Importance:Molecular residual disease (MRD) detection has potential to transform the selection of adjuvant therapy. Most evidence is from tumor-informed assays. Tissue-free assays not requiring sequencing of the primary tumor may simplify workflows if they offer accuracy similar to that of tumor-informed assays. Objective:To evaluate tissue-free circulating tumor DNA (ctDNA) analysis in patients with triple negative breast cancer (TNBC) and compare the tissue-free assay with tumor-informed assay results. Design, Setting, and Participants:This was a prognostic and exploratory analysis of patients with TNBC at moderate to high risk of recurrence who were participating in the ctDNA surveillance period of c-TRAK TN, a multicenter phase 2 clinical study. Plasma samples were collected from participants every 3 months for up to 2 years after the completion of adjuvant therapy and analyzed prospectively with digital polymerase chain reaction (dPCR). Data for the analysis were from a database lock on September 28, 2021, with subsequent follow-up through January 18, 2023. Data were analyzed from July 2025 to May 2026. Intervention or Exposure:Tissue-free assay leverages cancer differential methylation patterns to detect ctDNA. Main Outcome and Measures:Recurrence-free survival by tissue-free ctDNA detection status. Comparison between tissue-free ctDNA detection and both dPCR and whole-exome sequencing-powered multivariant tumor-informed assays. Results:The analysis included 1026 plasma samples from 159 patients (mean [SD; range] age, 51.4 [11.4; 25.0-78.0] years; 159 females [100%]). The tissue-free assay detected ctDNA in 54 patients (34.0%), with detection strongly associated with risk of recurrence (HR, 27.2; 95% CI, 13.7-54.2; P < .001). Among patients with ctDNA detected by both the tissue-free assay and dPCR (42 patients [26%]), tissue-free detection occurred at an earlier time point in 14 patients (33.3%); dPCR detection occurred before tissue-free detection in no patients. Median lead time to recurrence was 7.9 (95% CI, 6.1-10.5) months for tissue-free vs 5.8 (95% CI, 3.3-10.0) months for dPCR (HR, 0.57; 95% CI, 0.34-0.95; P = .03). Concordance between tissue-free and multivariant tumor-informed assays was good. Among patients detected by both assays (41 of 133), 12 of 41 (29.3%) had ctDNA detected earlier by multivariant tumor-informed assay with 1 (2.4%) earlier by the tissue-free assay. Median lead times to recurrence were 7.6 (95% CI, 4.6-10.5) months with tissue-free and 7.1 (95% CI, 5.7-10.0) months with multivariant tumor-informed assay (HR, 1.46; 95% CI, 0.87-2.44; P = .15). Conclusion and Relevance:In this prognostic study, tissue-free ctDNA detection during surveillance was strongly prognostic for recurrence in patients with TNBC. Comparable lead times between the tissue-free and multivariant tumor-informed assays support tissue-free MRD detection in a clinical trial setting, permitting ctDNA testing when tissue is not available.

Baseline characteristics comparison between our ctDNA analysis population and the overall plasmaMATCH population, (A) Cohorts A-D and (B) (Cohort E)

