PE-PCR, a novel method for detection of mutations in blood-cell-free DNA.

Abstract

e12016 Background: During the past few years, the “personalized” therapy based on specific tumor molecular alterations has become the standard of care of cancer patients . Recently, it has been shown that blood DNA is released from normal as well as tumor cells as a high molecular weight nucleic acid and carries the same genetic abnormalities as the cells of origin. However, the identification of these DNA alterations is difficult since the blood tumor DNA is found in very low concentration and it is mixed with a huge amount of normal homologous DNA. Therefore, most methods able to detect such abnormalities in cells are not applicable for blood cell free DNA. We report here a simple novel technology for mutation detection in blood DNA. Methods: The serum DNA is subject to two PCR rounds; in the first round, only DNA fragments containing the mutation are amplified by an extension primer encoding the mutation at its 3’ terminus. The DNA is then re-amplified by nested PCR for 40 cycles and the extension of mutated primer is detected by gel electrophoresis. The method was tested with DNA from the human lung cancer cell line A549 (ATCC # CCL-185) and was found to detect mutations in the presence of 10 12 [OP1] fold excess of normal DNA. Results: The sera from 10 patients with localized and metastatic lung (6) and pancreatic carcinomas (4) were tested for the presence of mutations in Kras and P53 genes. Seven patients had a mutation in a least one gene. Eleven patients without any clinical evidence of cancer tested negative. Conclusions: PE-PCR is a simple sensitive method for mutation detection in blood cell-free DNA, requiring only one ml blood and less than a 48 hour work. This method can potentially be used for mutation detection in most of the genes of clinical interest, as a non-invasive research tool, for early detection of tumor recurrence and possibly for early diagnosis of cancer when surgery can be curative.