BACKGROUND AND PURPOSE:Brain [18F]Fluoroestradiol (FES) PET enables noninvasive assessment of estrogen receptor (ER) expression and may improve detection of ER-positive (ER+) breast cancer brain metastases (BC-BM), with pilot studies demonstrating clinical utility. Our aim was to leverage our dynamic brain [18F]FES PET acquisition protocol to characterize BC-BM uptake kinetics across different post-injection intervals, with the goal of improving lesion detectability. MATERIALS AND METHODS:Patients with ER+ primary disease and BC-BM were enrolled prospectively and underwent dynamic [18F]FES PET/CT and contemporaneous standard-of-care contrast-enhanced brain MRI. PET was acquired over 90 minutes using a dynamic protocol (34 frames). Four 15-minute static time windows were reconstructed including 30-45, 45-60, 60-75, and 75-90 minutes post-injection, respectively. MRI was used as gold standard for clinical lesion characterization and segmentation, with subsequent [18F]FES PET co-registration. Time-window-specific PET-MRI lesion matching rate (LMR) and mean standardized uptake value ratio (SUVR, normalized to cerebellar cortex) were calculated. RESULTS:Eleven patients with a total of 92 MRI-confirmed lesions were included (73 parenchymal, 8 leptomeningeal, 6 calvarial, 4 dural-based, and 1 pituitary stalk). Time window-3 yielded the highest LMR at 68% (63/92 lesions), followed by time window-2 (62%), time window-4 (60%), and time window-1 (57%). Mean SUVR increased across time, with means of 1.83 ± 1.46, 2.08 ± 1.88, 2.29 ± 2.37, and 2.38 ± 2.50 for time window-1, -2, -3, and -4, respectively. Despite higher SUVR at later time points, 8 lesions (6 parenchymal, 1 leptomeningeal, and 1 calvarial) that were visible at 60-75 min were undetectable at 75-90 min. CONCLUSION:Time window-3 at 60-75 minutes post-injection provided optimal LMR for brain [18F]FES PET in ER+ BC and suspected BM. Imaging beyond this interval may not further improve diagnostic yield and may reduce visibility. Limitations of this study include small cohort size, population heterogeneity with regard to prior treatment, and lack of pathologic confirmation. Our findings suggest that 60-75 minutes post-injection may represent the optimal acquisition time window and highlight the importance of timing standardization for neuro-oncologic applications of brain [18F]FES PET.

e15060 Background: In metastatic Breast Cancer (mBC) Circulating Tumor Cells (CTCs) are an established prognostic indicator of patients (pts) with highly aggressive disease, poor clinical outcomes, and poor response rates to therapy. However, CTCs are typically found in < 20% of mBC pts and many pts without CTCs also progress. Cancer associated macrophage-like cells (CAMLs) are inflammatory tumor macrophages also found in blood, in > 90% of mBC, and are indicators of poor clinical outcomes independent of CTCs. We conducted a prospective study to model CTC & CAML subtypes from 212 mBC pt blood samples prior to induction of new systemic therapies to optimize and validate risk models based on 2-year outcomes of Progression-free survival (PFS) & Overall survival (OS). Methods: An observational multi-institutional prospective study was conducted on 212 mBC pts prior to induction of a new line of therapy who were progressing on their current therapy. Anonymized (7.5ml) whole blood samples were taken prior to therapy induction and filtered by CellSieve filtration. Quantities and subtypes of CTCs & CAMLs were categorized by their expression of CK+/CD45+/CD14+ for CAMLs and CK+/CD45-/CD14- for CTCs. PFS (RECIST v1.1) and OS hazard ratios (HRs) were calculated by censored univariate & multivariate analysis at 2 years. Results: Pt median age was 51 years (range 25-92); TNBC (44%), ER/PR+ (30%), HER2+ (15%). Pts were treated with chemotherapy alone (18%), immune checkpoint inhibitor (46%), targeted (40%), hormone (8%) and unknown (5%). 38% (81/212) pts had ≥1 CTCs which correlated with significantly poorer PFS (HR = 1.9; 95%CI 1.3-2.7; p = 0.0016) and OS (HR = 2.0; 95%CI, 1.3-3.0; p = 00025), with the most significant risk at ≥8 CTCs (14% pts), PFS (HR = 3.8; 95%CI, 2.1-6.9; p < 0.0001) and OS (HR = 7.2; 95% CI, 3.4-14.9; p < 0.0001). Independently, 93% (n = 196/212) of pts had ≥1 CAML, with a threshold of >40um size (84% pts) had significantly poorer PFS (HR = 2.0; 95% CI, 1.2-3.1; p = 0.0032), but not OS (HR = 1.5; 95%CI, 0.9-2.7; p = 0.1169). However, a threshold of >125um size CAML (43% pts) also had significantly poorer PFS (HR = 1.8; 95% CI, 1.2-2.7; p = 0.0013) and OS (HR = 1.6; 95% CI, 1.0-2.5; p = 0.0233). By combining models, the best outcomes were seen in pts with 0 CTCs & 0 CAMLs (mPFS = 12.6 & mOS = 21.3), followed by 0 CTCs & ≥40um CAMLs (mPFS = 5.2 & mOS = 17.5) and 0 CTCs & ≥125um CAMLs (mPFS = 4.9 & mOS = 15.2). Poorer outcomes were seen with ≥1 CTCs (mPFS = 3.3 & mOS = 13.5) and the worst outcomes with ≥8 CTCs (mPFS = 2.5 & mOS = 4.8). Conclusions: CTCs were uncommon in mBC pts but correlated with very poor clinical outcomes. In parallel analysis, CAMLs were common with CAML size correlating with increasingly poorer outcomes. By combining CTC & CAML subtypes, mBC pts were more accurately stratified by risk of progression and death. Additional multivariate studies correlating treatment class and tumor response rates are ongoing.

