Cell-free DNA methylation analysis as a marker of malignancy in pleural fluid

Abstract

Diagnosis of malignant pleural effusion (MPE) is made by cytological examination of pleural fluid or histological examination of pleural tissue from biopsy. Unfortunately, detection of malignancy using cytology has an overall sensitivity of 50%, and is dependent upon tumor load, volume of fluid assessed, and cytopathologist experience. The diagnostic yield of pleural fluid cytology is also compromised by low abundance of tumor cells or when morphology is obscured by inflammation or reactive mesothelial cells. A reliable molecular marker that may complement fluid cytology for the diagnosis of malignant pleural effusion is needed. The purpose of this study was to establish a molecular diagnostic approach based on pleural effusion cell-free DNA methylation analysis for the differential diagnosis of malignant pleural effusion and benign pleural effusion. This was a blind, prospective case-control biomarker study. We recruited 104 patients with pleural effusion for the study. We collected pleural fluid from patients with: MPE (n = 48), indeterminate pleural effusion in subjects with known malignancy or IPE (n = 28), and benign PE (n = 28), and performed the Sentinel-MPE liquid biopsy assay. The methylation level of Sentinel-MPE was markedly higher in the MPE samples compared to BPE control samples (p < 0.0001) and the same tendency was observed relative to IPE (p = 0.004). We also noted that the methylation signal was significantly higher in IPE relative to BPE (p < 0.001). We also assessed the diagnostic efficiency of the Sentinel-MPE test by performing receiver operating characteristic analysis (ROC). For the ROC analysis we combined the malignant and indeterminate pleural effusion groups (n = 76) and compared against the benign group (n = 28). The detection sensitivity and specificity of the Sentinel-MPE test was high (AUC = 0.912). The Sentinel-MPE appears to have better performance characteristics than cytology analysis. However, combining Sentinel-MPE with cytology analysis could be an even more effective approach for the diagnosis of MPE. The Sentinel-MPE test can discriminate between BPE and MPE. The Sentinel-MPE liquid biopsy test can detect aberrant DNA in several different tumor types. The Sentinel-MPE test can be a complementary tool to cytology in the diagnosis of MPE.

Figure 1

Study design for “all comers” pleural effusion for Sentinel-MPE liquid biopsy test. This was a prospective case control study with three different cohorts of pleural effusion disease benign pleural effusion subjects, paramalignant pleural effusion cases, and malignant pleural effusion individuals.

Figure 2

Performance of Sentinel-MPE liquid biopsy test in detecting malignancy in pleural effusion. (A) Waterfall plot displaying the methylation signal from the Sentinel-MPE liquid biopsy test for all 104 participants in the study. (B) Box plots displaying the mean methylation signal of benign pleural effusion (BPE), indeterminate pleural effusion (IPE), and malignant pleural effusion cohorts (MPE). (C) Receiver operating curve analysis of combined (MPE) and indeterminate effusion (IPE) groups compared to (BPE) group. The methylation signal levels were markedly higher in MPE and IPE groups relative to BPE group. (D) Receiver operating curve analysis of MPE versus BPE. There was an increase in the performance of the Sentinel-MPE liquid biopsy test. (E) Receiver operating curve analysis of combined MPE and IPE breast and lung cancer subjects compared to BPE subjects. Note the there was a further improvement in the performance of the Sentinel-MPE test for these two common cancer types.