Abstract: Provided are new and improved methods for detecting circulating tumor cells and tumor cell DNA in patient blood or other biofluid samples. Particular aspects comprise three steps: DNA extraction from patient samples, DNA digestion with multiple selected methylation-sensitive enzymes, and target amplification by a conventional or a real-time PCR with specific probe and/or primers. Also provided are a total of 40 tumor-specific DNA methylation loci as biomarkers having substantial utility and specificity in major types of human malignancies including hematopoietic and solid tumors.

Abstract: The present invention provides a detecting method of detecting malignant cells in a patient's specimen or a biological sample. Specifically, the inventive method includes the steps of extracting a genomic DNA, digesting said genomic DNA with one or multiple methylation sensitive restriction enzymes, and amplifying by PCR with one or multiple selected primers. The PCR can be performed in a conventional or a real-time platform. The inventive method can detect leukemia cells in 90% ALL patients at a sensitivity of up to 10?6. The inventive method also provides broad clinical applications in cancer (including hematopoietic and solid tumors) screening and risk assessment, early detection and diagnosis confirmation, and therapeutic monitoring, minimal residual disease detection and prognostic prediction.