Digital Competitive Allele-Specific TaqMan-Based Reverse Transcription-qPCR (castRT-qPCR) for Direct Detection and Enumeration of Circulating Tumor Cells

David Xingfei Deng, Yun Bao, Scott Sproul, Yu Wang, Fawn Wang, David Merrill, Pius Brzoska, Caifu Chen
Life Technologies, United States of America

Molecular characterization and enumeration of circulating tumor cells (CTCs) promise to be valuable for cancer cell diagnosis, survival prognosis, and treatment guidance. However, current molecular assays require extensive blood sample enrichment process before analyzing extraordinarily rare CTCs. Here we reported a new approach for direct CTC molecular detection in whole blood samples without prior biophysical processing by combining sample partition and digital assay of a highly specific castRT-qPCR. CastPCR can detect rare copies of mutant alleles with a wide dynamic range of more than 6-log orders and < 5-copy sensitivity. Whole blood samples with spiked-in known mutation lung cancer cell lines and from lung cancer patients were partitioned in aliquots of 2.5 μL – 50 μL onto 96- or 384-well plate(s), such that each well contains either one cancer cell or none with per well 20,000 – 400,000 normal white blood cells and 10 – 200 million red blood cells. RNAs and/or DNAs were extracted by magnetic beads and directly or were pre-amplified prior to mutation detection. Genetic mutations and cell type specific markers (such as CK19 and/or CEA) for CTC identification and enumeration were determined by castRT-qPCR and/or TaqMan Gene expression assays. 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. CastPCR clearly identified known mutation and CK19 in spiked-in samples of about 10 – 30 cells per mL whole blood, but detected no mutation signals in any sample well without cell spiked-in. Furthermore, cell type specific markers (CK19) and known EGFR mutation(s) were identified in the same sample wells, suggesting identified mutation is specific and from cancer cells not from normal cells. In five blood samples from lung cancer patients, EGFR mutation (p.L858R) was detected in all samples. Approximately 50% of circulating lung tumor cells in a patient with positive EGFR p.L858R mutation had also positive EGFR p.T790M mutation, an inducible drug-resistant CTC marker. For those samples with negative detection of EGFR mutation, corresponding wild type sequences were detected in all sample wells, suggesting the normal DNA amplification of those mutation-negative wells. Our data suggest that combination of digital sample enrichment and castRT-qPCR can be used to directly enumerate CTCs and detect cancer-related mutations in whole blood without prior biophysical sample enrichment. The new approach paves way for noninvasively CTC monitoring and individualized therapy.

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