Abstract B177: Non-invasive tumor mutation detection of cell-free urinary DNA using massively parallel deep DNA sequencing.

Abstract

Abstract Background: Previous studies have demonstrated the ability to detect tumor DNA mutations by PCR methodologies within cell-free urinary DNA in metastatic cancer patients. This has opened up the possibility to use massively parallel deep sequencing approaches for more global profiling of tumor mutations using cell free urinary DNA from these patients. Methods: In order to achieve clinical utility the sequencing of cell-free urinary DNA must overcome two technical hurdles: 1) the need for extremely high sensitivity, between 0.01-0.05% and 2) an ultra-short DNA footprint (∼30 bp). Here we report the development of a method using cell free DNA extracted from urine that enriches for extremely low levels of mutated tumor DNA and thereby provides a high detection sensitivity. Detection of the KRAS gene mutations were used as a model system; the developed assay utilizes a 31bp footprint, contains a pre-amplification step that specifically enriches mutated DNA fragments and detects 8 KRAS mutations at the codon 12 and 13 sites. Spiking experiments with DNA derived from cell lines harboring the KRAS G12D mutation were completed to determine the limits of sensitivity of our method. Results: Mutant DNA input at amounts of 0.2%, 0.05%, 0.01% and 0.0% of the total DNA returned observed detection levels of 18.25%, 4.45%, 1.84% and 0.54% respectively as a percentage of the total sequence reads with the detection of mutant DNA of 0.01% within the theoretical limit of the assay. To test our assay, we sequenced 30 ng of extracted urinary DNA from a stage IV colorectal carcinoma patient with a known mutation at the G12D site. Enrichment of the mutation presented at a level of 13.06% of our total sequence, corresponding to an input amount of approximately 0.14% mutational load in the patient's urine. Ongoing analysis of a larger number of patients will be presented to further evaluate the clinical sensitivity and specificity of KRAS mutations. Conclusion: Further development of massively parallel DNA sequencing to detect mutations from cell-free urinary DNA has the potential to monitor metastatic patients for response, non-response and the emergence of resistance mechanisms to molecularly targeted therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B177. Citation Format: Jason C. Poole, Filip Janku, Veronica R. Holley, Jennifer J. Wheler, Funda Meric-Bernstam, Rayjalakshmi Luthra, Lorieta Leppin, Lafifa Hassaine, Karena Kosco, Mark G. Erlander. Non-invasive tumor mutation detection of cell-free urinary DNA using massively parallel deep DNA sequencing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B177.