Background: Identifying mechanisms that govern the shedding of ctDNA in blood could inform the use of liquid biopsy in individual patients. Previous studies in the I-SPY2 neoadjuvant trial involving high-risk breast cancer showed that the detection of ctDNA before treatment was associated with aggressive clinical characteristics and residual ctDNA after treatment was associated with poor outcomes. Moreover, ctDNA positivity rates significantly varied across breast cancer subtypes suggesting that ctDNA shedding may in part be driven by subtype-specific etiology. We performed genome-wide transcriptomic analysis to identify genes and biological processes associated with increased ctDNA shedding within and across receptor subtypes. Methods: Our study involved 227 patients in I-SPY2 with tumor gene expression and ctDNA data at pretreatment. All patients were at high risk for recurrence (MammaPrint high). Each subtype: HR+HER2- (n=109), HER2+ (n=19), and triple negative breast cancer (TNBC, n=99) was evaluated independently. We performed differential expression (DE) analysis on the global transcriptome (m=19,134 genes) and curated gene signature (cGS, m=31 signatures developed in I-SPY2) data between ctDNA+ and ctDNA- patients at baseline. Gene-set enrichment analysis (GSEA) was also performed across hallmark (H, m=50), canonical pathway (CP, m=5,501), gene ontology (GO, m=9,996) and immunologic (IM, m=4,872) gene sets. Features were associated with ctDNA shedding if Benjamini-Hochberg adjusted p < 0.05. For subtypes with smaller sample size and unbalanced groups, we also report features with nominally significant p < 0.05. Results: ctDNA positivity rate was significantly higher in TNBC (91%) than in HR+HER2- and HER2+ (65% and 74% respectively, Fisher p <0.001). The HR+HER2- subtype had the most significant hits for DE analysis between ctDNA+ and ctDNA- patients, with 0.2% of genes and 3.2% of cGS. No genes or cGS were differentially expressed in TNBC and HER2+, likely due to imbalance or small size of these groups. For GSEA, we observed the most significant number of enrichments in HR+HER2- subtype, with 58%, 21.8%, 4.4% and 40.3% of H, CP, GO, and IM gene sets enriched, respectively. In the HER2+ subtype, 40% H, 15.7% CP and 36.4% IM gene sets were significantly enriched, while no gene sets were enriched in TNBC. To identify common mechanistic themes across subtypes, we also considered nominally significant features in DE and GSEA. Processes associated with infection and innate immune responses were enriched in ctDNA+ patients, while adaptive immune response and antigen presentation—e.g., T-cell, TCR and MHC II protein complex, and downregulation of MYC targets were enriched in ctDNA- patients. HR+HER2- and HER2+ subtypes shared the most common modulated features with 134 genes and 2,165 gene sets, including up-regulation of cell cycle and proliferation in ctDNA+ patients, as well as up- or down-regulation of specific immunologic and metabolic processes. In contrast, TNBC gene set enrichment was associated with more distinct biologic processes, sharing common enrichment of 113 and 27 gene sets with HR+HER2- and HER2+ subtype, respectively. Conclusions: Findings from our exploratory analysis suggest a key role of immune response pathways in the control of ctDNA release. Additionally, tumor cell proliferation was associated with increased shedding in HR+HER2- and HER2+ subtypes, while down regulation of MYC targets was associated with ctDNA- patients across all subtypes. These suggest an important role of cell cycle in ctDNA shedding. Overall, our analysis revealed common and unique mechanisms potentially associated with ctDNA shedding across and within subtypes. However, due to the unbalanced groups and limited sample sizes, validation in a larger cohort is warranted.

Citation Format: Rosalyn W. Sayaman, Denise M. Wolf, Christina Yau, Lamorna Brown Swigart, Gillian L. Hirst, Laura Sit, Nicholas O'Grady, Amy L. Delson, I-SPY2 Investigators, Laura Esserman, Laura J. van 't Veer, Mark Jesus M. Magbanua. Elucidating the biology of circulating tumor DNA (ctDNA) shedding across receptor subtypes in high-risk early-stage breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-03.