Abstract 2406: Validation of a simple low-cost method to monitor ctDNA in patients with solid cancers
Authors: Benesova Lucie , Belsanova Barbora , Minarikova Petra , Halkova Tereza , Pudil Jiri , Pazdirek Filip , Pesek Milos , Fiala Ondrej , Hoch Jiri , Zavoral Miroslav , Bunganic Bohus , Levy Miroslav , Lipska Ludmila , Petruzelka Lubos , Ryska Miroslav , Minarik Marek 
Published: August 2015

Abstract
Background: Detection of circulation tumor DNA (ctDNA) in plasma has become a viable option for non-invasive monitoring of patients. Also termed “liquid biopsy” the approach is applicable for pre-diction of response and prediction of resistance to biological therapy (1, 2). Various techniques have been used for ctDNA detection, frequently employing clonal amplification on a digital PCR format (3) with limits of detection (LOD) below 0.01% of mutant alleles. However, these techniques suffer from high complexity, expensive instrumentation, and a considerable cost per sample. We hereby present a simple low-cost alternative that is implementable to routine ctDNA testing.
Methods: A panel of PCR amplicons (106 - 174bp) was resolved by denaturing capillary electrophoresis (DCE) revealing minute presence of mutation specific hetero-duplexes. The final panel consisted of clinically relevant oncogenic mutations KRAS, NRAS, BRAF, PIK3CA and EGFR as well as cancer-related mutations in tumor suppressors TP53, APC and CTNNB1. A total of 299 patients was subsequently examined for presence of ctDNA in plasma including 194 with colorectal cancer (CRC), 26 with NSCLC and 79 with pancreatic cancer (PanC). CtDNA status was correlated to TNM stage and tumor markers (CEA and Ca19-9). In a subset of CRC patients (n = 20) the ctDNA was monitored in 2 - 6 month intervals and correlated to the therapy response.
Results: The experimental LOD value was in the range between 0.03 - 1% for all tested mutations within the panel. A minimum input amount of DNA was 5 pg (0,005 ng).. The overall rate of ctDNA detection was 32% for CRC (stages I - IV), 31% for NSCLC (stages III - IV) and 27% for PanC (stages II - IV). The highest detection rate, 69%, was observed in Stage IV CRC patients. Comparison with tumor markers (TM) revealed 62% of cases positive for both TM and ctDNA and 13% TM-negative cases with ctDNA positivity. Post-operative absence or persistence of ctDNA was related to the radicality of the surgical treatment and the ctDNA levels were concordant with the response to adjuvant chemotherapy. In several patients a disease progression was signalized based on ctDNA even prior to actual clinical detection by CT imaging.
Conclusion: DCE is a simple technique applicable for detection of ctDNA in cancer patients without a need for costly hardware/software equipment. The detection rates are 10 - 15% lower compared to the dedicated dPCR techniques, however, the method requires ca 100x less input DNA, the cost per patient is about 10-fold lower and the turnaround time per test is under 5 hours.
Supported by the Czech Ministry of Health Grant 14383.
Literature
1. Bettegowda C et al. Sci Transl Med. 2014,6(224):224ra24
2. Douillard JY et al. J Thorac Oncol. 2014, 9(9):1345-1353.
3. Benesova L et al. Anal Biochem 2013,433(2):227-234.
Citation Format: Lucie Benesova, Barbora Belsanova, Petra Minarikova, Tereza Halkova, Jiri Pudil, Filip Pazdirek, Milos Pesek, Ondrej Fiala, Jiri Hoch, Miroslav Zavoral, Bohus Bunganic, Miroslav Levy, Ludmila Lipska, Lubos Petruzelka, Miroslav Ryska, Marek Minarik. Validation of a simple low-cost method to monitor ctDNA in patients with solid cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2406. doi:10.1158/1538-7445.AM2015-2406