e13140 Background: ESR1 mutations emerge during endocrine therapy and are associated with endocrine resistance in HR+/HER2-metastatic breast cancer (mBC). The independent prognostic value of ESR1 mutations in patients starting second-line (2L) therapy remains unclear in real-world settings. Methods: We conducted a retrospective cohort study using Flatiron Health EHR-derived de-identified database (range: 02/01/2015-12/31/2024) of patients with HR+/HER2- mBC receiving first-line (1L) CDK4/6 inhibitor plus aromatase inhibitor and had ESR1 testing within 90 days of 2L therapy initiation. ESR1 status was classified as Mutated (any positive test within window), or Not Detected (ND, negative test with no prior positive). Primary endpoints were real-world progression-free survival (rwPFS; composite of progression or death) and overall survival (rwOS). Multivariable Cox proportional hazards models adjusted for age, ECOG, calendar year, demographics, metastatic burden, prior adjuvant AI, treatment-free interval,1L duration, practice setting, and sample type (Model A), with additional adjustment for 2L treatment (Model B). Stratified analyses by sample type (blood vs tissue) were also performed. Results: Of 40,437 patients identified, 480 met eligibility criteria with valid ESR1 results (170 ESR1 Mutated [35.4%], 310 ESR1 ND [64.6%]). Blood samples comprised 56% of tests, tissue 42%, other 2%. Patients with ESR1 mutation detected had significantly longer treatment-free interval (median 34 vs 10 months, p = 0.023) and 1L duration (20 vs 12 months, p < 0.001) than patients without ESR1 mutation detected. Median rwPFS was 5.9 months (95% CI: 5.1-9.0) for ESR1 Mutated vs 6.2 months (5.3-8.5) for ESR1 ND; median rwOS was 19.4 months (17.0-26.4) vs 24.0 months (19.3-30.5). In multivariable models, ESR1 Mutated status was associated with significantly worse rwPFS (Model A: HR = 1.60 [1.11-2.31], p = 0.011; Model B: HR = 1.79, [1.21-2.64], p = 0.004) and rwOS (Model A: HR = 1.76, [1.14-2.72], p = 0.011; Model B: HR = 1.93, [1.21-3.09], p = 0.006). Sample type stratification demonstrated the prognostic effect was significant in blood samples (rwPFS Model B: HR = 2.27, [1.36-3.78], p = 0.002; rwOS Model B: HR = 2.09, [1.26-3.47], p = 0.004) but not in tissue samples (rwPFS Model B: HR = 1.20, [0.60-2.39],p = 0.60; rwOS Model B: HR = 1.77, [0.77-4.09], p = 0.18). Conclusions: In this real-world cohort of patients with HR+/HER2- mBC, ESR1 Mutated status demonstrated significant prognostic value for worse rwPFS and rwOS after 1L CDK4/6i plus AI therapy, with effects independent of 2L treatment choice. Sample type stratification revealed that the prognostic signal was driven by blood-based testing, with no effect observed in tissue samples. These results have important implications for interpretation of ESR1 status.

11130 Background: Accurate ctDNA-based MRD detection in early breast cancer is limited by tumor heterogeneity, clonal evolution and methodological issues, limiting tumor-informed (TI) assays reliant on predefined variants. Tumor-agnostic (TA) approaches, while broader, are susceptible to false positives from clonal hematopoiesis and reduced sensitivity in low–tumor burden settings. Methods: We analyzed a single-center cohort of 74 female patients with stage I–III breast cancer undergoing post-treatment surveillance after completion of curative-intent therapy (surgery, radiation, and adjuvant systemic therapy as appropriate). Plasma samples collected post-treatment were analyzed using an integrated MRD assay combining TI ddPCR and TA NGS ctDNA detection strategies. CHIP filtering was incorporated to improve analytical robustness. MRD status was correlated with clinicopathologic features and contemporaneous imaging. MRD positivity was defined as detection by either TI or TA component. Results: At the time of ctDNA assessment, which was performed during standard post-treatment surveillance following completion of curative-intent therapy, 94.6% (70/74) of patients were imaging-negative. MRD was detected in 31.1% (23/74) of patients overall. All imaging-positive patients (4/4) were MRD-positive. Notably, among imaging-negative patients, 27.1% (19/70) were MRD-positive, indicating molecular residual disease not captured by conventional imaging. No imaging-positive cases were MRD-negative. MRD positivity was observed across all stages, including early-stage disease (I–II: 26%, 14/54), with the highest prevalence in stage III (47.4%, 9/19); one case had unknown staging. MRD was detected in 33.3% (18/54) of HR + /HER2 - , 25% (2/8) of TNBC, and 25% (3/12) of HER2 + tumors, demonstrating applicability across biological subtypes. Importantly, the hybrid approach increased detection sensitivity significantly. Among MRD-positive patients (n=23), 47.8% were detected exclusively by TA component, 43.5% by the TI component alone, and only 8.7% were detected by both. Reliance on a single MRD strategy would have failed to identify 91.3% (21/23) of MRD-positive cases, highlighting substantial biological complementarity between the two approaches. At early follow-up (median 113.5 days), MRD was detected in 23 patients, including 19 who were imaging-negative; among those with subsequent imaging-confirmed recurrence (n=4), the median MRD–imaging interval was 9.75 months. Conclusions: A hybrid TI and TA ctDNA MRD platform detects clinically occult molecular disease beyond imaging in stage I–III breast cancer. The complementary performance of TI and TA approaches substantially improves MRD detection and supports the use of hybrid MRD testing for early risk stratification and MRD-guided clinical trials.

