence was 93% overall and 99% for squamous lung tumors. The proteins in the classifier function in destruction of the extracellular matrix, metabolic homeostasis and inflammation. CONCLUSIONS: Selecting biomarkers resistant to sample processing variation led to robust lung cancer biomarkers that performed consistently in independent validations. They form a sensitive signature for detection of lung cancer, especially squamous cell histology. This non-invasive test could be used to improve the positive predictive value of CT screening, with the potential to avoid invasive evaluation of nonmalignant pulmonary nodules.

Early detection of malignant pleural mesothelioma in asbestos-exposed individuals with a noninvasive proteomics-based surveillance tool.

BACKGROUND: Malignant pleural mesothelioma (MM) is an aggressive, asbestos-related pulmonary cancer that is increasing in incidence. Because diagnosis is difficult and the disease is relatively rare, most patients present at a clinically advanced stage where possibility of cure is minimal. To improve surveillance and detection of MM in the high-risk population, we completed a series of clinical studies to develop a noninvasive test for early detection. METHODOLOGY/PRINCIPAL FINDINGS: We conducted multi-center case-control studies in serum from 117 MM cases and 142 asbestos-exposed control individuals. Biomarker discovery, verification, and validation were performed using SOMAmer proteomic technology, which simultaneously measures over 1000 proteins in unfractionated biologic samples. Using univariate and multivariate approaches we discovered 64 candidate protein biomarkers and derived a 13-marker random forest classifier with an AUC of 0.99±0.01 in training, 0.98±0.04 in independent blinded verification and 0.95±0.04 in blinded validation studies. Sensitivity and specificity at our pre-specified decision threshold were 97%/92% in training and 90%/95% in blinded verification. This classifier accuracy was maintained in a second blinded validation set with a sensitivity/specificity of 90%/89% and combined accuracy of 92%. Sensitivity correlated with pathologic stage; 77% of Stage I, 93% of Stage II, 96% of Stage III and 96% of Stage IV cases were detected. An alternative decision threshold in the validation study yielding 98% specificity would still detect 60% of MM cases. In a paired sample set the classifier AUC of 0.99 and 91%/94% sensitivity/specificity was superior to that of mesothelin with an AUC of 0.82 and 66%/88% sensitivity/specificity. The candidate biomarker panel consists of both inflammatory and proliferative proteins, processes strongly associated with asbestos-induced malignancy. SIGNIFICANCE: The SOMAmer biomarker panel discovered and validated in these studies provides a solid foundation for surveillance and diagnosis of MM in those at highest risk for this disease.

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.

Protein signature of lung cancer tissues.

Lung cancer remains the most common cause of cancer-related mortality. We applied a highly multiplexed proteomic technology (SOMAscan) to compare protein expression signatures of non small-cell lung cancer (NSCLC) tissues with healthy adjacent and distant tissues from surgical resections. In this first report of SOMAscan applied to tissues, we highlight 36 proteins that exhibit the largest expression differences between matched tumor and non-tumor tissues. The concentrations of twenty proteins increased and sixteen decreased in tumor tissue, thirteen of which are novel for NSCLC. NSCLC tissue biomarkers identified here overlap with a core set identified in a large serum-based NSCLC study with SOMAscan. We show that large-scale comparative analysis of protein expression can be used to develop novel histochemical probes. As expected, relative differences in protein expression are greater in tissues than in serum. The combined results from tissue and serum present the most extensive view to date of the complex changes in NSCLC protein expression and provide important implications for diagnosis and treatment.

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.