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Background  Cytology remains the gold standard for the detection of malignant cells in ascites. However, its sensitivity is limited. The aim of this study was to evaluate DNA methylation biomarkers for the differential diagnosis of benign (ascites in patients without malignancy), malignant (ascites in cancer patients directly caused by malignancy), and paramalignant (ascites in cancer patients caused by comorbidities but not by malignancy) ascites.    Methods  A cohort of 283 patients (134 cancer patients, 149 patients with benign diseases) presenting with ascites was prospectively enrolled. Ascites was evaluated by means of cytopathological investigation and DNA methylation of  SHOX2  and  SEPT9  in the cell-free and cellular fraction. DNA methylation in bisulfite-converted DNA was determined using quantitative methylation specific real-time PCR. Cytopathological and DNA methylation results were evaluated with regard to diagnosis and overall survival (OS).    Results  Patients with positive DNA methylation had a poor overall survival compared to methylation-negative patients (hazard ratio: HR = 1.97,  p  = 0.001). In multivariate survival analysis, DNA methylation was an independent prognostic parameter ( p  = 0.003) together with age (HR = 1.03,  p  < 0.001) and the presence of malignant disease (HR = 1.87,  p  < 0.001).  The combination of methylation with cytopathological analyses led to a 42 % increase in the detection rate of malignant ascites, resulting in 37 % positively diagnosed cancer patients and a specificity of 97 %. Among cancer patients, patients with DNA methylation-positive ascites showed an adverse clinical course (HR = 1.63,  p  = 0.039).    Conclusions  DNA methylation testing adds diagnostic and prognostic information and might constitute an effective ancillary method for the differential diagnosis of malignant, paramalignant, and benign ascites.    Electronic supplementary material  The online version of this article (doi:10.1186/s13148-016-0192-7) contains supplementary material, which is available to authorized users.

Diagnostic and prognostic value of SHOX2 and SEPT9 DNA methylation and cytology in benign, paramalignant, and malignant ascites