High RASSF1A promoter methylation levels are predictive of poor prognosis in fine-needle aspirate washings of breast cancer lesions

Previously, we reported that the accuracy of cytological diagnosis of breast lesions could be augmented through the quantitative assessment of DNA methylation of fine-needle aspirate (FNA) washings. Herein, we aimed at the evaluation of the prognostic value of quantitative promoter methylation at three gene loci (APC, CCND2, and RASSF1A) in a large series of FNA washings from breast lesions. Methylation levels of three gene promoters were assessed by quantitative methylation-specific PCR in bisulfite-modified DNA from 211 FNA washings, comprising 178 carcinomas and 33 benign lesions, both histopathologically confirmed. Receiver operator characteristic (ROC) curve analysis was used to determine the diagnostic performance of the gene panel in distinguishing cancer from non-cancerous lesions. Relevant clinicopathologic data and time to progression and/ or death from breast cancer were correlated with methylation findings. Log-rank test and Cox-regression model identified independent predictors of prognosis. APC, CCND2, and RASSF1A methylation levels differed significantly between malignant and benign lesions. ROC curve analysis confirmed the diagnostic performance of the gene panel. In univariate analysis, stage was significantly associated with overall, disease- specific and disease-free survival, whereas tumor grade was associated with disease-specific and disease-free survival. Remarkably, RASSF1A methylation was significantly and independently associated with worse disease-free survival in the final multivariate analysis. We confirmed that quantitative gene promoter methylation augments the diagnostic performance of cytopathology. Importantly, and in addition to standard clinicopathologic parameters, RASSF1A high-methylation levels are independent predictors of worse outcome in breast cancer. Thus, epigenetic biomarkers provide valuable tools for breast cancer patient management.