Mitosis in cell treated with eribulin+CENP-E inhibitor. Although the multipolar spindle is focused into a bipolar spindle early in mitosis, the cell enters anaphase with multiple polar chromosomes and both daughter cells die.

Abstract Background: Standard treatment of ductal carcinoma in situ (DCIS) in post-menopausal women is breast conserving surgery/mastectomy, radiotherapy, and adjuvant endocrine therapy (ET) with either tamoxifen or an aromatase inhibitor. Despite the need to individualize selection of ET based on patient characteristics and side effects, limited tools exist for this purpose. NSABP-B-35, a phase III trial evaluating anastrozole vs tamoxifen in post-menopausal women with DCIS, found that younger patients may benefit more from anastrozole. We hypothesized that causal machine learning methods, which predict treatment effect by accounting for complex interactions between baseline characteristics, would identify treatment benefit more accurately than age alone. Methods: Individual-level data from the NSABP-B-35 trial (n = 3104) were obtained from the NCI NCTN Data Archive, with administrative approval from NCI. Trial eligibility included: DCIS with no invasive component, hormone receptor positive, and previous lumpectomy (with clear margins and negative nodes), followed by whole-breast irradiation. Baseline variables included age, tumor palpability, presence of comedo necrosis, body mass index (BMI), and black race. The outcome was disease-free survival (DFS). The causal machine learning method, a T-learner with accelerated failure time-Bayesian Additive Regression Trees (AFT-BART), predicted the individualized treatment effect (ITE) of anastrozole vs tamoxifen on the difference in restricted mean survival time at 116 months, conditional on patient characteristics. Three repeats of five-fold cross-validation were used to generate out-of-sample predictions for each patient. Statistical significance (p < 0.05) was determined by an aggregate Cauchy association test (ACAT) calculated on the out-of-sample Qini coefficient p-values, a metric that quantifies how well the model orders patients by most to least benefit. Variable importance was quantified using kernelSHAP. Results: The AFT-BART ITE model was able to significantly prioritize patients in order of most to least benefit from anastrozole vs tamoxifen (p-value = 0.03). Predicted ITE ranged from an increase of DFS by 2.9 months to a decrease in DFS by 4.5 months when on anastrozole vs tamoxifen. Younger age, presence of comedo necrosis, and black race best predicted anastrozole benefit. No statistically significant heterogeneity was found when patient age alone was used to rank patients from most to least benefit (p-value = 0.6). Conclusions: Our causal machine learning model accurately predicted who would benefit from anastrozole vs tamoxifen, outperforming treatment selection based on age alone. Once validated, this model may help clinicians optimize treatment selection for post-menopausal women with DCIS. This work was supported by NLM 5T15LM007359. Citation Format: Emma Graham Linck, Alex Spicer, Marina N. Sharifi, Guanhua Chen, Mark Craven, Nataliya Uboha, Mark Burkard, Matthew Churpek. Causal machine learning personalizes endocrine therapy selection for ductal carcinoma in situ [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 2507.

Background The SMILE study is a multi-institutional phase II clinical trial to determine the efficacy and safety of an antiprogestin, onapristone, in combination with fulvestrant as second-line therapy for patients with ER+, PgR+/-, HER2- metastatic breast cancer. This study was terminated early and herein, we report patient characteristics, and outcomes. Methods Eligibility criteria included disease progression on >= 2 lines of prior therapy, ECOG performance status <= 2, measurable disease per RECIST 1.1 criteria, and optional F-18-fluorofuranylnorprogesterone (F-18-FFNP) PET/CT imaging. Results Consented subjects received standard-dose fulvestrant plus onapristone 50 mg orally, twice daily, until disease progression, or unacceptable toxicity. The study enrolled 11 women from 2 sites within the Wisconsin Oncology Network from November 2021 through March 2023. Mean age of the subjects was 58.5 years. Other than grade 1 toxicities, the treatment was well tolerated. None of the 11 subjects met RECIST 1.1 definition of response. The median time to progression was 63 days. A total of 4 of 11 patients had stable disease as best response and 2 of them were on treatment for 5.5 and 7.7 months. Two of the 11 subjects underwent functional imaging with F-18-FFNP PET/CT before and 10 or 14 days after starting treatment. For both subjects, tumor uptake of F-18-FFNP was stable or increased in all target lesions while F-18-FFNP uptake in the uterus, a normal PgR-rich internal control organ, was decreased. Conclusion The study regimen was well-tolerated with no significant toxicities. Future studies may evaluate antiprogestins with various combinations such as targeted therapies. (c) 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

