Abstract 3781: Tracking triple negative breast cancer (TNBC) evolution in the molecular residual disease (MRD) setting in the c-TRAK TN clinical trial
Authors: Coakley Maria , Cutts Rosalind J. , Sritharan Prithika , Hrebien Sarah , Swift Claire , Dunne Kathryn , Kilburn Lucy , Goddard Katie , Villacampa Guillermo , Rojas Patricia , Emmett Warren , Pipinikas Christodoulos , Hall Peter , Harper-Wynne Catherine , Hickish Tamas , Macpherson Iain , Okines Alicia , Wardley Andrew , Wheatley Duncan , Waters Simon , Bliss Judith M. , Garcia-Murillas Isaac , Turner Nicholas C. 
Published: March 2024

Abstract
Background: Circulating tumor DNA (ctDNA) to track early tumor clones and to identify the point of origin of clones in relapsed disease, could provide novel insights into the impact of heterogeneity on the development of MRD and relapse in TNBC.
Methods: c-TRAK TN recruited 161 patients (pts) with early TNBC into prospective ctDNA surveillance after curative therapy. Two distinct early tissue samples were provided, and a subset provided a relapse biopsy. Tissue was whole exome sequenced (WES) and relapse plasma DNA underwent error-corrected WES. Clonal evolution analysis was performed for pts who developed MRD with a minimum of 2 samples with WES (n=44), and non-relapse pts with samples before and after neoadjuvant chemotherapy (NAC) (n=16). Clonal structures were inferred with Pyclone; the founding clone as the cluster with highest cellular prevalence (CP). Clones were validated and tracked using the RaDaR ctDNA assay, supplemented with a median of 9 (range 6-45) variants specific to each clone. Cluster hierarchy and phylogenetic tree structure was derived using ClonEvol.
Results: A median of 3 (range 1-5) clones per pt validated. Non-validated clones (12.9%) and pts with only 1 clone validated were removed, resulting in 56 pts for analysis. To assess the impact of spatial heterogeneity, contemporaneous paired early tissue was available from 17 pts who developed MRD. Primary tumor subclones were detected in the MRD or relapse in 88.9% (8/9) pts with a subclone detected in all regions, versus 37.5% (3/8) pts with a subclone unique to 1 region (p = 0.049). To assess the impact of NAC, paired tissue before and after NAC, was available in 32 pts. Distinct sub-clonal evolutionary patterns were identified. Subclones increased in CP or were first detected after NAC in 75% (24/32) pts. Subclones present prior to NAC were reduced or lost in 62% (20/32) pts. Sub-clonal persistence was observed in 46.8% (15/32) pts. Subclones first detected following NAC were detected in the MRD in 60% (6/10) pts. Following NAC, subclones reduced or lost re-emerged in the MRD in 30% (3/10) pts, versus subclones that persisted which were detected in the MRD in 100% (9/9) pts (p=0.003). In 21 pts, relapse samples were available. Relapse-unique subclones were identified in 57% (12/21) pts, with 2 new subclones in 1 pt. Relapse-unique subclones were not detected in MRD in 41.6% (5/12) pts, and were detected in the MRD in 66.7% (8/12) of pts.
Conclusion: Polyclonal metastasis is a common mode of metastasis in TNBC, with frequent detection of primary tumor subclones in the metastasis. NAC provides an evolutionary bottleneck, with subclones detected post NAC being the most likely to persist into the MRD and subsequent relapse. Relapsed cancer frequently had subclones not detected in the primary cancer, with approximately half of these potentially late arising, and half detectable in MRD suggesting early establishment of diversity.
Citation Format: Maria Coakley, Rosalind J. Cutts, Prithika Sritharan, Sarah Hrebien, Claire Swift, Kathryn Dunne, Lucy Kilburn, Katie Goddard, Guillermo Villacampa, Patricia Rojas, Warren Emmett, Christodoulos Pipinikas, Peter Hall, Catherine Harper-Wynne, Tamas Hickish, Iain Macpherson, Alicia Okines, Andrew Wardley, Duncan Wheatley, Simon Waters, Judith M. Bliss, Isaac Garcia-Murillas, Nicholas C. Turner. Tracking triple negative breast cancer (TNBC) evolution in the molecular residual disease (MRD) setting in the c-TRAK TN clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3781.