Validation of the SHOX2 / PTGER4 DNA Methylation Marker Panel for Plasma-Based Discrimination between Patients with Malignant and Nonmalignant Lung Disease

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.

The interplay of circulating tumor DNA and chromatin modification, therapeutic resistance, and metastasis

“…Measurements of gene methylation in plasma DNA enable the early detection of primary cancer and metastases to other organs [82]. Plasma DNA SHOX2 and PTGER4 methylation [83] can differentiate lung cancer, nonmalignant lung diseases and the healthy state.…”

“…In addition to single gene methylation analysis, the detection of methylated gene panel could also be a promising marker for cancer diagnosis, prognosis and disease monitoring. Studies concerning the methylated gene panel have been reported in metastatic breast cancer [92], lung cancer [83], ovarian cancer [93] and primary non-small cell lung cancer [94].…”

DNA methylation biomarkers discovered in silico detect cancer in liquid biopsies from non-small cell lung cancer patients

“…These include markers or marker sets to detect e.g. lung [30][31][32][33][34][35], breast [12,13], colorectal [36,37] or pancreatic [38] cancers. A few DNA methylation cancer biomarkers are already in clinical use, e.g.…”

Whole blood FPR1 mRNA expression predicts both non‐small cell and small cell lung cancer

“…Other groups have explored the utility of blood and nonblood‐based biomarkers for lung cancer screening. A representative list include using gene expression of peripheral blood mononuclear cells, serum proteins, microRNAs, immunoassay, methylation, exhaled volatile organic compounds and urine protein biomarkers . Unlike other blood‐based lung cancer detection methods, the FPR1 ratio has been demonstrated to detect both NSCLC and SCLC.…”