New treatments for prostate cancer (PC) offer promise but challenge clinicians to optimize sequential selection. Subgroup analyses may identify factors, such as disease volume, that predict treatment benefit, but these analyses overlook the impact of the combined effect of multiple factors. To address this gap, we applied causal machine learning to phase III clinical trials to predict each patient’s individualized treatment effects (ITEs) given their unique multivariable baseline characteristics. Here, we quantify how frequently ITE models can identify significant treatment effect variation in trials of radiotherapy (RT) or systemic interventions for localized or metastatic PC. We included four PC phase III trials that met the following criteria: in the NCI NCTN Data Archive, >300 participants, and met target enrollment. One trial evaluated RT for localized PC (NCIC-PR.3), two evaluated systemic therapy for localized PC (RTOG-96-01 and RTOG-0521), and one evaluated systemic therapy for metastatic PC (CHAARTED). Trial-specific data included baseline characteristics from Table 1 of the primary publication and the outcome with the shortest median time to event across the trial’s population. Causal Survival Forest, an algorithm to estimate ITE, was iteratively trained in 4/5 of each trial to predict Restricted Mean Survival Time conditional on patient characteristics (cRMST) in the remaining 1/5, resulting in predictions for all trial participants. The median p-value for the Qini coefficient, a metric that quantifies how well the model orders patients by most to least benefit, across five repeats of model training was used to discern statistically significant performance. The variable importance of each model was quantified using Friedman’s H-statistic. Trial-specific ITE models accurately identified those who experienced benefit or harm in 2/4 trials: CHAARTED, a trial evaluating the addition of systemic therapy (docetaxel) to androgen deprivation therapy (ADT) for metastatic hormone-sensitive PC (Qini p-value < 0.05) and NCIC-PR.3, a trial evaluating pelvic RT for localized PC (Qini p-value < 0.05). In CHAARTED, all patients were predicted to benefit from docetaxel, but to varying degrees (cRMST range 0.19 to 1.42 years). The variables most predictive of treatment response were time from ADT treatment to randomization, baseline PSA, age, and volume of metastatic disease. In NCIC-PR.3, most patients benefited from pelvic irradiation, but some experienced harm (cRMST range -0.05 to 0.36 years). The variables most predictive of treatment response were age, PSA < 20 or > 50, Gleason score > 8, and prior hormone therapy. Conclusions: With clinical characteristics alone, ITE models accurately predicted personalized treatment effect estimates in 2/4 trials. Once validated, ITE modeling could help clinicians optimize treatment selection in PC. This work was supported by the NLM (5T15LM007359). Data was shared through NCI’s NCTN Data Archive, with approval from sponsors: NCI, ECOG, SWOG, NRG Oncology, RTOG, MRC, and the NCIC Clinical Trials Group. Emma Graham Linck MS, Alex Spicer MS, Marina Sharifi MD PhD, Guanhua Chen PhD, Mark Craven PhD, Nataliya Uboha MD PhD, Mark Burkard MD PhD, Matthew Churpek MD MPH PhD. Personalizing treatment selection for prostate cancer using causal machine learning [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Artificial Intelligence and Machine Learning; 2025 Jul 10-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(13_Suppl):Abstract nr A017.

