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

Abstract

Introduction: Low-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.

Methods: 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). 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.

Results: A 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%.

Conclusions: Measurement 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.

Keywords: Circulating tumor DNA; DNA methylation; Liquid biopsy; Lung cancer early detection; PTGER4; SHOX2.

Figure 1

Sample disposition, study setup, and polymerase chain reaction (PCR) assay formats. Boxes in the bottom line indicate number of valid results, number of PCR replicates, and bisDNA input volume (in parenthesis) per PCR assay. For more details, see Materials and Methods section. ACTB, actin, beta gene; FOXL2, forkhead box L2 gene; PTGER4, prostaglandin E receptor 4 gene; SHOX2, short stature homeobox 2 gene.

Figure 2

Receiver operating characteristic and area under the curve (AUC) analysis of pilot study 1 (AUC = 0.98) (A), study 2 (AUC = 0.91) (B), and study 3 (AUC = 0.95) (C).

Figure 3

Receiver operating characteristic and area under the curve (AUC) analysis of validation study: (A) Lung cancer (LC) versus all controls for training (AUC = 0.93) and validation study (AUC = 0.88), (B) LC versus nonmalignant disease (AUC = 0.86), (C) LC versus healthy controls (AUC = 0.91), (D) nonmalignant disease versus healthy controls (AUC = 0.58).

Figure 4

Comparison of protein (area under the curve [AUC] = 0.79) and methylation (AUC = 0.91) marker panel. The difference in the AUCs was statistically significant (p value = 0.004).