PURPOSE:Gedatolisib potently targets all four class I PI3K isoforms and mTORC1 and mTORC2 to comprehensively block the PI3K/AKT/mTOR pathway and has shown compelling activity in early clinical trials with palbociclib and fulvestrant. METHODS:This phase III randomized trial (VIKTORIA-1; ClinicalTrials.gov identifier: NCT05501886) evaluated the efficacy of gedatolisib-based therapy, comparing gedatolisib, palbociclib, and fulvestrant (gedatolisib triplet) and gedatolisib plus fulvestrant (gedatolisib doublet) with fulvestrant monotherapy in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative (HER2-), PIK3CA wild-type (WT) advanced breast cancer. Eligible patients had disease progression during or after CDK4/6 inhibitor and aromatase inhibitor treatment. Comparison of progression-free survival as assessed by blinded independent central review for gedatolisib triplet versus fulvestrant and gedatolisib doublet versus fulvestrant was the primary objective. RESULTS:A total of 392 patients were randomly assigned 1:1:1. The median study follow-up was 10.1 months. The median progression-free survival was 9.3 months in the gedatolisib-triplet group, 2.0 months in the fulvestrant group (hazard ratio [HR] for progression or death, 0.24 [95% CI, 0.17 to 0.35]; P < .001), and 7.4 months in the gedatolisib-doublet group (HR, 0.33 [95% CI, 0.24 to 0.48]; P < .001 v fulvestrant). Grade ≥3 treatment-related adverse events (TRAEs) reported in the gedatolisib-triplet and gedatolisib-doublet groups, respectively, included neutropenia (62.3%, 0.8%), stomatitis (19.2%, 12.3%), rash (4.6%, 5.4%), hyperglycemia (2.3%, 2.3%), and diarrhea (1.5%, 0.8%). Study treatment discontinuation because of TRAEs was reported in 2.3% (triplet) and 3.1% (doublet) of patients. CONCLUSION:The addition of gedatolisib to fulvestrant, with or without palbociclib, significantly reduced the risk of disease progression or death in patients with hormone receptor-positive/HER2-, PIK3CA WT advanced breast cancer.

Metastasis is the leading cause of death in patients with breast cancer (BC), but the mechanisms underlying metastasis formation are still poorly understood. Circulating tumor cells (CTCs) are considered the main seed of metastasis with demonstrated prognostic impact in patients with BC. They are conventionally identified as cells positive for epithelial markers and lacking leukocyte markers. Nonetheless, circulating cells expressing both markers [dual-positive cells (DPcells)] have been reported but poorly investigated. Here, we evaluated, in a cohort of 340 patients with advanced BC, the prognostic impact of DPcells, showing their association with worse survival, particularly in patients with less than five CTCs. Their prognostic value varied among BC subtypes, with greater relevance observed in triple-negative and HER2-positive BC. Moreover, by performing single-cell genomic profiling of DPcells isolated from patients, we detected genomic aberrations in 28 and 93% of analyzed DPcells and CTCs, respectively. In vivo, DPcells were detected only in the blood of immunocompetent but not immunodeficient mice and no differences in the lung metastatic colonization ability of DPcells versus control cancer cells were observed. Our findings highlight the importance of studying this overlooked subpopulation of CTCs as a prognostic biomarker in BC, which might be particularly important in specific BC subtypes. Moreover, our results support the malignancy and metastasis-forming capability of DPcells and underline the need for future studies better defining the origin of these cells.

Endocrine resistance is a complex phenomenon, including alterations of the ESR1 gene. The aim of this study was to simultaneously analyze ESR1 promoter methylation and ESR1 hotspot mutations in circulating tumor cells (CTCs) and paired plasma‐circulating tumor DNA (ctDNA) from patients with estrogen receptor‐positive (ER+) advanced breast cancer (BC). We retrospectively analyzed samples from 42 ER+ advanced BC patients characterized for CTCs and ctDNA at Northwestern University. CTCs were enumerated using the CellSearch® system, while ctDNA was analyzed with the Guardant360 NGS platform. Genomic DNA from CellSearch‐enriched CTC fractions was amplified and analyzed using the ESR1‐NAPA assay. ESR1 methylation analysis was performed in 34 samples. ESR1 mutations were detected in 59.5% CTC‐derived samples, a significantly higher proportion than in paired plasma ctDNA (29.6%). ESR1 methylation was observed in 26.5% patients. Concurrent ESR1 mutations and methylation were identified in six cases, suggesting combined genetic and epigenetic mechanisms of endocrine resistance. Overall, CTC‐derived genomic DNA showed higher sensitivity for detecting ESR1 mutations than plasma ctDNA, supporting the potential value of CTC analysis for characterizing endocrine resistance in advanced BC.

