Abstract 1507: Highly sensitive and specific digital quantification of cancer genetic aberrations
Authors: Miotke Laura K. , Lau Billy , Rumma Rowza , Ji Hanlee 
Published: May 2015

Abstract
Genetic aberrations play a major role in tumor development and progression. As these clinically relevant genetic aberrations are discovered, it becomes of pressing urgency to efficiently, rapidly and sensitively detect these targets in individual patient tumor samples.
Droplet digital PCR (ddPCR) is a robust technology for quantitating genetic variation with high sensitivity from minimal amounts of nucleic acid. Compared to the highly variable quantitative real-time PCR (qRT-PCR), ddPCR eliminates relative standards and has the advantage of measuring mutant and wild-type targets within the same well. Commonly, this is achieved with the use of a two-color fluorescent oligonucleotide probe (TaqMan) design, where the mutant is represented by a FAM probe and the wild type by a VIC or HEX probe. However, this approach is cumbersome and requires a significant amount of optimization.
In this study we present an alternate quantification method for assessing CNVs and SNVs using a non-specific DNA binding dye. Instead of two colored probes, our design manipulates the length of the region of interest (ROI) and control amplicons to distinguish between their fluorescent signals. The dye binds in greater amount to the longer length target giving a higher fluorescent signal than the mutant target and both populations are easily quantifiable. This flexible and cost-effective method of independent DNA quantification proves to be a robust alternative to the commercialized TaqMan assay.
The ability to customize this assay for a variety of functions is a major advantage of using a single-color dye and thus we can create highly sensitive assays for any gene. Here, we demonstrated the effectiveness of this method by assessing copy number of the proto-oncogene FLT3, a target for the small molecule inhibitor Sunitinib. We established accurate quantitation of a FLT3 copy number change in a tumor sample harboring a 1.5-fold amplification diluted to 20% in a normal sample. Additionally we explored the common V600E point mutation in BRAF and an activating mutation in HER2, a potential therapeutic target for cancers lacking HER2 amplification. Our assay was able to detect a mutation comprising less than 1% of an otherwise wild-type sample and in the case of HER2, distinguish between wild-type and two different mutant alleles. Aside from single base pair mutations and copy number analysis we used this assay to detect single base pair insertions and deletions. Finally, due to the sensitivity of ddPCR, the applications of this technique include the targeted analysis of small biopsies and circulating nucleic acid. We are also determining the improvement in performance with the incorporation of locked nucleic acids (LNA) in increasing specificity and sensitivity in lower quality samples.
Citation Format: Laura K. Miotke, Billy Lau, Rowza Rumma, Hanlee Ji. Highly sensitive and specific digital quantification of cancer genetic aberrations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1507. doi:10.1158/1538-7445.AM2014-1507