Metastatic triple negative breast cancer has a poor prognosis with limited targeted treatment options. In preclinical studies PI3K inhibition led to increased DNA damage and subsequent sensitization to PARP inhibition. This study aimed to investigate the safety and efficacy of the combination of an mTOR/pan-PI3K inhibitor, gedatolisib, with the PARP inhibitor, talazoparib, in patients with advanced triple negative breast cancer or advanced HER2 negative breast cancer and a germline BRCA1/2 mutation. The primary objective of the safety run-in was safety and tolerability of the combination and for dose escalation to find the maximum tolerated dose. A 3 + 3 design was utilized for dose escalation. The primary objective of the phase II study was objective response rate (ORR) in the patients with wildtype germline BRCA1/2. The prespecified efficacy threshold was 20

Hormone receptor (HR)-positive, HER2-positive breast cancers often develop resistance to endocrine and anti-HER2 therapies due to the heterogeneous expression of estrogen receptor (ER) and HER2 and the crosstalk of these growth-promoting pathways. However, how anti-HER2 agents activate ER and other growth-promoting pathways remains unknown. Single-cell RNA sequencing of BT474 breast cancer cells identified Bromodomain Containing Protein 8 (BRD8), an acetyl-lysine reader protein in the histone acetylase EP400 complex, as a pivotal mediator to activate ER in response to neratinib treatment. BRD8 expression was rapidly induced by various anti-HER2 agents (neratinib, lapatinib, and trastuzumab), and its depletion disrupted the crosstalk between ER and HER2 signaling pathways and rendered HR+/HER2+ cells and PDxOs more sensitive to anti-HER2 agents. BRD8, ER, and ER target genes are co-induced by neratinib in single-nucleus RNA and ATAC sequencing of a patient-derived xenograft (PDX). SnATAC-seq also reveals that the activated genes share open chromatin regions enriched in ER, forkhead box (FOX), and ETS family transcription factors (TF) binding motifs. FOX family TFs are well-known for regulating estrogen signaling, and ETS proteins have been shown to promote tumorigenesis. Since EP400 enhances H2AZ deposition and acetylation on chromatin, we performed H2AZ and H2AZac ChIP-sequencing in the presence or absence of BRD8 and neratinib treatment. We found that, in response to neratinib treatment, BRD8 activates ER, FOX, and ETS target genes through modulating H2AZac deposition and chromatin decompaction. This finding coincides with RNA-sequencing where BRD8 promotes cell growth in an ER-dependent and -independent manner. In line with these findings, patients who responded poorly to the anti-HER2 therapies exhibited higher levels of BRD8 target gene signature as compared to the responders. Furthermore, BRD8 knockout ablates the ER and HER2 signaling crosstalk and re-sensitizes neratinib-resistant HR+/HER2+ cells to neratinib. In summary, this work not only explains why ER signaling is activated upon anti-HER2 therapies but also identifies BRD8 as a druggable vulnerability for treating HR+/HER2+ breast cancer. Ang Gao, Parth Khatri, Gui Ma, Peng Liu, Mark Burkard, Kari Wisinski, Charles Perou, Huy Dinh, Wei Xu. BRD8 is a therapeutic vulnerability for overcoming resistance to dual ER/HER2 blockade therapy in HR+/HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4308.

Tumor heterogeneity is predicted to confer inferior clinical outcomes with precision-based strategies, however, modeling heterogeneity in a manner that still represents the tumor of origin remains a formidable challenge. Sequencing technologies are limited in their ability to identify rare subclonal populations and predict response to treatments for patients. Patient-derived organotypic cultures have significantly improved the modeling of cancer biology by faithfully representing the molecular features of primary malignant tissues. Patient-derived cancer organoid (PCO) cultures contain subclonal populations with the potential to recapitulate heterogeneity, although treatment response assessments commonly ignore diversity in the molecular profile or treatment response. Here, we demonstrate the advantage of evaluating individual PCO heterogeneity to enhance the sensitivity of these assays for predicting clinical response. Additionally, organoid subcultures identify subclonal populations with altered treatment response. Finally, dose escalation studies of PCOs to targeted anti-EGFR therapy are utilized which reveal divergent pathway expression when compared to pretreatment cultures. Overall, these studies demonstrate the importance of population-based organoid response assessments, the use of PCOs to identify molecular heterogeneity not observed with bulk tumor sequencing, and PCO heterogeneity for understanding therapeutic resistance mechanisms.

