Abstract Capivasertib, a pan-AKT inhibitor, has recently been approved in combination with fulvestrant to treat ER+ HER2- breast cancer patients with one or more of PI3KCA/AKT1/PTEN alterations. However, as with many targeted therapies acquired drug resistance remains a challenge. In this study, we investigated the mechanisms of acquired capivasertib resistance in AKT1 mutant breast cancer models. We established two estrogen receptor positive (ER+) AKT1 E17K mutant breast cancer patient derived organoids (PDOs), that were sensitive to capivasertib in vitro. PDOs were chronically exposed to 1µM capivasertib. PDOs were sequenced (DNA & RNA) at multiple timepoints of treatment (3 days, 4 and 9 weeks after development of resistance). Using CRISPR-Cas9 editing we also engineered CAMA1 (ER+) cells to express AKT1 E17K, and generated derived resistant AKT1 mutant and parental models in response to continuous capivasertib exposure. CAMA1 AKT1 mutant and parental resistant were screened with an siRNA Kinome library supplemented with genes from the PDO RNA-sequencing. Phosphoproteomics was performed on capivasertib resistant E17K models in the presence and absence of capivasertib. PDO models persisting after either 4 or 9 weeks of capivasertib treatment showed no acquisition of genetic driver events, or changes in AKT copy number. Resistant organoids at 9 weeks treatment showed increased expression of ERBB3 (HER3), EGFR3 and LPAR5 as well as increased expression of MAPK signalling components. In siRNA screens on CAMA1 derived resistant models, siRNA targeting ERBB2, CDK4, PLK4 and CHEK1, ESR1 and LPAR5 modulated sensitivity to capivasertib. Analysis of phosphoproteins in capivasertib resistant models revealed reactivation of mTORC1 signalling despite sustained AKT inhibition, with dramatic upregulation of PDK1 and mTORC2. Consistent with the siRNA screens, drug combinations identified fulvestrant as a synergistic agent with capivasertib in the resistant E17K models. Sensitivity to mTOR inhibition was maintained in capivasertib resistant models, with mTOR inhibition re-sensitizing cells to capivasertib. In these models, acquired resistance to capivasertib occurs through upregulation of ER signalling and rewiring of pathway signalling to bypass AKT inhibition and reactivate mTORC1. Targeting these mechanisms could prolong the use of capivasertib, with future work focusing on validating these mechanisms and re-sensitizing drug combinations in other AKT1 mutant models & PDOs. Citation Format: Sarah Mearns, Alex Pearson, Li-Xuan Sim, Rosalind Cutts, Prithika Sritharan, Heena Shah, Aditi Gulati, Stuart Williamson, Simon Barry, Elza De-Bruin, Nicholas C. Turner. Mechanisms of acquired resistance to AKT inhibitor capivasertib in AKT1 mutant patient derived breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB450.

Diagnostic performance of RaDaR assay at 24 months.

Baseline information (n=141).

AbstractPurpose: Detection of circulating tumor DNA (ctDNA) in patients who have completed treatment for early-stage breast cancer is associated with a high risk of relapse, yet the optimal assay for ctDNA detection is unknown. Experimental Design: The cTRAK-TN clinical trial prospectively used tumor-informed digital PCR (dPCR) assays for ctDNA molecular residual disease (MRD) detection in early-stage triple-negative breast cancer. We compared tumor-informed dPCR assays with tumor-informed personalized multimutation sequencing assays in 141 patients from cTRAK-TN. Results: MRD was first detected by personalized sequencing in 47.9% of patients, 0% first detected by dPCR, and 52.1% with both assays simultaneously (P < 0.001; Fisher exact test). The median lead time from ctDNA detection to relapse was 6.1 months with personalized sequencing and 3.9 months with dPCR (P = 0.004, mixed-effects Cox model). Detection of MRD at the first time point was associated with a shorter time to relapse compared with detection at subsequent time points (median lead time 4.2 vs. 7.1 months; P = 0.02). Conclusions: Personalized multimutation sequencing assays have potential clinically important improvements in clinical outcome in the early detection of MRD.

Characteristics of the ChemoNear patient population selected for pilot study.

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.

