Cancers often exhibit aberrant methylation of gene promoter regions associated with loss of tumor suppressor and/or DNA repair gene function. Such methylation constitutes an excellent marker for the molecular detection of micro-metastases and the diagnosis of tumor recurrences. We have developed a multiplex methylation-specific PCR (MSP) procedure for rapid and simultaneous assessment of the methylation of 5 loci: the tumor suppressor genes p16INK4a, death-associated protein kinase (DAPK) and p14ARF, and the DNA repair genes hMLH1 and O6-methylguanine-DNA-methyltransferase (MGMT). This multiplex test uses one single PCR reaction and only one electrophoretic run. In 98 samples of colorectal cancer studied, methylation of MGMT, DAPK, p16, hMLH1 and p14 was present in 31, 20, 17, 16 and 14% of tumors, respectively. In 58% of the tumors at least one methylated gene was found. This multiplex MSP constitutes a simple and inexpensive method for screening of molecular signatures in colorectal cancer and can be used profitably before employing more expensive and complex techniques such as microarray testing.

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