Abstract 1736: Digital castPCR for direct detection of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) in peripheral blood of lung cancer patients
Authors: Deng David Xingfei , Wu Yalei , Bao Yun 
Published: August 2012

[OBJECTIVES] Genetic alternations of EGFR and KRAS are frequently found in lung cancer cells. Accurate detection of these mutations from lung cancer peripheral blood may provide a noninvasive means for early cancer detection and disease monitoring. The goal of this study is to develop a quantitative analysis of EGFR and KRAS mutations both of ctDNA in plasma and of CTCs in blood cells from lung cancers by digital castPCR (competitive allele specific TaqMan-base PCR) technology. [METHODS] Molecular analysis and enumeration of CTCs in peripheral blood cells were analyzed by combining sample partition and digital castRT-qPCR assays without prior biophysical processing. The castPCR can detect rare copies of mutant alleles with a 6-log dynamic range and &lt; 5-copy detection sensitivity for EGFR and KRAS mutations. Quantitative analysis of EGFR and KRAS mutations in ctDNA was done in OpenArray platform by digital castPCR. For CTC detection, whole blood samples with spiked-in known mutation and gen marker lung cancer cell lines were partitioned onto 96- or 384-well plate(s), such that each well contains either one cancer cell or none together with normal white blood cells and red blood cells. RNAs and/or DNAs were extracted by magnetic beads and were pre-amplified prior to molecular detection. Genetic mutations and cell type specific markers (CK19) for CTC identification and enumeration were determined by castRT-qPCR and TaqMan Gene expression (GEx) assays, respectively. Blood samples from lung cancer patients were first separated into blood cells and plasma portions before CTC detections. The sample plasma portions were used to detect EGFR and KRAS mutations of ctDNA by digital castPCR in OpenArray platform. [RESULTS] The sample partition process resulted in a relative CTC enrichment or digital enrichment of 20 - 400 folds (the relative ratio of CTC to normal cells) in a CTC-positive well. The RT-castPCR and TaqMan GEx assays accurately quantify the number of spiked-in tumor cells (10 - 60 cells/mL) in whole blood with known EGFR and/or KRAS mutations and CK19. For 10 blood samples from lung cancer patients, CTCs were detected from all patients even with stage I and II of 3 patients. In the plasma portions of the same samples, digital castPCR by OpenArray was able to detect EGFR mutations (p.L858R) and 19 mutations of EFGR exon 9. The results of EGFR mutations are consistent between intact CTCs and ctDNA. [CONCLUSION] Our preliminary data suggest that combination of digital sample enrichment and castRT-qPCR can be used to directly enumerate CTCs and detect cancer-related mutations in blood cells without prior biophysical sample enrichment. Digital castPCR by OpenArray can be used to assess circulating tumor DNA. Further test in larger clinical samples are warranted.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1736. doi:1538-7445.AM2012-1736