Abstract B175: Detection and monitoring of BRAF and KRAS mutations in cell-free urinary DNA of metastatic cancer patients by droplet digital PCR.

Abstract

Abstract Background: Detection and monitoring of oncogenic mutations in cell-free urinary DNA opens the possibility of a new paradigm for a truly non-invasive method of individualized care for metastatic cancer patients, enabling the quantitation of mutational tumor load and respective concordance to therapeutic responsiveness followed by detection of emerging genomic alterations underlying acquired resistance. Methods: Cell-free DNA was isolated from single and/or multiple sequential urine samples from patients with advanced cancers and BRAF V600E, KRAS G12D or G12V mutations in the tumor tissue from a CLIA-certified laboratory, who progressed on systemic therapy. Assays for quantitative assessment of BRAF V600E, KRAS G12D and G12V mutations in cell-free urinary DNA were developed using droplet digital PCR methodology (RainDance, MA) with enrichment of mutation-containing DNA fragments by pre-amplification of BRAF and KRAS genes. Mutation sensitivity of at least 0.03% was achieved by spike-in experiments of input DNA from cell-lines containing BRAF and KRAS mutations. Healthy controls (N=6) yielded baseline signals that were ∼10-fold less than observed for 0.03% sensitivity. Results: Cell-free DNA was extracted from urine of 25 patients with diverse advanced cancers (colorectal cancer, n=8; melanoma, n=7; non-small cell lung cancer, n=6; papillary thyroid carcinoma, n=2; appendiceal carcinoma, n=1; and glioblastoma, n=1) with BRAF V600E (N=18), KRAS G12D (N=5) and KRAS G12V (N=2) in the tumor tissue. Of 18 patients with BRAF V600E mutations in the tumor, 17 (94%) had the same mutation in urinary cell-free DNA. In addition, all 5 (100%) patients with KRAS mutations (G12D, n=5; G12V, n=2) in the tumor tissue DNA had these same mutations in urinary cell-free DNA.A total of 5 patients with BRAF V600E mutations had longitudinal analysis of percentage of cell-free urinary DNA BRAF V600E mutation to wild-type in sequentially collected urine samples. Although the numbers are small the detected amount of BRAF mutant copies are in agreement with a clinical course. Conclusion: Our preliminary data suggest that detecting BRAF V600E, KRAS G12D, and G12V mutations in cell-free DNA from urine can offer a noninvasive alternative to mutation testing of tumor tissue with excellent concordance, and should be investigated further for testing and monitoring of mutation status in patients with cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B175. Citation Format: Filip Janku, Gerald S. Falchook, Sarina A. Piha-Paul, Aung Naing, Apostolia M. Tsimberidou, Veronica R. Holley, Daniel D. Karp, Ralph G. Zinner, Siqing Fu, Jennifer J. Wheler, David S. Hong, Funda Meric-Bernstam, Vanda M. Stepanek, Rayjalakshmi Luthra, Lorieta Leppin, Latifa Hassaine, Karena Kosco, Jason C. Poole, Mark G. Erlander. Detection and monitoring of BRAF and KRAS mutations in cell-free urinary DNA of metastatic cancer patients by droplet digital PCR. [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 B175.