500 Background: Tumor gene expression tests are widely used to assist adjuvant chemotherapy decisions for women with early breast cancer (EBC). OPTIMA (Optimal Personalised Treatment of early breast cancer using Multi-parameter Analysis) is an international RCT comparing chemotherapy decisions made with the Prosigna (PAM50) gene expression test with standard treatment in mostly node-positive patients. Methods: Women and men aged >40 recommended to receive chemotherapy for ER+ HER2- EBC with 0-9 involved axillary nodes and T size >30mm if node negative were eligible. Randomization was between standard chemotherapy followed by endocrine therapy (CET) or to a Prosigna test directed chemotherapy decision. Patients with Prosigna ROR score > 60 tumors were assigned CET whilst those with low ROR score (≤60) tumors received endocrine therapy (ET) alone. ET for premenopausal women included ovarian function suppression (OFS) in the absence of chemotherapy-induced ovarian insufficiency. ROR scores were not disclosed, and patients receiving CET were blinded to their randomization. OPTIMA was designed to demonstrate non-inferiority (NI) of 5-year invasive breast cancer free survival (IBCFS) in the test-directed arm with a 3% margin in the per protocol (PP) population using a 5% 1-sided alpha. Control arm testing allowed treatment comparison within the low ROR score population at a 3.5% NI margin. Results: From 16 th Jan 2017 to 12 th Dec 2025 4429 patients were randomized, 2215 to the control arm and 2214 to the test-directed arm of whom 2061 (93%) and 2097 (95%) were included in the respective PP group. Patient characteristics in the PP population were well-balanced; 62% were postmenopausal, 37% premenopausal and 0.8% male. 73% had pN1/pN1sn, 8% had pN0/pN1mi and 19% had pN2 tumors. 68% had low ROR score tumors. With a median follow-up of 3.9 years (interquartile range 2.0-5.9), 280 IBCFS events occurred (141 on control arm; 139 on test directed arm) of which 66% were distant recurrences. The 5-year IBCFS rate in the control arm was 91.5% [95% CI 89.7- 92.9%] and 90.4% [95% CI 88.6- 92.0%] in the test-directed arm, Hazard Ratio (HR) 0.99 [90% CI 0.81- 1.20], NI p = 0.013, thereby meeting the pre-defined NI margin. The corresponding 5-year IBCFS rates for the low ROR score population were 94.9% [95% CI 92.9- 96.4%] and 93.7% [95% CI 91.8- 95.2%] for the two arms respectively, HR 1.06 [90% CI 0.78- 1.46] NI p = 0.0051 again demonstrating non-inferiority. There was no significant outcome heterogeneity between subgroups including for menopausal and nodal status. Conclusions: The OPTIMA trial demonstrates that women and men with ER+ HER2- EBC and ROR score ≤60 tumors can safely avoid chemotherapy. It provides evidence for the utility of test-directed chemotherapy in premenopausal women treated with OFS and patients with high levels of nodal involvement. Clinical trial information: ISRCTN42400492.

Prediction of treatment benefit from on treatment ctDNA dynamics optimal cutpoint in cohorts A-D

Boxplot graph showing distribution of CDR C2D1

Supplementary Figure 5: Boxplot graph showing distribution of CDR C2D1 values in patients in cohort E by BRCA status

C1D1 variants for BRCA1/2 wildtype patients with undetectable ctDNA at C2D1 (CDR = 0) (N=6)

PURPOSE:ctDNA dynamic levels may identify patients who will respond to therapy. We assessed ctDNA baseline levels and on-treatment dynamics in patients with advanced breast cancer on the plasmaMATCH trial with mutation-targeted therapies (cohorts A-D) and triple-negative breast cancer on olaparib and ceralasertib combination (cohort E). EXPERIMENTAL DESIGN:Blood samples were collected at baseline [cycle 1 day 1 (C1D1)] and before treatment on cycle 2 day 1 (C2D1). Samples were sequenced using error-corrected targeted panels (Guardant360/GuardantOMNI). Circulating DNA ratio was calculated as the ratio of C2D1/C1D1 circulating DNA ratio, and baseline ctDNA levels were associated with progression-free survival (PFS) and confirmed objective response rates (ORR). RESULTS:A total of 167 patients had assessable C1D1-C2D1 ctDNA results. There was a strong association between baseline ctDNA levels and response in cohort E; low baseline levels were associated with longer PFS (HR, 0.33; P = 0.001) and higher ORR (40% vs. 9.7%; P = 0.02). In cohorts A to D, there was a weaker association with PFS (HR, 0.60; P = 0.03) and ORR (15.2% vs. 5.7%; P = 0.17). Associations of baseline ctDNA level and ORR were validated in the independent PEARL study. For on-treatment dynamics, suppression of ctDNA below median was predictive in cohorts A to D (HR, 0.47; P = 0.001) but not in cohort E (HR, 1.02; P = 0.94). Undetectable ctDNA levels at C2D1 were associated with good outcomes in both cohorts: in cohort E with improved PFS (HR, 0.25; P = 0.01) and improved ORR (86% vs. 11%; P = 0.01). Six of seven patients with undetectable on-treatment ctDNA were BRCA1/BRCA2/PALB2 wild type. CONCLUSIONS:Baseline low ctDNA levels predict response to targeted therapy, potentially suggesting shared mechanisms between high ctDNA release and resistance to therapy. Both baseline ctDNA levels and on-treatment dynamics are a promising surrogate endpoint for drug development, with clearance of ctDNA being a robust cross-therapy surrogate for outcomes.