Background/Objectives: Circulating tumor-associated cells (CTACs) are rare among peripheral blood nucleated cells (PBNCs), creating a challenge for image-based multi-cancer detection. We evaluated a predefined CTAC-detection pipeline incorporating Attention U-Net segmentation, post-processing, cytological feature extraction, and Random Forest classification. Methods: Model suitability was explored in asymptomatic individuals and patients with advanced solid tumors. Clinical performance was assessed in a case-control cohort of therapy-naive stage I/II cancers, benign conditions, and asymptomatic individuals, followed by four prospective cohort evaluations performed within the same laboratory and imaging workflow: recurrent cancer with low radiological tumor burden, peri-operative solid tumors, suspected cancer, and asymptomatic screening. PBNCs were stained with EpCAM/Hoechst 33342 and imaged. Pathologists' review established ground truth annotations. Results: The model had 90.68% sensitivity and 99.53% specificity in the exploratory study. In the case-control cohort, sensitivity was 88.65% in therapy-naive stage I/II cancers, while specificity was 78.95% in benign conditions and >99.9% in asymptomatic individuals. In the prospective cohorts, CTAC detection sensitivity was 91.96% in pretreated low tumor burden cases; CTACs were detected in 100% of pre-surgery specimens and 29.41% of post-surgery specimens; and in suspected cancer cases, the Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were 96.34% and 32.35%, respectively. In the asymptomatic screening cohort, 44/7183 participants were CTAC-positive; 16 had confirmed Stage I/II cancer, 10 had no radiologically detectable disease at the available assessment, and 18 remained unresolved. The conservative lower-bound PPV was 36.36%, and the NPV was 99.97%; estimates remain provisional pending complete follow-up. Conclusions: The integrated Attention U-Net/feature-based classification pipeline demonstrated consistent CTAC detection across the evaluated cohorts and supports its potential clinical utility for cancer detection.

LBA4 Background: ESR1 mutations ( ESR1 m) constitutively activate the estrogen receptor (ER) and are the most common mechanism of acquired resistance to aromatase inhibitor (AI) + CDK4/6i. Molecular monitoring by ctDNA analysis can detect the emergence of ESR1 m during 1L AI + CDK4/6i. Camizestrant, the next-generation selective ER degrader (SERD) and complete ER antagonist, has shown anti-tumor activity in pts with and without detectable ESR1 m. SERENA-6 is the first global registrational Phase 3 trial assessing a ctDNA-guided approach to detect the emergence of ESR1 m during 1L AI + CDK4/6i to inform a switch in therapy ahead of disease progression. Methods: Pts with HR+/HER2– ABC who had received ≥6 months of 1L AI (anastrozole/letrozole) + CDK4/6i (abemaciclib/palbociclib/ribociclib) were enrolled and had ctDNA tested for ESR1 m every 2–3 months, coinciding with routine imaging. At ESR1 m detection, pts without evidence of disease progression were randomized 1:1 to switch to camizestrant (75 mg) with continued CDK4/6i (type and dose maintained) + placebo for AI vs continuing AI + CDK4/6i + placebo for camizestrant. The primary endpoint was investigator-assessed PFS (per RECIST v1.1). Prespecified interim analysis data cutoff was Nov 28, 2024. Results: 3,256 eligible pts were surveilled for ESR1 m using ctDNA until 315 eligible pts were randomized to switch to camizestrant (n=157) or continue with AI (n=158). All pts remained on the same CDK4/6i. ~50% of randomized pts had ESR1 m detected at the first ctDNA test. Baseline characteristics were well balanced between treatments. After 171 PFS events, hazard ratio for PFS was 0.44 (95% CI 0.31–0.60, p<0.00001; median PFS 16.0 vs 9.2 months). PFS benefit was consistent across subgroups. PFS rate at 12 months was 60.7% (95% CI 51.1–69.0) vs 33.4% (95% CI 24.9–42.2) and at 24 months was 29.7% (95% CI 19.0–41.2) vs 5.4% (95% CI 0.7–18.2). PFS2 hazard ratio was 0.52 (95% CI 0.33–0.81; 27% maturity). OS is immature (12%). Camizestrant + CDK4/6i was well tolerated with safety consistent with the known profiles of camizestrant, and of each CDK4/6i. Rates of treatment discontinuation due to adverse events were 1.3% for camizestrant and 1.9% for AI. Conclusions: Camizestrant + CDK4/6i guided by emergence of ESR1 m during 1L AI + CDK4/6i in pts with HR+/HER2– ABC resulted in a statistically significant and clinically meaningful improvement in PFS. SERENA-6 is the first global Phase 3 trial to demonstrate clinical utility of using ctDNA to detect and treat emerging resistance, ahead of disease progression. These findings represent a potential new treatment strategy to optimize and improve 1L patient outcomes. Clinical trial information: NCT04964934 .

