Non-invasive approaches for cell-free DNA (cfDNA) assessment provide an opportunity for cancer detection and intervention. Here, we use a machine learning model for detecting tumor-derived cfDNA through genome-wide analyses of cfDNA fragmentation in a prospective study of 365 individuals at risk for lung cancer. We validate the cancer detection model using an independent cohort of 385 non-cancer individuals and 46 lung cancer patients. Combining fragmentation features, clinical risk factors, and CEA levels, followed by CT imaging, detected 94% of patients with cancer across stages and subtypes, including 91% of stage I/II and 96% of stage III/IV, at 80% specificity. Genome-wide fragmentation profiles across ~13,000 ASCL1 transcription factor binding sites distinguished individuals with small cell lung cancer from those with non-small cell lung cancer with high accuracy (AUC = 0.98). A higher fragmentation score represented an independent prognostic indicator of survival. This approach provides a facile avenue for non-invasive detection of lung cancer.

Each year millions of pulmonary nodules are discovered by computed tomography and subsequently biopsied. As the majority of these nodules are benign, many patients undergo unnecessary and costly invasive procedures. We present a 13-protein blood-based classifier that differentiates malignant and benign nodules with high confidence, thereby providing a diagnostic tool to avoid invasive biopsy on benign nodules. Using a systems biology strategy, 371 protein candidates were identified and a multiple reaction monitoring (MRM) assay was developed for each. The MRM assays were applied in a three-site discovery study (n = 143) on plasma samples from patients with benign and Stage IA cancer matched on nodule size, age, gender and clinical site, producing a 13-protein classifier. The classifier was validated on an independent set of plasma samples (n = 104), exhibiting a high negative predictive value (NPV) of 90%. Validation performance on samples from a non-discovery clinical site showed NPV of 94%, indicating the general effectiveness of the classifier. A pathway analysis demonstrated that the classifier proteins are likely modulated by a few transcription regulators (NF2L2, AHR, MYC, FOS) that are associated with lung cancer, lung inflammation and oxidative stress networks. The classifier score was independent of patient nodule size, smoking history and age, which are risk factors used for clinical management of pulmonary nodules. Thus this molecular test can provide a powerful complementary tool for physicians in lung cancer diagnosis.

Background-Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine expressed widely by vascular cells. However, scant in vivo evidence supports direct participation of MIF in atherogenesis. Therefore, we investigated whether deficiency of MIF modulates atherosclerotic lesion formation and composition in low-density lipoprotein receptor-deficient (LDLr Ϫ/Ϫ ) mice. Methods and Results-MIFϪ/Ϫ LDLr Ϫ/Ϫ and LDLr Ϫ/Ϫ mice were generated and consumed an atherogenic diet for 12 or 26 weeks. MIF Ϫ/Ϫ LDLr Ϫ/Ϫ mice had significantly reduced abdominal aorta lipid deposition and intimal thickening from aortic arch throughout the abdominal aorta compared with LDLr Ϫ/Ϫ mice. Marked retardation of atherosclerosis over time in MIF-deficient mice accompanied decreased lesion cell proliferation. At 26 weeks, 20% of MIF-deficient mice developed only early, fatty streak-like lesions, whereas Ͼ80% of LDLr Ϫ/Ϫ mice developed advanced lesions containing calcification and lipid cores. Analysis of smooth muscle cells from mouse aortae demonstrated that MIF deficiency reduced smooth muscle cell proliferation, cysteine protease expression, and elastinolytic and collagenolytic activities. Conclusions-Deficiency of MIF reduces atherogenesis in LDLrϪ/Ϫ mice. These results provide novel insight into inflammatory pathways operating in atheromata and identify a new potential target for modulating atherogenesis.

Abstract-The pathogenesis of atherosclerosis and abdominal aortic aneurysm involves substantial proteolysis of the arterial extracellular matrix. The lysosomal cysteine proteases can exert potent elastolytic and collagenolytic activity. Human atherosclerotic plaques have increased cysteine protease content and decreased levels of the endogenous inhibitor cystatin C, suggesting an imbalance that would favor matrix degradation in the arterial wall. This study tested directly the hypothesis that impaired expression of cystatin C alters arterial structure. Cystatin C-deficient mice (Cyst C Ϫ/Ϫ ) were crossbred with apolipoprotein E-deficient mice (ApoE Ϫ/Ϫ ) to generate cystatin C and apolipoprotein E-double deficient mice (Cyst C Ϫ/Ϫ ApoE Ϫ/Ϫ ). After 12 weeks on an atherogenic diet, cystatin C deficiency yielded significantly increased tunica media elastic lamina fragmentation, decreased medial size, and increased smooth muscle cell and collagen content in aortic lesions of ApoE Ϫ/Ϫ mice. Cyst C Ϫ/Ϫ ApoE Ϫ/Ϫ mice also showed dilated thoracic and abdominal aortae compared with control ApoE Ϫ/Ϫ mice, although atheroma lesion size, intimal macrophage accumulation, and lipid core size did not differ between these mice. These findings demonstrate directly the importance of cysteine protease/protease inhibitor balance in dysregulated arterial integrity and remodeling during experimental atherogenesis. (Circ Res. 2005;96:368-375.) Key Words: cystatin C Ⅲ apolipoprotein E Ⅲ cysteine protease Ⅲ atherosclerosis Ⅲ smooth muscle cells A therogenesis involves substantial remodeling of the arterial extracellular matrix. 1 Considerable evidence links degradation of elastic laminae and of interstitial collagen with migration of leukocytes and smooth muscle cells (SMCs), neointima formation, atherosclerotic plaque rupture, and to arterial ectasia and aneurysm formation.We previously proposed a role for cysteine proteases, in particular cathepsins S and K, in atherosclerosis. Human atheromata display substantially increased expression of cathepsins S and K compared with normal vessels, and cathepsin S-positive SMCs colocalize with sites of elastic lamina fragmentation in the tunica media. 2 Human vascular SMCs and endothelial cells express cathepsin S-dependent elastolytic and collagenolytic activity in response to proinflammatory cytokines or growth factors in vitro. 2,3 Our recent finding of attenuated atherosclerosis in cathepsin S-deficient mice provided direct evidence for cysteine protease involvement in atherogenesis. 4 Mice deficient in cathepsin S had 50% less atherosclerosis than controls at both 8 and 12 weeks of atherogenic diet.Importantly, we found a reciprocal relationship between the levels of cathepsins tested and their most abundant endogenous inhibitor cystatin C in diseased human arterial tissue. Both atherosclerotic and aneurysmal human lesions show reduction of cystatin C compared with normal arteries, 5 indicating an imbalance in cysteine proteases and their inhibitor in human arterial disease. 2 Eriksson et ...