A Dual-Marker DNA Methylation Assay Enables High-Sensitivity Detection of Malignant Effusions Across Cancer Types.

Objective: This study evaluated the diagnostic value of a combined SHOX2 and RASSF1A methylation assay (LungMe®) in differentiating malignant from benign pleural and peritoneal effusions. Methods: A total of 696 patients with pleural or peritoneal effusions were prospectively enrolled. Effusion sediment samples were analyzed by liquid-based cytology (TCT), cell block histology, and methylation-specific PCR for both SHOX2 and RASSF1A. The final diagnosis was confirmed by clinical, imaging, and pathological follow-up. Results: Lung cancer was the predominant cause of malignant pleural effusions (71.96%), while malignant peritoneal effusions mainly resulted from ovarian, gastric, and liver cancers. The optimal ΔCt cutoffs for SHOX2 and RASSF1A were ΔCt ≤ 9 and ΔCt ≤ 12, respectively. The combined methylation assay achieved a sensitivity of 79.1% and specificity of 90.4% for malignant effusions. When combined with cytology, the overall sensitivity increased from 34.7% (TCT alone) to 91.7%, with a positive predictive value (PPV) of 93.9% and a negative predictive value (NPV) of 86.7%. The assay showed high sensitivity across multiple tumor types, including 100% for esophageal and breast cancers, and ≥80% for lung cancer, gastric cancer, lymphoma, and cholangiocarcinoma. Conclusions: The combined SHOX2 and RASSF1A methylation assay has high diagnostic value for benign-malignant discrimination of pleural and peritoneal effusions, improving diagnostic sensitivity when combined with conventional cytology. Thus, within the context of this single-center study, the LungMe® assay demonstrates promise in expanding pan-cancer diagnostics, but will require further multicenter external validation and prospective clinical studies before routine clinical adoption.