Abstract
IntroductionLow-dose computed tomography (LDCT) is used for screening for lung cancer (LC) in high-risk patients in the United States. The definition of high risk and the impact of frequent false-positive results of low-dose computed tomography remains a challenge. DNA methylation biomarkers are valuable noninvasive diagnostic tools for cancer detection. This study reports on the evaluation of methylation markers in plasma DNA for LC detection and discrimination of malignant from nonmalignant lung disease.MethodsCirculating DNA was extracted from 3.5-mL plasma samples, treated with bisulfite using a commercially available kit, purified, and assayed by real-time polymerase chain reaction for assessment of DNA methylation of short stature homeobox 2 gene (SHOX2), prostaglandin E receptor 4 gene (PTGER4), and forkhead box L2 gene (FOXL2). In three independent case-control studies these assays were evaluated and optimized. The resultant assay, a triplex polymerase chain reaction combining SHOX2, PTGER4, and the reference gene actin, beta gene (ACTB), was validated using plasma from patients with and without malignant disease.ResultsA panel of SHOX2 and PTGER4 provided promising results in three independent case-control studies examining a total of 330 plasma specimens (area under the receiver operating characteristic curve = 91%–98%). A validation study with 172 patient samples demonstrated significant discriminatory performance in distinguishing patients with LC from subjects without malignancy (area under the curve = 0.88). At a fixed specificity of 90%, sensitivity for LC was 67%; at a fixed sensitivity of 90%, specificity was 73%.ConclusionsMeasurement of SHOX2 and PTGER4 methylation in plasma DNA allowed detection of LC and differentiation of nonmalignant diseases. Development of a diagnostic test based on this panel may provide clinical utility in combination with current imaging techniques to improve LC risk stratification.
Highlights
Low-dose computed tomography (LDCT) is used for screening for lung cancer (LC) in high-risk patients in the United States
Circulating DNA was extracted from 3.5-mL plasma samples, treated with bisulfite using a commercially available kit, purified, and assayed by real-time polymerase chain reaction for assessment of DNA methylation of short stature homeobox 2 gene (SHOX2), prostaglandin E receptor 4 gene (PTGER4), and forkhead box L2 gene (FOXL2)
A panel of SHOX2 and PTGER4 provided promising results in three independent case-control studies examining a total of 330 plasma specimens
Summary
Low-dose computed tomography (LDCT) is used for screening for lung cancer (LC) in high-risk patients in the United States. Journal of Thoracic Oncology Vol 12 No 1 from a substantial number of positive calls (27%), of which 96% have been determined to be false positives.[4] As a consequence, the NELSON trial used a substantially different definition of a positive screening result, which led to a 10-fold decrease in the positive rate (2.7%), reducing the proportion of false positives to 60% at the expense of some reduction in sensitivity for LC detection.[5,6] The need for better definition of the screeningeligible population led to risk assessment models developed from large trials.[7,8] algorithms for management of so-called intermediate nodules have been published.[9,10,11] Because of the imperfections of each of these methods, there is an ongoing quest for sensitive and reliable biomarkers with the potential to complement current cancer risk assessments.[12]
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