Variant Allele Fraction of CHIP genes at different timepoints

Alternative methodologies evaluated for the calculation of CDR and PFS predictions (cohorts A-D)

List of the 74 genes included in the Guardant360 panel used for sequencing

Background De-escalation trials aim to balance clinical outcomes and quality of life (QoL), especially for premenopausal women with early breast cancer (BC), who often experience a greater treatment burden affecting their physical and psychosocial well-being. We sought to understand their patient journey—barriers, facilitators, and expectations—when joining de-escalation trials to inform patient-centered design and implementation. Methods OPTIMA-YOUNG, an EU-co-funded international trial, investigates a genomic assay-guided approach to de-escalate chemotherapy in premenopausal patients with early-stage hormone receptor (HR)+ BC. A Co-creation Board of patients and healthcare professionals (HCPs) ensured stakeholder involvement throughout the trial. Focus groups (FGs) explored decision-making, needs, and QoL priorities related to joining a de-escalation clinical trial. Discussions were recorded, transcribed, anonymized, and analyzed using MAXQDA software. Results The three FGs included 20 participants (11 patients, 9 HCPs) from 14 countries. Three themes emerged: 1) emotional and cognitive responses to BC diagnosis/treatment; 2) environmental, HCP, and social influences on choices; 3) coping with side effects and QoL challenges. Barriers to joining a de-escalation trial included limited emotional support at diagnosis, cross-country variations in shared decision-making, poor communication on long-term side effects, fear of recurrence, and HCPs’ tendency to overtreat younger patients. Developing training for patients and HCPs was seen essential for improving communication, shared decision-making skills, and enhancing symptom management and QoL. Conclusions This pre-implementation study identified factors at the patient, HCP, and system levels that, if addressed, could improve the trial experience. Insights helped refine the OPTIMA-YOUNG protocol and implementation plan and may inform future de-escalation trials.

Understanding mechanisms that facilitate early events in metastatic seeding is key to developing therapeutic approaches to reduce metastasis. Here we identify uracil as a metastasis-associated metabolite in genetically engineered mouse models of cancer and in patients with metastatic breast cancer. Uracil is generated by the enzyme uridine phosphorylase-1 (UPP1), and we find that neutrophils are a significant source of UPP1 in metastatic cancer. Mammary tumours increase expression of adhesion molecules on the neutrophil surface, in a UPP1-dependent manner, leading to decreased neutrophil motility in the pre-metastatic lung. UPP1-expressing neutrophils suppress T-cell proliferation, and the UPP1 product uracil increases fibronectin deposition in the extracellular microenvironment. Knockout or inhibition of UPP1 in mice with mammary tumours increases T-cell numbers and reduces fibronectin content in the lung, and decreases the proportion of mice that develop lung metastasis. These data indicate that UPP1 influences neutrophil behaviour and extracellular matrix deposition in the lung, and suggest that circulating uracil could be a marker of metastasis, and that pharmacological inhibition of UPP1 could be a strategy to reduce recurrence.