Background/Objectives: Recent clinical trials in breast cancer have demonstrated that some patients benefit from immune checkpoint blockade, though better predictive markers are needed. The accumulation of the immunomodulatory matrix proteoglycan versican (VCAN) can predict the exclusion of CD8+ tumor-infiltrating lymphocytes (TILs) in some settings and, thus, is evaluated in breast cancer here. Methods: A total of 230 breast cancers were analyzed for VCAN accumulation, VCAN proteolysis, and CD8+ TILs. CD8+ TILs were categorized based on their localization in the tumor epithelial or stromal compartments. Results: VCAN accumulation was detected in 90% of breast cancers, more commonly in ER+ tumors (93% vs. 77%; p < 0.001). MCF7 cells treated with estrogen upregulate VCAN without an enhanced expression of ADAMTS-proteases. VCAN-undetectable tumors demonstrate greater CD8+ TILs compared to VCAN-detectable tumors (p = 0.012). CD8+ T cells within TNBC tumors with high VCAN proteolysis infiltrated the epithelial compartment more often than in tumors with low VCAN proteolysis (91% vs. 42% respectively; p = 0.008). In the TCGA cohort, a strong inverse correlation between CD8A and VCAN expression was observed across subtypes. Conclusions: VCAN accumulation correlates with the exclusion of CD8+ TILs across subtypes of breast cancer, warranting further validation of VCAN accumulation and proteolysis as predictive biomarkers for breast cancer immunotherapy.

Background: Multiple single institution studies have shown that FES-PET predicts clinical benefit of endocrine based therapy for patients with metastatic breast cancer (MBC). ECOG-ACRIN trial EAI142 is a multicenter imaging biomarker study of FES-PET to assess baseline FES uptake qualitatively and quantitatively prior to initiation of first-line endocrine therapy with or without CDK4/6 inhibitors in MBC. The primary objective is to determine the negative-predictive value (NPV) of FES uptake for predicting clinical benefit in patients with estrogen-receptor positive (ER+) MBC at 6 months after treatment with endocrine based therapy. In this analysis, we present the primary analysis: the imaging results and 6-month follow-up data in the fully enrolled cohort. Methods: From 2016-2022, 14 centers enrolled 105 female patients; 98 completed study procedures and were treated with endocrine based therapy. [18F]Fluorodeoxyglucose (FDG)-PET at baseline was encouraged, but not required, and was obtained in 66 patients. SUVmax of up to 5 of the most prominent lesions were recorded and qualitatively assessed for FES uptake in all patients. A sub-set analysis was done using FDG to guide lesion selection in the 66 patients who had both FES and FDG scans with the goal to better identify FES-negative lesions and heterogeneity in FES uptake. Patients were followed for progression-free survival (PFS) at 6 months (primary endpoint) and at 2 years (secondary endpoint). To determine the reproducibility of quantitative assessment of tumor FES uptake, 10 patients underwent a second FES-PET scan within 12 days of the first scan and prior to initiation of treatment in a test-retest sub-study. Results: Of the 450 lesions identified at baseline, the majority were in bone or soft tissue (262 bone, 124 lymph node, 40 visceral). Most patients had 5 or more evaluable lesions (83/98, 85%). The majority of patients were postmenopausal (80/98, 82%) and treated with an aromatase inhibitor (69/98, 70%) and CDK4/6 inhibitor (73/98, 74%). We identified a wide range in uptake (SUVmax of 1.2-44.7), but only 19/98 (19%) patients had any lesions with qualitatively mild, minimal or absent FES uptake. No patient had an average SUVmax for the most prominent lesions less than the pre-specified cut-off of 1.5, therefore an NPV was unattainable. A total of 16/98 (16%) patients progressed at 6 months. Of 4 patients with heterogenous uptake, 1 (25%) progressed at 6 months. In the sub-set analysis, FDG -guided lesion location did not identify any additional FES-negative lesions. We observed excellent reproducibility in 79 test-retest lesions (SUVmax 1.5-20.2) with a lesion level intra-class correlation coefficient of 0.94. Two-year PFS will be updated at the time of presentation. Conclusions: In patients undergoing first-line therapy for ER+ MBC, we evaluated ER expression by both qualitative and quantitative FES-PET. NPV could not be assessed due to a lower than expected proportion of FES-negative scans and rate of progressive disease at 6 months needed to meet the assumptions used for power calculations. Test-retest studies confirmed quantitative overall and lesional reproducibility of FES-PET. Citation Format: Hannah Linden, Fenghai Duan, Farrokh Dehdashti, Amy M Fowler, Amy S Clark, Jennifer M Specht, Jian Q Yu, Hsiaoj Lee, Lanell M Peterson, Mark Muzi, Jennifer S Winn, Hayley Knollman, Mark E Burkard, Foluso O Ademuyiwa, Rathan M Subramaniam, Lucy Hanna, Angela M DeMichele, Jonathan E McConathy, Antonio C Wolff, David A Mankoff. [18F]Fluoroestradiol (FES)-PET as a predictive measure for endocrine therapy in patients with newly diagnosed metastatic breast cancer: Results from EAI142 [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 PS11-01.

