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Circulating-tumour DNA (ctDNA) detection using an ultra-sensitive next generation sequencing (NGS)-based assay in patients with resected colorectal cancer (CRC) in the phase III ASCOLT trial
Background
Identification of somatic mutations in ctDNA may detect minimal residual disease (MRD) in patients with curatively treated CRC who may eventually recur. Highly sensitive NGS-based assays are known to have comparable sensitivity to digital PCR approaches, with the added benefit of broader coverage and no requirement for a priori knowledge of tumour-based mutations. The ASpirin for Dukes C and high risk Dukes B COLorecTal cancer trial (ASCOLT, NCT00565708) is a randomised, double-blinded, phase III fully accrued study investigating the benefit and safety of aspirin in the adjuvant setting. In the Australian and New Zealand cohort, serial plasma samples were prospectively collected at study enrolment (within 90 days of completing adjuvant chemotherapy), then at 6 and 12 months, along with baseline tumour tissue. In this pilot, we evaluated the utility of an ultra-sensitive ctDNA NGS assay (SafeSEQ, Sysmex Inostics), comparing plasma and tumour-based sequencing.
Methods
Extracted cell-free DNA (cfDNA, up to 20,000 genomic equivalents [GE, validated maximum DNA input]) was analysed using a SafeSEQ CRC MRD assay that detects mutations in 14 genes relevant in CRC, including AKT1, APC, BRAF, CTNNB1, ERBB3, FBXW7, KRAS, NRAS, PIK3CA, POLE, PPP2R1A, RNF43, SMAD4 and TP53. Tumour sequencing used an amplicon-based NGS 59-gene custom panel. cfDNA results were correlated with tumour sequencing and recurrence data.
Results
Twenty-nine plasma samples (median 3.6 mL, median DNA 71.7 GE/μL) from the first 10 patients were analysed (6 female; median age 65; 2 with high-risk stage II, 8 stage III). All patients had received adjuvant 5-Fluorouracil-based chemotherapy (7 also with oxaliplatin; 1 also with neoadjuvant chemoradiation for rectal cancer). Median follow-up was 59.9 months, with 3 known recurrences (median time to recurrence 23.3 months). Two of 3 patients who experienced recurrence had detectable ctDNA: 1 at the 6-month timepoint and the other at 12 months (negative at 6 months). In relation to the timing of ctDNA positivity, cancer recurrence was detected by CT 16.5 months after (lung metastases) for 1 patient and within 4 weeks (lung and nodal metastases) for the other. Both patients’ plasma mutation profiles (KRAS G12V and KRAS G13D/ SMAD4 L540P, respectively) were concordant with their tumour analysis. A third patient experienced disease recurrence 59.2 months after study commencement (peritoneal metastases) but did not have detectable ctDNA at any of the 3 timepoints. CEA did not predict recurrence in any of the 3 patients. Another patient with detectable cfDNA at the 6-month timepoint (TP53 R248W, seen in plasma only, negative at 12 months) has not had a recorded recurrence; analysis of matched peripheral blood mononuclear cells is awaited.
Conclusions
In this pilot cohort of 10 ASCOLT trial patients with high-risk resected CRC following adjuvant chemotherapy, 2 of 3 patients who recurred had early detectable ctDNA using an NGS-based SafeSEQ assay. A third patient with late recurrence did not have detectable ctDNA. Plasma and tumour sequencing results were concordant in 2 of 3 patients with detectable cfDNA. Analyses in the larger cohort (n=368) are underway.
Clinical trial identification
NCT00565708.
Legal entity responsible for the study
Australasian Gastro-Intestinal Trial Group.
Funding
This work is supported by an Australasian Gastro-Intestinal Cancer Trials Group Innovation Grant. D. Day is a recipient of the Royal Australasian College of Physicians Foundation 2022 Basser Research Entry Scholarship.
Disclosures
A. Starus: Full / Part-time employment: Sysmex Inostics, Inc. D. Prasse: Full / Part-time employment: Sysmex Inostics GmbH. A. Lamik: Full / Part-time employment: Sysmex Inostics GmbH. J. Fredebohm: Full / Part-time employment: Sysmex Inostics GmbH. J. Simes: Research grant / Funding (institution): Bayer. S. Li: Research grant / Funding (institution): WEHI, WEHI, WEHI. F. Jones: Full / Part-time employment: Sysmex Inostics, Inc.. All other authors have declared no conflicts of interest.