Introduction: Activation of the PI3K-AKT and mTOR pathways is a major feature of the biology of breast cancer (BC), with these pathways altered or dysregulated in most BC. Oncogenic mutations in AKT1, most frequently E17K, are found in ∼5% of advanced BC and are targetable by the approved AKT inhibitor capivasertib. We conducted genomic and transcriptomic analysis of a cohort of AKT1 mutant metastatic BCs with the aim to identify determinants of response to capivasertib. Methods: We identified 39 AKT1 mutant BC from the plasmaMATCH clinical trial (18/39; NCT03182634), the FAKTION clinical trial (7/39 NCT01992952), and the ABC-BIO tissue collection study (14/39; CCR3991). FFPE extracted DNA was subject to whole exome sequencing (WES), and RNA to Truseq RNA sequencing. Clonal dominance was assessed in circulating tumour DNA (ctDNA) using Guardant360. Allelic imbalance at the AKT1 locus and clonal dominance of the AKT1 mutation were associated with progression free survival (PFS) in plasmaMATCH Cohorts C (capivasertib plus fulvestrant) and D (capivasertib alone) combined. Results:WES at median coverage 93x (range 55-169x) reconfirmed AKT1 mutations in 33/39 (85%) samples: 25/33 (76%) E17K; 3/33 (12%) L52R; 1/33 (3%) E49K; 1/33 (3%) L52R+C77F; 1/33 (3%) L52R+E375K; 1/33 (3%) S129L; 1/33 (3%) Q79K. 6/39 discordant samples principally reflected AKT1 mutations identified in ctDNA analysis which were not present in the single site tissue biopsy. Most common pathogenic alterations found in the AKT1 mutated cohort were TP53 9/33 (27%), CDH1 5/33 (15%), GATA3 3/33 (9%) and MAP3K1 2/33 (6%). Rates of mutations in PIK3CA (2/33; 6%) and ESR1 (2/33; 6%) were reduced compared to those expected in ER+HER- metastatic BC sets. Analysis of the AKT1 locus revealed amplification of AKT1 gene in 14/33 (42%) and loss of heterozygosity (LOH) of the wild type allele in 10/33 (30%). Overall LOH and/or amplification (allelic imbalance) was observed in 18/33 (55%) of AKT1 mutant tumours. 15/33 (46%) of the patients with confirmed AKT1 mutations had received treatment with capivasertib in PlasmaMATCH. Median PFS in patients was 13.08 months (10/15 patients) with allelic imbalance and 3.19 months without allelic imbalance (5/15 patients; HR 8.798, 95% CI1.58-49.00, P=0.004, log rank test). RNA sequencing data was obtained for 36/39 samples with median 13M reads (range 11-49M) including 31/33 of the confirmed mutant samples in WES. PAM50 subtypes for the cohort were LumA (12/31), LumB (7/31), HER2Enriched (7/31), Basal (1/31) and normal-like (2/31). RNAseq showed increased (0.87 log fold change, P=0.035) expression of AKT1 in tumours with allelic imbalance compared to those without, and high expression levels of mutant transcript. Finally, in plasmaMATCH, clonality of the AKT1 mutation was assessed in ctDNA, with 16/24 (67%) patients having clonally dominant AKT1 mutations. Median PFS in patients with clonally dominant mutations was 10.2 months and subclonal mutations 3.2 months (HR 3.1, 95% CI0.9-10.5, P=0.014, log rank test). Conclusions: Breast cancers with mutations in AKT1 frequently have allelic imbalance favouring the mutation, resulting in increased expression of mutant transcript. Allelic imbalance of AKT1, and/or clonally dominant AKT1 mutations, were prognostic for significantly greater PFS of patients treated with capivasertib. Citation Format: Sarah Mearns, Alex Pearson, Ros Cutts, Heena Shah, Li-Xuan Sim, Belinda Kingston, Kathryn Dunne, Marta Lubowiecki, Andriani Hadjiconstanti, Hannah Johnson, Lucy Kilburn, Laura Moretti, Andrew M. Wardley, Iain R. Macpherson, Richard D. Baird, Rebecca Roylance, Angela Casbard, Margherita Carucci, Sacha J Howell, Robert H Jones, Judith Bliss, Alistair Ring, Alistair Ring. Predicting response to capivasertib in AKT1 mutant advanced breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-01-19.

Altered cellular metabolism has been associated with the acquisition of invasive phenotypes during metastasis. To study this, we combined a genetically engineered mouse model of mammary carcinoma with syngeneic transplantation and primary tumor resection to generate isogenic cells from primary tumors and their corresponding lung micrometastases. Metabolic analyses indicated that micrometastatic cells increase proline production at the expense of glutathione synthesis, leading to a reduction in total glutathione levels. Micrometastatic cells also have altered sphingomyelin metabolism, leading to increased intracellular levels of specific ceramides. The combination of these metabolic adaptations alters extracellular vesicle (EV) production to render the microenvironment more permissive for invasion. Indeed, micrometastatic cells shut down Rab27-dependent production of EVs and, instead, switch on neutral sphingomyelinase-2 (nSM2)-dependent EV release. EVs released in an nSM2-dependent manner from micrometastatic cells, in turn, influence the ability of fibroblasts to deposit extracellular matrix, which promotes cancer cell invasiveness. These data provide evidence that metabolic rewiring drives invasive processes in metastasis by influencing EV release.

Background When taken as prescribed, endocrine therapy is effective in reducing risk of recurrence and mortality in the treatment of patients with breast cancer. However, treatment side effects can act as a barrier to medication adherence. Existing research has not identified any specific side effects as consistent predictors of nonadherence. Our aim was to explore the influence of symptom clusters on self-reported adherence in patients with breast cancer. Methods A cross-sectional online survey was conducted, including patients with breast cancer currently or previously prescribed endocrine therapy (N=1051). This included measures of self-reported endocrine therapy adherence and common symptoms among this population (insomnia, depression, anxiety, fatigue, musculoskeletal, and vasomotor symptoms). Results Unintentional nonadherence was higher than intentional nonadherence (50.8% vs 31.01%). The most troublesome symptom was insomnia (73.83% displayed probable insomnia disorder). K-means cluster analysis identified 2 symptom clusters: overall High symptoms, and overall Low symptoms. Participants in the Low symptoms cluster were significantly more likely to be classed as adherent based on unintentional and intentional items. Conclusions Nonadherence was high in the current sample, and significantly more likely in participants reporting overall severe symptoms. Clinicians should be aware of the scale of common side effects and facilitate open conversation about potential barriers to adherence. Follow-up care should include assessment of common symptoms and signpost patients to appropriate support or treatment when required. Future research should explore potential for a central symptom to act as a target for intervention, to relieve overall side effect burden and facilitate better medication adherence.

