With the advent of second generation sequencing (SGS), for the first time there is a truly viable possibility of sequencing a substantial portion of an individual cancer patient's genome within a short time period and at relatively low cost, thus detecting mutations that can have prognostic or predictive value, or can serve as a fingerprint for tumor follow-up in a particular patient. However, there is still a missing link in providing a truly reliable identification of mutation fingerprints in patient tumor samples, as clinically-relevant mutations in tumors with heterogeneity, stromal contamination or in bodily fluids is problematic due to admixture with wild type alleles and can still be missed. And yet, the clinical significance of identifying these low-level mutation fingerprints is major in several situations as frequently these are the mutations that confer resistance, offer prognostic and predictive information and that would be useful for treatment follow-up. Unfortunately the new sequencing technologies 'lose steam'when it comes to detecting low-level mutations, and for SGS currently it's either deep sequencing or high-throughput capability, but not both. Thus integration of SGS with clinical practice cannot be effectively exploited. We developed Co-amplification at Lower Denaturation temperature (COLD-PCR), a new form of PCR that amplifies preferentially the minority alleles from mixtures of wild type and mutation-containing sequences, irrespective of where the mutation lies, providing a strong enrichment of the mutated sequences during PCR. We propose to establish massively-parallel COLD-PCR to enrich mutant sequences prior to their screening via SGA (Illumina), thus enabling 'deep'sequencing while also retaining high-throughput capability. To enable massively-parallel COLD-PCR, a micro-fluidic device that dispenses DNA and PCR reagents within individual nano-droplets (RainDance"""""""") prior to PCR will be adapted to perform COLD-PCR in millions of separate nano- reactions simultaneously. The novel combination of technologies will be used to identify mutational fingerprints in tumors from 20 colon cancer patients, including low-level mutations, and then follow these fingerprints in plasma in the course of radio-chemo-therapy, to provide a molecular surrogate to therapy response. The proposed use of Novel Transformative Emerging Technologies is also applicable to other types of cancer and provides a solution bridging the gap in technology and enabling SGS to be applied to clinical oncology practice. Therefore relevance to Public Health is high.

Public Health Relevance

Screening of individual patients'tumors for genetic alterations over many genes in a 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 clinical integration of the most powerful platforms for molecular profiling and follow-up of individual patients treatment. We propose an answer to this challenge by combining 3 cutting edge technologies, COLD-PCR, RainDance nano-droplets and Next Generation Sequencing. The novel combination of these technologies bridges the existing technology gap and enables reliable mutation screening in multiple genes simultaneously, in surgical cancer samples or bodily fluids. 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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZCA1-SRLB-V (M1))
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Thurin, Magdalena
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Dana-Farber Cancer Institute
United States
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Castellanos-Rizaldos, Elena; Milbury, Coren A; Guha, Minakshi et al. (2014) COLD-PCR enriches low-level variant DNA sequences and increases the sensitivity of genetic testing. Methods Mol Biol 1102:623-39
Murphy, D M; Bejar, R; Stevenson, K et al. (2013) NRAS mutations with low allele burden have independent prognostic significance for patients with lower risk myelodysplastic syndromes. Leukemia 27:2077-81
Milbury, Coren A; Correll, Mick; Quackenbush, John et al. (2012) COLD-PCR enrichment of rare cancer mutations prior to targeted amplicon resequencing. Clin Chem 58:580-9
Castellanos-Rizaldos, E; Liu, Pingfang; Milbury, Coren A et al. (2012) Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment. Clin Chem 58:1130-8
Castellanos-Rizaldos, Elena; Milbury, Coren Audrey; Makrigiorgos, G Mike (2012) Enrichment of mutations in multiple DNA sequences using COLD-PCR in emulsion. PLoS One 7:e51362