Abstract LB-170: Droplet digital PCR detection and longitudinal monitoring of BRAF mutations in cell-free urinary DNA of patients with metastatic cancers or Erdheim-Chester disease
Authors: Janku Filip , Falchook Gerald S. , Piha-Paul Sarina A. , Naing Aung , Holley Veronica R. , Hong David S. , Subbiah Vivek , Karp Daniel D. , Zinner Ralph G. , Fu Siqing , Wheler Jennifer J. , Meric-Bernstam Funda , Stepanek Vanda M. , Luthra Rajyalakshmi , Leppin Lorieta , Hassaine Latifa , Kosco Karena , Poole Jason C. , Vibat Cecile Rose T. , Erlander Mark G. 
Published: May 2015

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, which would enable 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 or Erdheim-Chester disease and BRAF V600E mutation in the tumor tissue from a CLIA-certified laboratory, who progressed on systemic therapy. Assays for quantitative assessment of BRAF V600E mutation in cell-free urinary DNA were developed using digital droplet PCR methodology (RainDance, MA) with enrichment of mutation-containing DNA fragments by pre-amplification of the BRAF gene. Detection limits were established as wild-type (&lt;0.05% of mutant copies), low-mutant (0.05%-0.107%), mutant (&gt;0.107%).
Results: Cell-free DNA was extracted from urine of 33 patients with diverse advanced cancers (melanoma, n=11; colorectal cancer, n=8; non-small cell lung cancer, n=4; papillary thyroid carcinoma, n=4; other, n=4) or Erdheim-Chester disease (n=1) with BRAF V600E mutation in the tumor tissue. Of these 33 patients, 25 (76%) had the same mutation in urinary cell-free DNA (mutant, n=14; low-mutant, n=11). In addition, 17 patients had longitudinal analysis of percentage of BRAF V600E mutation to wild-type in sequentially collected urine samples and the dynamics of BRAF V600E mutation in the urine correlated with response to therapy in 13 (76%) of them.
Conclusion: Our data suggest that detecting BRAF V600E mutation in cell-free DNA from urine can offer a noninvasive alternative to mutation testing of tumor tissue with acceptable concordance and should be investigated further for testing and monitoring of mutation status in patients with cancer and Erdheim-Chester disease.
Citation Format: Filip Janku, Gerald S. Falchook, Sarina A. Piha-Paul, Aung Naing, Veronica R. Holley, David S. Hong, Vivek Subbiah, Daniel D. Karp, Ralph G. Zinner, Siqing Fu, Jennifer J. Wheler, Funda Meric-Bernstam, Vanda M. Stepanek, Rajyalakshmi Luthra, Lorieta Leppin, Latifa Hassaine, Karena Kosco, Jason C. Poole, Cecile Rose T. Vibat, Mark G. Erlander. Droplet digital PCR detection and longitudinal monitoring of BRAF mutations in cell-free urinary DNA of patients with metastatic cancers or Erdheim-Chester disease. [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 LB-170. doi:10.1158/1538-7445.AM2014-LB-170