Circulating tumor cells (CTCs) and immune cells form dynamic multicellular ecosystems in blood, but their spatial organization and clinical relevance have not been systematically characterized. We developed the Cell and Cluster Identification Program (CCIP), an artificial intelligence–based framework that analyzes routine multiplex immunofluorescence blood scans to segment cells, identify CTCs and five immune lineages with high accuracy, and quantify multicellular clusters and tumor–immune interactions. Applying CCIP to 2,693 blood scans from 1,399 patients, we profiled over 60 million cells (>7 million multi–cell clusters) and linked imaging–derived features to patient outcomes. Correlated with circulating–tumor DNA mutation burdens, a 14–feature image model predicted overall survival in breast cancer, outperformed clinicopathologic variables and CTC enumeration, and generalized to prostate cancer. Prognostic imaging signatures were also associated with therapy response-related progression–free survival as well as with single–cell RNA sequencing–derived immune suppression states, connecting circulating tumor–immune architecture with systemic immune dysfunction.

1015 Background: Patients living in food deserts or experiencing food insecurity have higher cancer mortality rates. While neighborhood poverty correlates with more aggressive tumor biology, including higher OncotypeDX scores and rates of TP53 mutations, the associations among food access, somatic genomic profiles, and clinical outcomes in metastatic breast cancer (MBC) remain understudied. Methods: This multi-institutional retrospective cohort study included patients with MBC and circulating tumor DNA (ctDNA) testing through the Guardant360 assay from Washington University in St. Louis (N = 651), Massachusetts General Hospital (N = 349), Weill Cornell Medicine (N = 187), and Northwestern University (N = 86). Patient census tracts were linked with the USDA Food Access Research Atlas, which provides data on census-level food access throughout the United States. Deidentified data were shared across sites. We categorized patients by: Food access: Low access (LA) if ≥33% of the census tract lived > 1 mile (urban) or > 10 miles (rural) from a food store Income and food access: Low-income and low access (LILA) if the census tract was both LA and poverty rate was ≥20% We assessed descriptive differences and determined associations between LA/LILA and ctDNA profiles using multivariate analysis adjusted for race, stage, and subtype. Results: Of the 851 patients with census tract data, 46.8% lived in LA and 8.0% lived in LILA areas. Although White patients were more likely to live farther from food stores than Black patients (48.9% vs 34.4%, p = 0.003), Black patients were significantly more likely to live in LILA areas (19.2% vs 6.1%, p = < 0.001). On multivariate analysis, patients in the overall cohort living in LA areas were more likely to have RTK/RAS pathway mutations (Odds ratio [OR] 2.05, 95% confidence interval [CI] 1.36-3.09, p = 0.001), confirmed in the hormone receptor positive, HER2 negative (HR+/HER2-) cohort (OR 2.18, 95% CI 1.34-3.55, p = 0.002). Patients in the overall cohort living in LILA areas were more likely to have CCNE1 copy number variants [cnv] (OR 2.81, 95% CI 1.18-6.73, p = 0.02). Patients in LA areas had significantly shorter overall survival (OS) after first ctDNA test (24 months [mos] vs 31 mos, p = 0.01). In HR+/HER2- MBC, Black patients in LA areas had the shortest OS (11 mos) compared to those in high access areas (38 mos) and White patients in either setting (27-33 mos, p = 0.02). Conclusions: Patients with MBC living in food deserts appear to have distinct ctDNA profiles, including higher rates of RTK/RAS pathway mutations, which drive tumor growth, and CCNE1 cnv, associated with poor prognosis. We also found a significant survival disadvantage associated with living in LA areas, particularly among Black patients. These findings demonstrate the necessity of considering the intersection of tumor biology and social determinants of health in understanding and treating MBC.

1036 Background: Activating PIK3CA mutations are established oncogenic drivers and predictive biomarkers in MBC. In contrast, PIK3CA amplifications are rare, poorly characterized, and of unclear clinical significance. Tissue-based studies report a prevalence of 1–7%, but their detection and relevance in ctDNA are largely unexplored. Moreover, whether amplifications interact with co-occurring mutations to define a distinct high-risk subset is unknown. We therefore aimed to characterize the impact of PIK3CA amplifications detected in ctDNA through a multi-institutional cohort of patients (pts) with MBC. Methods: This retrospective study analyzed a multi-institutional cohort of 1579 pts with MBC and baseline ctDNA testing with the Guardant360 NGS panel within a large academic consortium (PMAC). Hormone Receptor positivity (HR+) and HER2 status were defined based on the most recent biopsy. Associations between SNVs, CNVs, and clinical characteristics were assessed using multivariable logistic regression. The impact of prognosis, adjusted for the number of prior treatment lines, was evaluated through Cox regression for overall survival (OS), defined from time of baseline ctDNA collection. Results: Among 1,579 pts, 1,121 (71%) were HR+/HER2–, 214 (13.5%) HER2+, and 244 (15.5%) had triple-negative breast cancer (TNBC). PIK3CA CNVs were detected in 7.7% of pts, and 44.7% harbored a concomitant PIK3CA SNVs. PIK3CA CNVs were significantly enriched in TNBC compared with other subtypes (Odds Ratio [OR] 2.02, p=0.011). In the overall population, PIK3CA CNVs were associated with significantly worse OS (HR 2.46, p<0.001), an effect observed across all subtypes, including HR+/HER2– (HR 2.21, p<0.001), HER2+ (HR 5.23, p<0.001), and TNBC (HR 1.81, p=0.018). In multivariable analysis, PIK3CA CNVs remained independently associated with inferior OS in the overall cohort (HR 1.61, p=0.015) and in HR+/HER2– disease (HR 1.38, p=0.048). Notably, the coexistence of a PIK3CA CNV and mutation identified a subset with particularly poor prognosis. Compared with patients harboring PIK3CA SNVs alone, those with concurrent SNVs and CNVs had significantly worse OS in the overall population (HR 1.80, p=0.002), in HR+/HER2– disease (HR 1.51, p=0.037), and in TNBC (HR 5.72, p<0.001). Conclusions: PIK3CA amplifications detected in ctDNA are relatively frequent in MBC and are associated with significantly worse survival across molecular subtypes. The coexistence of PIK3CA amplification and mutation identifies a distinct high-risk subset with particularly poor prognosis, beyond the effect of mutations alone. Further mechanistic understanding and real-world outcome analyses are needed to determine whether PIK3CA amplifications, alone or in combination with mutations, have predictive relevance for response or resistance to PI3K-pathway inhibitors.