Human epidermal growth factor receptor 2 (HER2, also known as ERBB2) signaling promotes cell growth and differentiation, and is overexpressed in several tumor types, including breast, gastric and colorectal cancer. HER2-targeted therapies have shown clinical activity against these tumor types, resulting in regulatory approvals. However, the efficacy of HER2 therapies in tumors with HER2 mutations has not been widely investigated. SGNTUC-019 is an open-label, phase 2 basket study evaluating tucatinib, a HER2-targeted tyrosine kinase inhibitor, in combination with trastuzumab in patients with HER2-altered solid tumors. The study included a cohort of 31 heavily pretreated female patients with HER2-mutated metastatic breast cancer who were also HER2 negative per local testing. Hormone receptor (HR)-positive patients also received fulvestrant. The overall response rate (primary endpoint) was 41.9% (90% confidence interval (CI): 26.9-58.2). Secondary endpoints of duration of response and progression-free survival were 12.6 months (90% CI: 4.7 to not estimable) and 9.5 months (90% CI: 5.4-13.8), respectively. No new safety signals were detected. Responses were observed across various HER2 mutations, including mutations in the tyrosine kinase and extracellular domains. The chemotherapy-free regimen of tucatinib and trastuzumab showed clinically meaningful antitumor activity with durable responses and favorable tolerability in heavily pretreated patients with HER2 mutations. These data support further investigation of HER2-targeted therapies in this patient population. ClinicalTrials.gov registration: NCT04579380.

