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 emerged, 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 circulating DNA, urine, or heterogeneous cancers. We intend to use COLD-PCR, a recently developed method that enriches unknown mutation-containing sequences over wild-type, normal alleles during PCR amplification. In previous work we showed COLD-PCR- NGS-based sequencing for mutations down to 0.02% abundance. However, COLD-PCR was only applicable with a single amplicon per reaction, limiting its efficient combination with NGS. This STTR proposes a simple and powerful modification that enables COLD-PCR to be applied to hundreds or thousands of DNA targets in a single reaction, thus enabling mutation enrichment in disease- specific gene panels prior to NGS. The new approach, temperature-tolerant-COLD-PCR (TT-COLD-PCR) converts the rare mutations to high abundance mutations, overcoming the `noise' and avoiding the costly need for repeated sequence reads during NGS. In Phase I we obtained proof of principle for TT-COLD-PCR. In Phase II, 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 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.
Screening of patients' tumors for genetic alterations over many genes in a minimally- invasive, rapid and cost-effective manner is a significant challenge that must be fulfilled in order to realize the promise of individualized cancer treatment. Although major advances have been made, there is still a significant gap in technology that prevents molecular profiling from repeated blood collections (`liquid biopsies'). This STTR project provides an answer to this challenge by employing a new technology, TT-COLD-PCR in combination with Next Generation Sequencing. This novel approach overcomes technical limitations and enables reliable mutation screening in multiple genes simultaneously, in bodily fluids or surgical cancer samples from individual patients. In view of the fundamental role of mutations in causing cancer and modulating tumor response to drug treatment, this project has significant implications for public health.