PURPOSE OF REVIEW:Liquid biopsy (LB) is rapidly transforming the management of hormone receptor-positive/HER2-negative (HR+/HER2-) metastatic breast cancer (mBC). In this review, we discuss the most recent findings on circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), exploring their roles across the treatment continuum, from treatment selection to resistance monitoring. RECENT FINDINGS:ctDNA-based detection of actionable mutations - including ESR1 and PIK3CA - achieved regulatory validation as a primary predictive biomarker for treatment selection, supporting the approval of novel endocrine agents and targeted therapies in HR+/HER2- mBC. Serial ctDNA monitoring demonstrated the ability to intercept emerging resistance mechanisms ahead of radiological progression, opening a window for preemptive therapeutic adaptation. CTCs enumeration maintains robust independent prognostic value, with emerging evidence supporting its role in guiding treatment escalation and de-escalation decisions. Integrated multiparametric LB approaches combining CTCs and ctDNA are crucial to improve prognostic accuracy and clinical decision-making. SUMMARY:LB is progressively shifting from a research tool to a clinical decision-making instrument in HR+/HER2- mBC. Prospective interventional trials, methodological standardization, and integrated strategies are needed to fully translate its potential into improved patient outcomes.

Supplementary Figure 1 Shows the distribution of ERBB2 mutations among metastatic cancer patients, survival outcomes according to metastatic sites (bone vs visceral), and detailed CTC counts from HR+/Her2- MBC patients. Supplementary Figure 2 Shows the therapeutic effect of Tras-ALN on metastasis spread from bone metastasis models of HR+ breast cancer via uCT, live and ex vivo bioluminescence profiling of metastatic organs. Supplementary Figure 3 shows the increased expression of ERBB2 in MSC 3D coculture and anti-ER treatment conditions. Supplementary Figure 4 shows the immune-independent effect of Tras-ALN in blocking metastasis progression. Supplementary Figure 5 shows that direct cell-cell interaction is not required for MSC-induced Her2 expression based on 3D transwell culture conditions. Supplementary 1 of Figure 6 shows genetic modulations of Her2 (ERBB2) in MCF7 and ZR75-1 breast cancer models. Supplementary 2 of Figure 6 shows that genetic alteration of Her2 impacts multiorgan metastasis seeding in HR+ breast cancer models. Supplementary 1 of Figure 7 shows that miRNAs with Her2 targeting potential are associated with overall survival in breast cancer. Supplementary 2 of Figure 7 shows that miR-133a/b abrogates brain metastasis seeding from bone-derived ZR75-1 cells.

Abstract Background: In metastatic cancer, Circulating Tumor cells (CTCs) are established prognostic indictors of patients (pts) less likely to respond to new lines of systemic therapy, with poor clinical outcomes, such as shorter progression free survival (PFS) and overall survival (OS). However, CTCs are typically found in specific malignancies (breast, prostate & colon), often in <20% of pts with metastatic disease, and pts without CTCs may also rapidly progress. Recently, an inflammatory pro-tumorigenic macrophage emanating from tumor stroma (i.e. Cancer associated macrophage-like cell [CAML]) was found in >90% of metastatic cancer pts, and whose phagocytic engorgement appears to correlate with poor outcomes, independent of CTCs. As CTCs and CAMLs are isolated in conjunction from a single blood sample, and both are prognostic for outcomes, we evaluated their utilization prior to induction of new systemic therapy in 6 types of metastatic cancer to model pt risk stratification based on 2 year outcomes. Methods: A prospective 2 year blind multi-institutional study was undertaken to model CTCs and CAMLs in prognosticating outcomes prior to induction of a new line of systemic therapy (n=233) in metastatic: Breast (n=60), Prostate (n=40), Pancreas (n=25), Colon (n=28), Renal Cell Carcinoma (RCC) (n=39), and Lung (n=40). Blood was filtered by CellSieveTM filters with subtypes of CTCs & hyper-enlarged CAMLs (≥100µm) enumerated. A machine learning algorithm was trained on this initial data set to develop predictive models which could stratify pt populations by risk for likelihood of PFS & OS over 2 years, including known clinical variables. Results: CTCs were absent in 80% (n=185/233) of pts, and their absence was prognostic for better PFS (HR=1.6, p=0.046), but not OS (HR=1.3, p=0.2045). In parallel, enlarged CAMLs (≥100µm) were found in 28% (n=51/185) of pts without CTCs, and were also prognostic for worse PFS (HR=2.0, p=0.0082) and OS (HR=1.9, p=0.0412). Specifically, CTCs were found in 55% (n=33) breast, 20% (n=8) prostate, 12% (n=3) pancreas, 11% (n=3) colon, 0% (n=0) RCC, and 0% (n=0) lung pts. Enlarged CAMLs were found in 45% (n=27) breast, 20% (n=8) prostate, 44% (n=11) pancreas, 44% (n=17) colon, 18% (n=7) RCC and 20% (n=8) lung pts. Overall, models indicated that ≥1 CTC (n=47) had mPFS=3.9 & mOS=14.9, while pts with 0 CTCs and ≥100µm CAMLs (n=51) had a mPFS=6.9 & mOS=13.8, and pts with 0 CTCs and <100µm CAMLs (n=134) had a mPFS=10.7 & mOS>24 months. Conclusions: These initial models confirm that both CTCs and enlarged CAMLs are prognostic indicators of worse PFS & OS. The simultaneous quantification both CTCs and CAMLs allows for more accurate pan-cancer risk stratification in an array of cancer pt populations. Additional clinical variables incorporated into the models may allow better risk subtyping and possibly forecast optimal treatment regimens. Citation Format: Daniel L. Adams, Steven H. Lin, Massimo Cristofanilli, Carolina Reduzzi, Saranya Chumsri, Martin J. Edelman, Susan Tsai, Raymond C. Bergan, Mohammed Aldakkak, Thai H. Ho, Cha-Mei Tang. Combining circulating tumor cells and cancer associated macrophage-like cells enhances risk stratification models in pan-cancer metastatic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1068.