Purpose: The gut microbiome has been shown to influence the efficacy of some systemic anti-cancer therapies. This prospective study explored the ability of the pre-treatment gut microbiome to predict pathological complete response (pCR) in patients receiving neoadjuvant chemotherapy (NACT) for early breast cancer.

Abstract Background The presence or absence of residual disease following neoadjuvant systemic anti-cancer therapy (neoSACT) for HER2-positive early breast cancer (HER2+ EBC) provides powerful prognostic information. Pathological complete response (pCR) following neoSACT, particularly in patients with earlier clinical TNM stage at diagnosis, identifies a population with excellent survival outcomes in whom the balance of toxicity associated with the current treatment pathway may be disproportionate to absolute clinical benefit. Aims >HER2-RADiCAL seeks to reduce the treatment burden and healthcare costs of treating HER2+ EBC by testing the hypothesis that pCR can be used as a functional response biomarker to select patients who can safely receive less systemic therapy with minimal/no loss of efficacy. Trial design and eligibility criteria HER2-RADiCAL is a response-directed interventional cohort/threshold-crossing design study embedded within a real-world data-driven clinical pathway model. Patients with ER-positive or negative HER2+ EBC with cT1N1 or cT2N0-1 stage at diagnosis are eligible after completion of standard of care neoSACT and locally determined pCR (ypT0/Tis ypN0). Patients with pathological features consistent with previous malignant involvement in >4 nodes are not eligible. Adjuvant trastuzumab +/- pertuzumab may have continued prior to study entry provided no more than 9 cycles of trastuzumab are received. After registration all participants receive a total of 9 cycles of trastuzumab including those administered prior to study entry. Participants receive no further pertuzumab and no adjuvant chemotherapy. Adjuvant endocrine therapy and radiotherapy are given as per standard of care. Central pathology review of pCR status will be conducted in a subset of cases. A Study Within a Trial (SWAT) will explore identification of factors influencing patient decision-making for participation in studies of response-adapted treatment. Statistical methods The primary endpoint is relapse-free interval. Recruitment of 720 participants over 3.5 years will provide 90% power to exclude an event rate >6.5% at 3 years (expected event rate ≤4%). Secondary endpoints include relapse-free survival, invasive breast cancer-free survival, invasive disease-free survival, distant recurrence-free interval, breast cancer-free interval, overall survival, treatment pathway adherence and cost-effectiveness. An interventional cohort is preferred to a randomised non-inferiority trial based on the known event rate in this population which is sufficiently low such that any deviation from this expected event rate would indicate failure of the reduced therapy strategy. Real-world data driven clinical pathway model Health economic modelling will compare the protocol-driven study cohort with two comparator pathways: a non-response adapted maximum therapy pathway (the standard clinical pathway prior to the study) and a real-world representative pathway derived from anonymised data for all patients treated for HER2+ EBC within the UK NHS. Patient and public involvement Our PPI partners continue to shape this research, holding key roles as trial management group members, overseeing progress of the study, contributing to protocol amendment development, and design and implementation of the SWAT. Current status The first patient was enrolled in December 2021. At 10th July 2023, 25 research sites have opened with 39 participants recruited. Site opening continues with a minimum target of 40. Responding to continued use of anthracycline-based regimens in UK practice, eligibility criteria have recently been extended to permit entry of patients who have received anthracycline-containing neoSACT. For further information contact her2radical-icrctsu@icr.ac.uk. Citation Format: Iain Macpherson, Stuart McIntosh, Lucy Kilburn, Holly Tovey, Laura Moretti, Katie Goddard, Indrani Bhattacharya, Clinton Boyd, Cliona Kirwan, Ciara O'Brien, Duncan Wheatley, Jennifer Glendenning, Mairead MacKenzie, Hilary Stobart, Claire Snowdon, Andrew Wardley, Abeer Shaaban, Peter Hall, David Cameron, Judith Bliss. The HER2-RADiCAL study (Response ADaptive CAre pLan) – Tailoring treatment for HER2 positive early breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-19-01.