Abstract 1380: Detecting cancer mutations at the resolution of individual DNA molecules for longitudinal monitoring
Authors: Wood Christina M. , Lau Billy , Miotke Laura , Ji Hanlee P. , Greer Stephanie 
Published: July 2016

Abstract
We have developed a new molecular assay that utilizes digital PCR to detect and quantify cancer mutations within poor quality and limited quantity samples such as archival tissue DNA and circulating tumor DNA (ctDNA). This digital PCR assay is highly sensitive and is capable of detecting the targeted mutant fraction at an individual DNA molecule resolution. Thus, this assay is ideal for applications where DNA is in low abundance. An example is where DNA is shed from tumors that can be extracted from the plasma fraction of routine blood draws. The resulting circulating DNA (ctDNA) is extremely low in concentration, which makes it a prime candidate for our highly sensitive molecular assay.
Our assay incorporates small amplicon PCR primers and can be configured for nearly any coding mutation; practically, this means that any cancer or DNA sample can be tested efficiently. Mutation quantitation relies on two DNA primer sets that are identical with the exception of the mutant or wild type specific base at the 3’ end of the “detecting” primer sets, to amplify the genomic region of interest. Through the addition of artificial 5’ non-complementary tails to our mutant and wild type specific “detection” primers, we are able to consistently differentiate between droplets that contain the mutant or wild type alleles based upon their differential amplicon lengths. The synthetic amplicon extension tails minimize bias within the PCR reaction by allowing our primers to target an identical region of genomic DNA, with the exception of the single nucleotide variant specific base at the 3’ end of the detecting primer. The dPCR technology allows the standard PCR reaction to be partitioned into 20,000 independent wells; we can then assay each individual droplet to assess whether the individual DNA molecule partitioned into the droplet is “wild-type” or the target “mutant”. The resulting data provides an absolute count of mutant and wild-type templates in a given patient sample.
We have optimized this new molecular assay technology for quantitatively measuring clinically actionable mutations including BRAF V600E, KRAS G12D among others. We have successfully validated the sensitivity, specificity and reproducibility of our assays through controlled cell line DNA mixed dilution samples ranging from 66% mutant to 0.1% mutant in each of our optimized primer sets. Our assay improves the limit of detection to seven DNA molecules containing a mutation among a total number of 7,000 genome equivalents. Because our assay is sensitive down to the single DNA molecule resolution, we have also been able to reduce the amount of clinical sample DNA required to determine the presence of clinically actionable mutations such as those occurring in KRAS or BRAF. We are testing this extremely low cost, highly sensitive diagnostic technology for detecting nearly any cancer mutation from longitudinal samples of ctDNA.
Citation Format: Christina M. Wood, Billy Lau, Laura Miotke, Hanlee P. Ji, Stephanie Greer. Detecting cancer mutations at the resolution of individual DNA molecules for longitudinal monitoring. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1380.