Background Circulating tumor associated cell (CTAC) detection-based multi-cancer early detection (MCED) strategies may be hindered by the rarity of CTACs among millions of peripheral blood nucleated cells (PBNCs). We developed an advanced U-Net-based encoder-decoder model for pixel-level CTAC discrimination that integrates attention-gated skip connections to preserve morphological and fluorescence details. Methods Model suitability was explored in an initial cohort of asymptomatic individuals (n = 428) and patients with advanced solid tumors (n = 354). A case-control study assessed clinical performance in therapy-naive stage I/II cancer patients (n = 185), individuals with benign conditions (n = 129), and asymptomatic individuals (n = 111). The model was then validated across four prospective studies on distinct populations: recurrent cancer cases with low tumor burden (n = 224); patients with solid tumors in the peri-operative setting (n = 17); suspected cancer cases (n = 259); and asymptomatic individuals (n = 7,183), respectively. All studies used blinded peripheral blood specimens from which PBNCs were isolated, stained for EpCAM / Hoechst 33342, and imaged. Ground truth annotations were established via pathologist review. The U-Net pipeline encoded spatial information in the images via convolutional and pooling layers and generated pixel-wise segmentation masks to identify CTACs. In all studies, sensitivity was based on CTAC detection rate in cancer specimens and CTAC undetectability rate in specimens from healthy asymptomatic individuals or those with benign conditions Results In the exploratory study, the model had 90.68% (95% CI: 87.16%, 93.50%) sensitivity and 99.53% (95% CI: 98.32%, 99.94%) specificity. In the case-control cohort, the model had 88.65% sensitivity (95% CI: 83.17%, 92.83%), 78.95% (95% CI: 71.03%, 85.53%) specificity in benign conditions, and >99.9% specificity in asymptomatic individuals. Among the four prospective studies, the model had: (a) 91.96% (95% CI: 87.60%, 95.17%) sensitivity in pretreated patients with low tumor burden; (b) 100% sensitivity in pre-surgery specimens, and 29.41% sensitivity in post-surgery specimens; (c) 96.34% PPV (95% CI: 93.22%, 98.05%) and a 32.35% NPV (95% CI: 25.58%, 39.95%) for diagnostic triaging; and, (d)11% PPV (95% CI: 31.72%, 53.24%) and 99.97% NPV (95% CI: 99.90%, 99.99%) for MCED in healthy asymptomatic individuals. Conclusions The attention-enhanced U-Net achieved robust, generalizable performance for CTAC-detection in case-control and prospective cohorts, supporting its clinical utility for accurate cancer detection. ### Competing Interest Statement HAS and AdS report no conflicts of interest associated with the scope of the present study. MC, SL, NR, TC, AG and RP declare fractional stock ownership of the study sponsor. DP, VD, DA, SS, PA, SP, SA, PS, SG, AjS are employees of the study sponsor. RD is the founder of the study sponsor. ### Funding Statement This study did not receive any funding ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Institutional Ethics Committee (IEC) of the Study Sponsor (Datar Cancer Genetics, DCG) gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Shallow whole-genome sequencing (sWGS) is a cost-effective method for rapidly detecting large-scale genome alterations like copy number alterations. The sWGS workflow involves DNA extraction, library preparation, and sequencing, followed by specialized bioinformatics analyses, which we carefully review at each step. This workflow includes data preprocessing and alignment to a reference genome, with each bioinformatics stage carefully detailed as it plays a pivotal role in ensuring the accuracy and biological relevance of the results. The application of this process enables reliable data collection and analysis for monitoring cancer evolution and treatment responses by identifying critical genome changes, providing significant insights for prognosis and therapeutic decision-making across various cancer types.

