In:
Cancer Research, American Association for Cancer Research (AACR), Vol. 78, No. 13_Supplement ( 2018-07-01), p. 1604-1604
Abstract:
With the emergence of precision cancer medicine, there is an increasing need for new longitudinal diagnostic tests to evaluate patients at the initial diagnosis, during treatment, and for routine monitoring. As a general solution, we developed a single-color digital PCR assay technology that detects and quantifies circulating DNA cancer mutations collected from the plasma of cancer patients. The assay has a sensitivity of 0.1% mutation allelic fraction and can be designed for nearly any cancer mutation. In addition, this assay demonstrates design flexibility, low cost, and robust performance, making it a powerful approach for longitudinal monitoring of cancer patients during treatment. Positive controls are a critical component for highly sensitive detection of circulating DNA cancer mutations. However, there are minimal resources for providing standard curves. Commercially available cancer cell lines cover a very limited number of cancer mutations. Amplicons or oligonucleotides pose a high risk of contamination due to the sheer number of molecules generated, an important consideration when working with rare molecular species such as cell-free DNA. To address this challenge, we developed a novel technology that enables a single copy of synthetic human genome sequence to be introduced into a non-mammalian eukaryotic genome. The system highly-scalable and capable of producing thousands of unique clones which can be archived for future use, accessed individually, or combined to create customizable target pools on-demand. These custom designed synthetic positive controls are therefore readily renewable, require no amplification to obtain, and most importantly allow precise control over the number of molar equivalents of each target in a reaction. Using these synthetic controls, we simulate low mutation-allele fractions to validate the specificity and sensitivity of our assay. As a proof of concept, we have designed approximately 400 cancer mutation controls in driver genes such as KRAS and EGFR, and developed a design process to generate multiplex digital PCR assays targeting these driver genes. We will validate the digital PCR assays with standard bulk PCR and will validate the template by comparing to results generated using cancer cell line derived DNA. In the era of circulating DNA diagnostics, these genome engineered synthetic controls meet a critical need for reliable and customizable positive controls. Citation Format: Christina M. Wood-Bouwens, Robert P. St.Onge, Hanlee P. Ji. Highly sensitive digital detection of circulating DNA cancer mutations using synthetic genome standards [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1604.
Type of Medium:
Online Resource
ISSN:
0008-5472
,
1538-7445
DOI:
10.1158/1538-7445.AM2018-1604
Language:
English
Publisher:
American Association for Cancer Research (AACR)
Publication Date:
2018
detail.hit.zdb_id:
2036785-5
detail.hit.zdb_id:
1432-1
detail.hit.zdb_id:
410466-3
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