DNA-based assay for EPHB6 expression in breast carcinoma cells as a potential diagnostic test for detecting tumor cells in circulation.

The early detection of breast cancer is critical for improved treatment and better management of the disease. The dissemination of tumor cells into the blood stream is known to occur early in tumor progression and these circulating tumor cells (CTCs) may be detectable before the occurrence of tumor metastasis. Methylation-specific polymerase chain reaction (MSP) can be exploited for detecting CTCs on the basis of differential methylation of numerous gene promoters in normal and carcinoma cells. In this study, we describe the relationship between loss of Ephrin receptor B6 (EPHB6) expression and the aggressiveness of breast carcinoma cell lines (BCCLs). The loss of EPHB6 expression in more aggressive BCCLs is regulated in a methylation-dependent manner. We demonstrate the ability of an EPHB6 MSP to distinguish between methylated and unmethylated EPHB6 promoters, and to predict expression of the EPHB6 transcript and protein. The sensitivity of MSP was related to the volume of blood processed for DNA isolation. As few as 50 tumor cells in 5 ml blood were detectable with a high efficiency. However, the detection of 10 tumor cells/5 ml was not as efficient. On the other hand, 5 tumor cells or 100 pg of free DNA in 200 microl of blood was also easily detectable. Our results suggest that MSP could be applied to detect even a single cell in 1 ml of blood by employing appropriate modifications. The EPHB6 MSP has clinical implications for the prognosis and/or diagnosis of breast and other cancer types including neuroblastoma, melanoma, and non-small cell lung carcinoma wherein EPHB6 expression is lost in more aggressive forms of the disease.