Abstract Introduction. Patients with metastatic triple-negative breast cancer (mTNBC) have poor survival but may be eligible for immune check point inhibitor (ICIs). CCR5 is overexpressed in ∼95% of TNBC. Data suggest that combining the CCR5 inhibitor leronlimab with an ICI may improve survival in mTNBC. Methods. Analysis of patient gene expression, tumor histology, cancer-associated macrophage-like cells/circulating tumor cells (CAML/CTC) from clinical studies and tissue cultures of TNBC were conducted. Findings. In breast cancer cohorts (N=1,096) CCR5 expression correlated with gene signatures of T cell immune exhaustion. Across public TNBC cohorts (N=73; after deduplication), CCR5 expression correlated with both GSEA and gene signatures of T cell infiltration and T cell immune exhaustion. TNBC subtype analysis showed CCR5 enrichment in epithelial cells of MLIA (Mesenchymal-like Immune-Altered, Jézéquel subtype) and IM (immune modulatory, Lehmann subtype). TNBC Subtype analyses showed higher CCR5-related signals in tumors classified as MLIA and IM subtypes. In cultured MDA-MB-231 TNBC cells, CCR5 expression suppressed glycosylated PDL1; CCR5 inhibition increased the abundance of PDL1 (18 kDa, 35 kDa, and glycosylated 55 kDa forms). To understand the mechanisms by which CCR5 may promote immune exhaustion, we investigated a heterotypic signal between TNBC cultured cells and the tumor microenvironment (TME) using a proteomic approach. CCR5 activity induced sB7-H3 (CD276), sTyro3 and the Tyro3 ligand Pros1. Both CD276 and Tyro3 are associated with ICI resistance; and abundance of both were attenuated by CCR5 blockade with leronlimab. In Vivo. Leronlimab induced PD-1 expression in CD8+ T cells in lymph node of rhesus macaques and had variable modulation on expression of several T cell exhaustion markers. In a retrospective analysis of data pooled from 28 patients with mTNBC leronlimab induced PD-L1 in CTC/CAMLs. Leronlimab was generally well tolerated. Higher leronlimab dose (550-700 mg once weekly), induction of PD-L1, and the formation of CCR5 dots in CTC/CAMLs, and treatment with leronlimab in combination, or subsequently, with an ICI were associated with improved survival. The median age of the 28 patients was 48.5 years (range 32-83), patients had a median of 2 prior lines of therapy in the metastatic setting (range 0-5), 18 patients had visceral metastases (64%), of which 8 had brain metastases, and 10 had non-visceral metastases, 17.9% of heavily pretreated mTNBC patients are currently alive after median >60 months of follow-up. Conclusions. Leronlimab is well tolerated, inducing PDL1 expression on CTC/CAMLs, which may prime tumors for PDL1 blockade. CCR5 may promote ICI resistance in TNBC by upregulating immune checkpoints (sB7-H3) and sTyro3. Overall, 17.9% (5/28) of patients with mTNBC treated with leronlimab are currently alive after a median of >60 months follow-up. Citation Format: Richard G. Pestell, Ritika Harish, Zhiping Li, Danni Li, Xuanmao Jiao, Hallgeir Rui, Massimo Cristofanilli, Daniel L. Adams, Neil E. Buss, Jonah B. Sacha, Jacob P. Lalezari. Leronlimab induces PD-L1 expression and is associated with long-term survival with an ICI in PD-L1 low metastatic TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1033.

Background Circulating tumor-associated cells (CTACs) are rare among peripheral blood nucleated cells (PBNCs), posing a challenge to use them as Multi Cancer Early Detection (MCED) tool. We developed an attention-gated U-Net model for pixel-level CTAC discrimination that preserves key morphological and fluorescence details. Methods Model suitability was explored in asymptomatic individuals (n = 428) and patients with advanced solid tumors (n = 354). Clinical performance was assessed in case-control cohort of therapy-naive stage I/II cancers (n = 185), benign conditions (n = 129), and asymptomatic (n = 111) individuals, followed by validation across four prospective studies on distinct patient populations: recurrent cancer with low tumor burden (n = 224); solid tumors in peri-operative setting (n = 17); suspected cancer (n = 259); and asymptomatic population (n = 7183). PBNCs were isolated using blinded peripheral blood specimens, stained with EpCAM/Hoechst33342, and imaged. Pathologists’ review established ground truth annotations. The U-Net pipeline encoded spatial features via convolutional and pooling layers to generate pixel-wise segmentation masks for CTAC identification. Sensitivity was determined by CTAC detection in cancer specimens and by their absence in healthy or benign samples. Results The model had 90.68% sensitivity and 99.53% specificity in exploratory study. In case-control cohort, sensitivity was 88.65% in benign conditions and > 99.9% in asymptomatic individuals, while specificity was 78.95% in benign conditions. Among four prospective studies, sensitivity was (a) 91.96% in pretreated low tumor burden patients; (b) 100% in pre-surgery, and 29.41% in post-surgery specimens; (c) 96.34% Positive Predictive Value (PPV) and 32.35% Negative Predictive Value (NPV) for diagnostic triaging; and (d)11% PPV and 99.97% NPV for MCED in asymptomatic individuals. Conclusion The attention-enhanced U-Net achieved robust performance in CTAC detection across case-control and prospective cohorts, supporting its clinical utility for cancer detection.