Abstract RF3-05: Tissue-free circulating tumour DNA detection in patients with early triple negative breast cancer from the c-TRAK-TN trial

Abstract Background: Detection of circulating tumour DNA (ctDNA) in plasma predicts future recurrence in early breast cancer (EBC). Previous studies have relied on tumour-informed approaches with personalised assays. In this study, we performed orthogonal testing on plasma samples collected prospectively in the c-TRAK-TN study to compare a tissue-free assay with digital droplet PCR (ddPCR) for ctDNA analysis. Methods: c-TRAK-TN recruited 161 patients with early triple negative breast cancer (TNBC) deemed clinically high risk of relapse. Patients underwent ctDNA analysis by ddPCR starting within 4 weeks of completion of treatment and continuing every 3 months for up to 2 years. ddPCR tracked 1 or 2 patient specific variants as previously described. We further analysed 1062 timepoints from a total of 159 patients (median 7 per patient, range 1-11) using Guardant Reveal assay, a tissue-free approach that exploits differential methylation patterns in breast cancer compared with normal tissue. Results: Samples were successfully analysed from 95.3% (1012/1062) timepoints from 159 patients with the tissue-free Reveal assay. CtDNA was detected in 34.0% (54/159) of patients in at least 1 timepoint, with a median methylation tumour fraction of 0.266% (range 0.002 to 51.0%). Detection of ctDNA with the tumour agnostic assay strongly associated with future risk of relapse (HR 20.62, p<0.0001; logrank). Of patients who relapsed by 24 months follow up, 89.5% (34/38) of relapses were detected by prior ctDNA analysis. There were 159 patients available for comparison between ddPCR and the tissue-free assay (Reveal), in 1005 timepoints (median 7 per patient, range 1-11). Concordant test results were observed in 950/1005 timepoints with an overall agreement rate of 94.5%. In this cohort, 63.5% (101/159) of patients did not have ctDNA detected by either assay; 26.4% (42/159) had ctDNA detected by both assays and 10.1% (16/159) had discordant results. Of those detected, 44.8% (26/58) were detected by the tissue-free assay first and 6.9% (4/58) by ddPCR (p<0.0001, Fisher exact test). The median time from first detection of ctDNA to relapse for the tissue-free assay was 7.9 months (95% confidence interval (CI): 5.7-10.5 months) compared to ddPCR assay with 5.7 months (95% CI: 2.9-9.7 months) (HR = 0.6, p=0.0574; mixed effects cox model). Conclusion: Detection of ctDNA with a tissue-free assay anticipated relapse with high accuracy, in patients with early TNBC. The tissue-free assay frequently detected ctDNA at an earlier timepoint than ddPCR, trending towards a longer lead time from detection of ctDNA to relapse. Tissue-free approaches might offer simpler workflows for ctDNA analysis than tumour-informed approaches, and further assessment in clinical trials is warranted. Citation Format: N. Cunningham, R. J. Cutts, C. Swift, K. Dunne, M. Dewan, L. Kilburn, K. Goddard, P. Hall, C. Harper-Wynne, T. Hickish, I. Macpherson, A. Okines, A. Wardley, S. Waters, C. Palmieri, M. Winter, J. Bliss, D. Dustin, M. Ellis, I. Garcia- Murillas, N. C. Turner. Tissue-free circulating tumour DNA detection in patients with early triple negative breast cancer from the c-TRAK-TN trial [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr RF3-05.