Cancers develop from the life-long accumulation of critical somatic mutations due to DNA-damaging agents that lead to cells transforming into tumor-forming cells. These low-level tumor-associated somatic DNA mutations can have profound implications for development of metastasis, prognosis, choice of treatment, follow-up or early cancer detection. Unless they are effectively detected, these low-level mutations can misinform patient management decisions or become missed opportunities for personalized medicine. Widely-used technologies such as sequencing are not sensitive enough to detect these mutations when they are at very low percentages compared to normal DNA. Likewise the next generation sequencing technologies (NGS) are promising technology advances that can effectively detect prevalent somatic mutations in targeted gene panels;however due to the limited quantity of DNA in most patient samples and the abundance of normal DNA when analyzing blood, NGS 'loses steam'and its integration with clinical practice is problematic. For mutations at an abundance of ~2-5% or below, NGS generates false positives ('noise') independent of sequencing depth;yet these are often the clinically relevant mutations causing resistance to drug treatments. Commercial sample preparation kits for targeted re-sequencing of cancer gene panels have emerged1-3, however they are uniformly unable to detect mutations below a 2% abundance level. Thus, while targeted re-sequencing provides an opportunity for integration of NGS with clinical oncology, the technology is ineffective in detecting DNA mutations in heterogeneous cancers or in circulating DNA. We intend to use COLD-PCR, a new method that enriches unknown mutation-containing sequences over wild-type, normal alleles during PCR amplification. We have been able to show sequencing of mutations down to 0.02% abundance. However in its current form this method only can be used with single amplicon per reaction, limiting its efficient combination with NGS. In this project we propose a simple and powerful modification that enables COLD-PCR to be applied on hundreds or thousands of DNA targets in a single reaction, thus enabling mutation enrichment in cancer- specific gene panels prior to NGS. This would convert the rare mutations to high abundance mutations, overcoming the 'noise'and avoid the costly need for repeated sequence reads during NGS. This method, known as temperature-tolerant-COLD-PCR (TT-COLD-PCR), will be developed into kits for cancer-specific gene panels, to magnify rare mutations in multiple DNA targets thus enabling expanded application of targeted re-sequencing for heterogeneous cancers or circulating DNA. This project meets one of the stated aims of the NCI to support the development of new methods of diagnosis for the detection, discovery and validation of biomarkers for cancer detection, diagnosis and prognosis.

Public Health Relevance

The selection of the best available cancer treatment is, in many instances, dependent on the genetic profile of the patient's cancer cells. This can be difficult to determine when there is limited availability of tumor DNA, as when analyzing blood, when obscured by far more abundant healthy cell DNA and when many DNA targets need to be analyzed. This project aims to develop a multiplexed DNA analysis technique that enriches low abundance cancer mutations in clinical specimens such as blood to improve cancer treatment selection.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Small Business Technology Transfer (STTR) Grants - Phase I (R41)
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Special Emphasis Panel (ZRG1-IMST-K (14))
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Lou, Xing-Jian
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Transgenomic, Inc.
United States
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Castellanos-Rizaldos, Elena; Richardson, Katherine; Lin, Rui et al. (2015) Single-tube, highly parallel mutation enrichment in cancer gene panels by use of temperature-tolerant COLD-PCR. Clin Chem 61:267-77