Abstract
The detection of plasma cell–free tumor DNA (ctDNA) is prognostic in colorectal cancer (CRC) and has potential for early prediction of disease recurrence. In clinical routine, ctDNA-based diagnostics are limited by the low concentration of ctDNA and error rates of standard next-generation sequencing (NGS) approaches. We evaluated the potential to increase the stability and yield of plasma cell–free DNA (cfDNA) for routine diagnostic purposes using different blood collection tubes and various manual or automated cfDNA extraction protocols. Sensitivity for low-level ctDNA was measured in KRAS-mutant cfDNA using an error-reduced NGS procedure. To test the applicability of rapid evaluation of ctDNA persistence in clinical routine, we prospectively analyzed postoperative samples of 67 CRC (stage II) patients. ctDNA detection was linear between 0.0045 and 45%, with high sensitivity (94%) and specificity (100%) for mutations at 0.1% VAF. The stability and yield of cfDNA were superior when using Streck BCT tubes and a protocol by Zymo Research. Sensitivity for ctDNA increased 1.5-fold by the integration of variant reads from triplicate PCRs and with PCR template concentration. In clinical samples, ctDNA persistence was found in ∼9% of samples, drawn 2 weeks after surgery. Moreover, in a retrospective analysis of 14 CRC patients with relapse during adjuvant therapy, we successfully detected ctDNA (median 0.38% VAF; range 0.18–5.04% VAF) in 92.85% of patients significantly prior (median 112 days) to imaging-based surveillance. Using optimized pre-analytical conditions, the detection of postoperative ctDNA is feasible with excellent sensitivity and allows the prediction of CRC recurrence in routine oncology testing.
Highlights
Cell-free DNA represents a fraction of molecules that are constantly released into the blood circulation and other body fluids by cellular processes of necrosis or apoptosis (Schwarzenbach et al, 2011)
Total yields of cell–free DNA (cfDNA) from plasma samples (1–3 ml) were significantly higher (p < 0.0001) using procedures by Zymo Research and Qiagen than those using the protocol of Analytik Jena
We provide an adaptable, cost-effective, and rapid approach for the sensitive detection of plasma cell–free tumor DNA (ctDNA) in clinical routine, using adequate pre-analytical cfDNA processing and subsequent error-reduced deep sequencing
Summary
Cell-free DNA (cfDNA) represents a fraction of molecules that are constantly released into the blood circulation and other body fluids by cellular processes of necrosis or apoptosis (Schwarzenbach et al, 2011). In malignant diseases such as cancer, plasma cfDNA is enriched with circulating cell-free tumor DNA (ctDNA) and may carry tumor-derived genetic and epigenetic aberrations, reflecting the ctDNA Detection in Colorectal Cancer clonal architecture and evolution of corresponding cancer cells in the primary tumor tissue (Schwarzenbach et al, 2011; Crowley et al, 2013). Due to its relevance as the second leading cause of cancer-related deaths and improvements in targeted therapies, plasma ctDNA has been evaluated in colorectal cancer (CRC) with respect to risk stratification (Tie et al, 2016; Luo et al, 2020), prognosis (El Messaoudi et al, 2016; Wang et al, 2019), and prediction of treatment response or progression during postoperative surveillance (Siravegna et al, 2015; Tie et al, 2015; Reinert et al, 2016; Tie et al, 2016)
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