PURPOSE Although patients with metastatic breast cancer (MBC) have been living longer with the advent of more effective treatments such as targeted therapy and immunotherapy, the disease remains incurable, and most patients will undergo therapy indefinitely. When beginning therapy, patients are typically prescribed dose often based upon the maximum tolerated dose identified in phase I clinical trials. However, patients' perspectives about tolerability and willingness to discuss individualized dosing of drugs upon initiation of a new regimen and throughout the course of treatment have not been comprehensively evaluated. METHODS Patient advocates and medical oncologists from the Patient-Centered Dosing Initiative (PCDI) developed a survey to ascertain the prevalence and severity of MBC patients' treatment-related side effects, the level of patient-physician communication, mitigation strategies, perception about the relative efficacy of higher versus lower doses, and willingness to discuss alternative dosing. The PCDI distributed the anonymous confidential online survey in August 2020 to individuals with self-reported MBC. RESULTS One thousand and two hundred twenty-one patients with MBC completed the survey. 86.1% (n = 1,051) reported experiencing at least one significant treatment-related side effect, and of these, 20.3% (n = 213) visited the emergency room/hospital and 43.2% (n = 454) missed at least one treatment. Nearly all patients with side effects (97.6%, n = 1,026) informed their doctor and 81.7% (n = 838) received assistance. Of the 556 patients given a dose reduction for side-effect mitigation, 82.6% (n = 459) reported relief. Notably, majority of patients (53.3%, n = 651) do not believe that higher dose is always more effective than lower dose, and 92.3% (n = 1,127) would be willing to discuss flexible dosing options with their physicians based upon personal characteristics to optimize quality of life. CONCLUSION Given that the majority of patients with MBC experienced at least one substantial treatment-related side effect and most patients given a dose reduction reported improvement, innovative dosage-related strategies are warranted to sustain and improve patients' well-being. Patient-physician discussions in which the patient's unique attributes and circumstances are assessed upon initiation of new treatment and throughout the course of therapy may facilitate the identification of the most favorable dose for each patient, and the majority of patients would be receptive to this approach.

Abstract Drugs that perturb microtubules are commonly used to treat breast cancers of all subtypes in both early stage and metastatic disease, but they are effective in only approximately 50% of patients. High concentrations of microtubule-targeting agents can elicit mitotic arrest in cell culture models; however, recent evidence from primary and metastatic breast cancers has revealed that these agents only accumulate at intratumoral levels capable of inducing abnormal multipolar mitotic spindles, not mitotic arrest. Although the maintenance of multipolar spindles can generate cytotoxic rates of chromosomal instability (CIN), focusing of aberrant multipolar spindles into normal bipolar spindles can dramatically reduce CIN and confer resistance to microtubule poisons. Here, we showed that inhibition of the mitotic kinesin centromeric-associated protein-E (CENP-E) overcomes resistance caused by focusing multipolar spindles. Clinically relevant microtubule-targeting agents used a mechanistically conserved pathway to induce multipolar spindles without requiring centrosome amplification. Focusing could occur at any point in mitosis, with earlier focusing conferring greater resistance to antimicrotubule agents. CENP-E inhibition increased CIN on focused spindles by generating chromosomes that remained misaligned at spindle poles during anaphase, which substantially increased death in the resulting daughter cells. CENP-E inhibition synergized with diverse, clinically relevant microtubule poisons to potentiate cell death in cell lines and suppress tumor growth in orthotopic tumor models. These results suggest that primary resistance to microtubule-targeting drugs can be overcome by simultaneous inhibition of CENP-E. Significance: The increased incidence of polar chromosomes induced by inhibition of the mitotic kinesin CENP-E exacerbates chromosomal instability, reduces daughter cell viability, and improves sensitivity to microtubule-targeting therapies.

Super-resolution microscopy has become an indispensable tool across diverse research fields, offering unprecedented insights into biological architectures with nanometer scale resolution. Compared with traditional nanometer-scale imaging methods such as electron microscopy, super-resolution microscopy offers several advantages, including the simultaneous labeling of multiple target biomolecules with high specificity and simpler sample preparation, making it accessible to most researchers. In this study, we introduce two optimized methods of super-resolution imaging: 4-fold and 12-fold 3D-isotropic and preserved Expansion Microscopy (4× and 12× 3D-ExM). 3D-ExM is a straightforward expansion microscopy technique featuring a single-step process, providing robust and reproducible 3D isotropic expansion for both 2D and 3D cell culture models. With standard confocal microscopy, 12× 3D-ExM achieves a lateral resolution of <30 nm, enabling the visualization of nanoscale structures, including chromosomes, kinetochores, nuclear pore complexes, and Epstein-Barr virus particles. These results demonstrate that 3D-ExM provides cost-effective and user-friendly super-resolution microscopy, making it highly suitable for a wide range of cell biology research, including studies on cellular and chromatin architectures.

