Background: Circulating tumor DNA (ctDNA) is a promising biomarker for the detection of minimal residual disease and monitoring treatment in patients with CRC. The performance demands of any technology used for this purpose, however, are tremendous. Here we aim to develop a high-performance multiplex NGS platform suitable for cancer MRD using ctDNA.
Methods: We have developed Firefly, a NGS method capable of detecting low-frequency variants with high precision in plasma cfDNA. In our protocol, denatured double-stranded cfDNA is circularized and converted into long tandem repeats using rolling-circle amplification enabling consensus-based concatemer error correction. We demonstrated Firefly’s performance sensitivity and specificity by testing our technology on cfDNA samples with known variant frequencies and cfDNA collected from healthy individuals (n = 82). Further analysis of Firefly as a tool for MRD and treatment monitoring was performed by tracking ctDNA mutation profile concordance between 81 CRC tumor samples and their corresponding plasma samples collected from patients before and after treatment.
Results: Performance sensitivity of Firefly NGS was 0.1% with an error-rate was 1 in 1 Million for 20ng of input ctDNA. Concordance analysis was performed on CRC tumor/plasma pairings derived from patients with CRC using, Accu-Act, a 61-gene assay. The number of tumor-matching mutations detected in plasma varied greatly on a per-patient basis (range, 0-28). Pre and post-treatment ctDNA profiling was performed on all 81 patients included in our study (surgery, n = 56; chemotherapy/radiotherapy, n = 30). Among patients who underwent surgery, 46% had detectable tumor-matching mutations in their plasma. Among patients who received neoadjuvant therapy, 70% ctDNA fluctuations consistent with tumor reduction based on surgical tumor regression grades evaluation(TRG1-3).
Conclusions: We report a novel ultra-accurate NGS-based ctDNA assay suitable for MRD and monitoring in CRC patients. Firefly should ultimately make a significant contribution in the development of personalized cancer treatment.