Abstract 3486: Detection of AR W741 mutation in circulating tumor DNA from plasma samples of castration-resistant prostate cancer patients using LNA clamp-mediated PCR.
Authors: Eggert Ines Verena , Schmitz Arndt , Koenig Frank , Asadullah Khusru , Krahn Thomas , Stresemann Antje 
Published: August 2014

Abstract
After one to two years of hormone therapy, most hormone-dependent prostate cancers become refractory and stop responding to castration treatment. Treatment-emergent mutations in the AR (androgen receptor) gene have been identified in these patients and present a possible mechanism underlying the development of castration-resistant prostate cancer (CRPC). However, since most patient have a biopsy performed only at the time of diagnosis, in general, representative tumor tissue sample giving real time information about the disease status from CRPC patients are missing. The detection of AR mutations in circulating tumor DNA (ctDNA) may be a potential alternative.
In this study we validated a locked nucleic acid (LNA) clamp assay for the detection of the AR mutation W741C, known to lead to bicalutamide resistance. The LNA clamp-mediated PCR is based on the blockade of the wildtype (wt) DNA by the LNA, RNA monomers with a modified backbone leading to an increased thermal stability. The identification of the qRT-PCR-enriched mutated DNA (mut) was subsequently performed by sequencing.
In a first step, we optimized the annealing and elongation temperatures as well as the concentrations of LNA construct and DNA necessary to achieve the optimal blocker efficiency for the wt, but avoiding blockage of the mut. Therefore we used different concentrations of the genomic wt DNA from the prostate cancer cell line LAPC4. A cell line (KuCaP-1) harboring the W741C mutation was obtained from the group of O. Ogawa (Kyoto University). Using optimized parameters, we were successful in detecting mutated DNA in a ≥ 1000x background of wt DNA. In a second step, we applied the developed assay to clinical plasma samples of 19 CRPC patients, all of whom were receiving bicalutamide treatment except patients 3, 15 and 18. The ctDNA was isolated from plasma using the QIAamp Circulating Nucleic Acid Kit from Qiagen. Three out of nineteen patients were classified as mutation-positive (PID 4, 5, 11). The availability and subsequent sequencing of ctDNA from patient 5 confirmed the existence of the bicalutamide resistance W741 mutation. Due to the small amount of available DNA, sequencing of the mutated ctDNA of patient 4 failed. The ctDNA of patient 11 harbored another SNP in codon 741 leading to a false positive result. In conclusion, our work led to the successful development of a potent LNA clamp-mediated qRT-PCR method for the detection of the bicalutamide resistance mutation W741 in ctDNA of plasma samples.
Citation Format: Ines Verena Eggert, Arndt Schmitz, Frank Koenig, Khusru Asadullah, Thomas Krahn, Antje Stresemann. Detection of AR W741 mutation in circulating tumor DNA from plasma samples of castration-resistant prostate cancer patients using LNA clamp-mediated PCR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3486. doi:10.1158/1538-7445.AM2013-3486