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Lung cancer detection by methylation-preserving PCR

Institution: University of Southern California
Investigator(s): Ite Offringa, Ph.D.
Award Cycle: 2011 (Cycle 20) Grant #: 20XT-0021 Award: $324,325
Subject Area: Cancer
Award Type: Exploratory/Developmental Award

Initial Award Abstract
This project focuses on the TRDRP priority area of LUNG CANCER, which is caused for an important part by exposure to primary and secondary tobacco smoke. BACKGROUND: Early detection is the cornerstone of saving the lives of lung cancer patients; once the cancer has spread it is very difficult to eradicate. DNA molecules with cancer-specific changes are very powerful tools to detect cancer, since it has been demonstrated that cancer patients shed cancer DNA into the blood. DNA hypermethylation at promoter-associated CpG islands is a hallmark of cancer. It has been demonstrated by many labs that different types of cancer carry distinct DNA methylation changes. Thus, detection of such hypermethylated DNA in blood plasma would be an ideal non-invasive method to identify cancer patients. Current epigenomic strategies will make it easy to identify good candidate markers. However, the most difficult step is sensitive detection. Here we are tackling this most crucial problem. The polymerase chain reaction (PCR), normally used to exponentially amplify DNA so that it can be easily detected, cannot be directly applied to DNA methylation. The PCR polymerase does not "see" the methylation and does not copy it. Therefore, to detect DNA hypermethylation, a chemical step called bisulfite conversion must first be carried out. This step does not change methylated cytosine nucleotides (methyl-C), but turns unmethylated cytosines into uracil, effectively altering the DNA sequence so that the methylation information can now be maintained during the PCR amplification. However, bisulfite treatment can destroy 50-90% of the DNA. When DNA is abundant, for example when it is isolated from a large number of cells, this is not a problem. But in the case of cancer patient blood, very little methylated DNA may be shed from the tumor, particularly if the tumor is small in early stage cancer patients. OBJECTIVE/HYPOTHESIS: We propose to develop a method that will allow us to amplify the DNA before bisulfite treatment is carried out, in such a way that the methylation information is maintained. We call this method 'methylation-preserving PCR'. To do this, we will use different combinations of enzymes and genetic engineering to modify these enzymes. We hypothesize that under the right conditions, it is possible to duplicate DNA while retaining sufficient methylation information. In combination with methods previously developed at USC, we expect to be able to detect the lung cancer-specific DNA methylation signatures after methylation-preserving PCR. Our SPECIFIC AIMS are: 1) To develop the best combination of engineered enzymes to copy methylated DNA. 2) To extensively test this method on different DNA templates that carry different densities of CpG dinucleotides and different amounts of methylation. 3) To test this method using plasma DNA from lung cancer patients and non-cancer controls. METHODS: First we will generate a series of recombinant enzymes that will be used alone or in combination to copy a synthetic methylated DNA template. The efficacy will be measured by bisulfite genomic sequencing. We will then extensively characterize the properties of the copied methylated DNA with respect to accuracy, using a variety of different templates. Lastly, we will apply the method to DNA purified from lung cancer patient plasma and plasma from control non-cancer subjects. We have a plasma collection in house that has been assembled from lung cancer patients and control subjects in Los Angeles specifically for DNA methylation studies. The Laird-Offringa lab is particularly well positioned to carry out the described studies because we have extensive in house experience with genetic engineering, recombinant protein production, the measurement of nucleic acid protein interaction using surface plasmon resonance (Biacore), and a variety of state-of-the-art techniques to detect DNA methylation. We have collected a large number of plasma samples from lung cancer patients and non-cancer controls and work very closely with the new USC/Epigenome Center and its director, providing further expertise and support in the analysis of DNA methylation.