Representativeness of the study population

Comparison of median lead time from ctDNA detection to relapse between dPCR and RaDaR assays using paired data in all individuals with relapse (n=40).

Background: Detection of circulating tumour DNA (ctDNA) in patients (pts) who have completed treatment for early-stage breast cancer is associated with a high risk of future relapse. Identifying those at high risk of subsequent relapse may allow tailoring of further therapy to delay or prevent recurrence. Previous analysis of this cohort showed that tools capable of detecting ctDNA at lower concentrations are needed to increase sensitivity and lengthen the lead time between ctDNA detection and relapse. We compared ctDNA detection via a personalised sequencing assay to dPCR in patients from the cTRAK TN clinical trial. Methods: The cTRAK-TN trial recruited 161 pts into prospective ctDNA surveillance with dPCR, with ctDNA positive pts randomised to 1) CT staging plus pembrolizumab therapy for patients without relapse or 2) observation. Pts had serial post-treatment surveillance plasma samples collected every 3 months for up to 2 years. Whole exome sequencing (WES) was performed on tumor DNA from FFPE samples to design personalised Residual Disease and Recurrence (RaDaR®) multiplex PCR based NGS assays. Retrospectively, plasma DNA extracted from a minimum of 2mls banked plasma, was sequenced with personalised RaDaR assays, and ctDNA detection identified with a proprietary algorithm. dPCR assays tracked 1-2 mutations, as previously described. Primary endpoint was rate of positive ctDNA detection by 12 months from start of surveillance in both assays. Secondary endpoints were agreement in ctDNA detection between RaDaR and dPCR assays and lead-time between ctDNA detection and disease recurrence. Results: Overall, 147 pts and 241 tissue samples were subject to WES, and RaDaR assays were developed for 142 pts with sufficient plasma for testing. RaDaR assays tracked a median of 47 variants (range 33-56) per patient, and a total of 907 timepoints were analysed (median 6 timepoints per pt, range 1-11). With RaDaR, 39.4% (56/142) patients tested ctDNA positive during follow-up, with a median ctDNA detected level of 0.081% estimated variant allele fraction (eVAF). With dPCR, 35.2% (50/142) pts tested ctDNA positive. The ctDNA detection rate by 12 months from the start of ctDNA surveillance was 36.2% (95% CI; 27.6% – 43.7%) with RaDaR and 29.9% (95%CI; 21.6% – 37.3%) with dPCR. The overall test agreement between RaDaR and dPCR assays was 92.7% (95%CI; 90.7% – 94.4%). From a patient perspective, 58.7% pts were ctDNA negative for both assays, 32.9% ctDNA were positive for both assays and 8.6% presented discrepancies. ctDNA was detected by RaDaR but not by dPCR in 9 pts and it was detected by dPCR but not by RaDaR in 3 pts. Among ctDNA positive pts, 55.2% were first detected positive by RaDaR, 5.2% by dPCR, and 39.6% were detected at the same time-point (test of proportions, p< 0.001). The median lead time from ctDNA detection to relapse was 7.1 months (95% CI 5.9 – 15.9%) with RaDaR and 5.7 months (95% CI 3.2% – 7.4%) with dPCR. Conclusion: The RaDaR personalised multi-mutation sequencing assay detected MRD with a longer median lead time prior to relapse, and with higher sensitivity, than dPCR mutation tracking assays. These findings have implications for the choice of ctDNA assay in clinical trials designed to treat patients at the point of MRD detection. Citation Format: Maria Coakley, Prithika Sritharan, Guillermo Villacampa, Claire Swift, Kathryn Dunne, Lucy Kilburn, Katie Goddard, Patricia Rojas, Andy Joad, Warren Emmett, Charlene Knape, Karen Howarth, Peter S. Hall, Catherine Harper-Wynne, Tamas Hickish, Iain Macpherson, Alicia F. Okines, Andrew M. Wardley, Duncan Wheatley, Simon Waters, Rosalind Cutts, Isaac Garcia-Murillas, Judith Bliss, Nicholas Turner. PD5-03 Comparison of a personalized sequencing assay and digital PCR for circulating tumor DNA based Molecular Residual Disease detection in early-stage triple negative breast cancer in the cTRAK-TN trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-03.