Abstract Background: ctDNA monitoring during adjuvant endocrine therapy provides an opportunity to detect molecular relapse before clinically apparent recurrence. The rate and dynamics of ctDNA positivity and the frequency of asymptomatic but imaging detectable metastatic disease at the time of ctDNA detection remain unknown in high-risk ER+/HER2- breast cancers. We present results of ctDNA positivity rates in 508 and imaging results in ctDNA+ patients from a prospective, multicenter, randomized ctDNA surveillance and intervention trial, DARE (NCT04567420). Patients and methods: Patients receiving adjuvant endocrine therapy for > 6 months but < 7 years, with either (i) risk of recurrence > 15% calculated by PREDICT, RSPC, or CTS5, or (ii) > 4 positive axillary lymph nodes, or (iii) primary tumor > 5 cm, or (iv) 1-3 positive nodes with grade 3 histology, or > 3 cm tumor, or Oncotype Dx RS > 26, MammaPrint high risk, EndoPredict > 4, Prosigna score > 60 were eligible for ctDNA surveillance with the SignateraTM assay (Natera Inc.) every 4-6 months during routine follow up visits. ctDNA+ patients underwent systemic staging with imaging and randomized to continuation of adjuvant therapy versus switching to fulvestrant plus palbociclib if there was no evidence of distant metastatic disease. The primary objectives are to assess the incidence of ctDNA positivity in the surveillance phase and to assess if palbociclib plus fulvestrant improves relapse-free survival in 100 randomized patients. This is an updated, protocol-driven interim report to determine if screening eligibility criteria needs to be revised to keep randomization rate > 15% of the screened population. Results: The trial is open at 15 sites and enrolled 508 patients between May 2021 and June 2023; 882 plasma ctDNA tests were performed successfully in 364 patients (72%). The most common reason for failure to generate a personalized ctDNA assay was insufficient tissue submitted, 78% of failed tests were due to preanalytical failure, the technical failure rate was 22%. Thirty patients, 8.2% of those with results available, had >1 positive ctDNA result, the overall positivity rate across all assays was 3.4% (n=30/882). Patient characteristics are shown in the table (not all patients have complete data), 47% of ctDNA+ cases had >4 + lymph nodes. ctDNA positivity rate in the first test was 3.8%, and anytime ctDNA detection rate among those with serial testing was 7.2%. Among ctDNA+ patients, the first ctDNA draw was positive in 23 of 30 cases (77%) with 36.5 months median time (range 6-102 months) from surgery to testing. Using 12 months interval brackets from surgery to 1st ctDNA positivity, annual detection rates were 2,3% (1/44), 8.5% (7/82), 10.8% (9/83), 7.5% (4/53), 13,2% (5/38), and 6.2% (4/64), at 1st, 2nd, 3rd, 4th and > 5th year post-surgery, respectively, due to small sample sizes, 95% confidence broadly overlap. Five ctDNA+ patients (16.7%) had asymptomatic, imaging-detectable metastatic disease, 22 ctDNA+ patients were randomized, the goal is to accrue a total of 100 patients. Conclusions: ctDNA surveillance of ER+/HER2- breast cancers during adjuvant endocrine therapy indicate 8.3% detection rate at patient level and 3.4% at assay level. Serial screening increases detection rates as 23% of positive ctDNA tests occurred after an initial negative result. 83% of ctDNA+ patients had true molecular relapse without imaging detectable metastatic disease. Eligibility for screening on the trial is now restricted to patients with >4 + lymph nodes, randomization is open for any patients who are ctDNA+ including routine commercial screening. Table of patient characteristics with ctDNA result (n=364) ctDNA+ (n=30) Age < 50 29% 23% Age >50 71% 77% PR + 78% 73% PR - 7% 17% HER2 IHC negative 79% 77% Grade 1 8% 7% Grade 2 53% 50% Grade 3 29% 37% T 1 19% 17% T2 43% 43% T3 27% 27% T4 2% 7% 0 +nodes 12% 12% 1 +node 24% 10% 2 +nodes 15% 13% 3 +nodes 8% 13% >4 +nodes 0 47% Citation Format: Lajos Pusztai, Ekaterina Kalashnikova, Evthokia Hobbs, Ursa Brown-Glaberman, Monica Mita, Paula Klein, Fengting Yan, Sima Ehsani, Wajeeha Razaq, Alison Stopeck, Manali Bhave, Michelle Loch, Sagar Sardesai, Evanthia Roussos Torres, Mark Burkard, Femi Okubanjo, Eric Gauthier, Angel Rodriguez, Minetta Liu, Peter Kabos. Circulating tumor DNA (ctDNA) monitoring of estrogen receptor-positive, human epidermal growth factor receptor 2-negative (ER+/HER2-) high risk breast cancer during adjuvant endocrine therapy [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 PS06-02.

Mitosis is carefully orchestrated by reversible phosphorylation events. Polo-like kinase 1 (Plk1) regulates multiple functions across the kinetochore during mitotic progression. Recently, Bub1 (outer kinetochore) and CENP-U (inner kinetochore) were described as two major sites of Plk1 recruitment to the kinetochore. Here, we report an additional dynamic site of Plk1 recruitment to the inner centromere. Inner centromere docking occurs during late prometaphase and metaphase, exhibiting transient residency at multiple chromosomes. Chromosomes with inner centromere-localized Plk1 have end-on attached microtubules, diminished Spindle Assembly Checkpoint (SAC) components, and low Shugoshin 1 (Sgo1) levels at the inner centromere. Mechanistically, recruitment is driven by Cdk1 activity and requires Plk1’s Polo-Box Domain (PBD). Moreover, inhibition of Bub1 or Protein Phosphatase 2A (PP2A) increases Plk1 recruitment and residency at the inner centromere. Collectively, our data identify a novel pathway for Plk1 recruitment to the inner centromere that is dynamically regulated by counteracting activities of Cdk1 and Bub1/PP2A. ### Competing Interest Statement M.E.B. declares the following: Research funding from Abbvie, Arcus, Apollomics, Elevation Oncology, Endeavor, Genentech, Puma, and Loxo Oncology, Seagen. The other authors declare no competing interests.

Quantitative measures of CIN are crucial to our understanding of its role in cancer. Technological advances have changed the way CIN is quantified, offering increased accuracy and insight. Here, we review measures of CIN through its rise as a field, discuss considerations for its measurement, and look forward to future quantification of CIN.

PURPOSE Somatic and germline testing are increasingly used to estimate risks for patients with cancer. Although both germline testing and somatic testing can identify genetic variants that could change a patient's care and eligible treatments, the aims of these tests and their technologies are fundamentally different and cannot be used interchangeably. This study examines the timing and results of somatic and germline genetic testing for patients with cancer at UW Health. METHODS Eight hundred and seventy-seven participants underwent somatic genetic testing, which was reviewed by the Precision Medicine Molecular Tumor Board (PMMTB). Patients were diagnosed with cancers, including breast, colorectal, endometrial, pancreatic, or ovarian cancer, and met National Comprehensive Cancer Network criteria for germline genetic testing. Germline testing details were collected by medical record review. RESULTS The results of this study found that only 310 patients (35%) had germline evaluation before PMMTB review. The percent of germline pathogenic/likely pathogenic variants identified in actionable genes was 28%. Most germline variants were identified in the BRCA1 (26%) and BRCA2 (28%) genes. In total, 65% (54/83) of germline variants were detected with both germline testing and somatic testing; however, 35% (29/83) of germline variants were not identified on somatic results. These results demonstrate the importance of combination germline and somatic testing. CONCLUSION This study highlights the differences in genetic testing types and demonstrates that conducting germline testing at earlier stages of diagnoses is necessary to identify potentially actionable and treatment-specific variants in patients with cancer.