Background: Circulating tumor DNA (ctDNA) testing in plasma offers the opportunity to identify early breast cancer (BC) recurrence in patients (pts) who, despite completion of definitive therapy, have molecular residual disease (MRD). ZEST is a randomized, phase 3, double-blind trial evaluating whether niraparib can enhance disease-free survival in pts with BC and ctDNA detection (ctDNA+) without evidence of radiographic recurrence after completion of curative intent therapy. Methods: Pts with stage I–III triple negative breast cancer (TNBC) or BRCA-mutated HER2− BC were eligible. A personalized, tumor-informed ctDNA assay (Signatera) was designed based on whole-exome sequencing of tumor tissue and matched normal blood. Testing for ctDNA-based MRD began any time after the end of definitive treatment (EODT); pts with HR+ disease were allowed concurrent endocrine therapy (stable regimen). Upon ctDNA+, radiographic staging was performed. Those without evidence of metastatic disease were randomized (1:1) to niraparib (200 or 300 mg/day, depending on weight and platelet count) or placebo (PBO). Imaging was performed every 12 weeks. The primary endpoint was safety of niraparib; disease-free survival (measured as time from randomization to recurrence or death from any cause) was also assessed. Results: As of May 8, 2024, a total of 2746 pts were prescreened; 1901 pts had ≥1 ctDNA test result (ctDNA+, 147 [8%]). Of the 1901 pts, median age was 52 (range, 22–88) years; 1683 pts (89%) had TNBC (ctDNA+, 135), and 218 (11%) had BRCA-mutated HR+ disease (ctDNA+, 12); 620 (33%) pts received neoadjuvant therapy, 643 (34%) received adjuvant therapy, and 593 (31%) received both neoadjuvant and adjuvant therapy. Due to low rates of ctDNA+, study enrollment was terminated early. ctDNA+ was 5.2% on first test of pts with ≥1 test, and 4.4% on second or subsequent test of pts with ≥2 tests. Of pts with ctDNA+ (n=147), 66% had ctDNA+ on the first test, 91% on the first or second test, and 59% were ≤6 months from EODT. Compared with pts with ctDNA undetected, pts with ctDNA+ tended to have positive lymph nodes, T3/T4 tumor size, stage III disease, residual disease after neoadjuvant therapy, and received both neoadjuvant and adjuvant therapy. Of the 147 pts with ctDNA+, 72 (49%) had radiographic recurrence upon initial staging before randomization. The rate of radiographic recurrence was 52% (51/98) at first ctDNA+ test and 44% (21/48) at second or later ctDNA+ test. Of pts with ctDNA+, 40 were enrolled and randomized (niraparib, 18; PBO, 22); 36 pts (90%) had TNBC, and 4 pts (10%) had BRCA-mutated HR+ disease. At data cutoff, 6 pts in the niraparib arm and 4 pts in the PBO arm remained on study without radiographic recurrence. Median disease-free survival was 11.4 (95% CI, 5.7–18.2) months for niraparib vs 5.4 (95% CI, 2.8–9.3) months for PBO (hazard ratio, 0.64; 95% CI, 0.30–1.39). No new safety signals were observed for niraparib. Conclusions: The ZEST study was terminated early because of infeasibility of completing enrollment due to a low rate of ctDNA+ and a high rate of metastatic disease at the time of ctDNA+. Although ctDNA testing began any time after EODT, ctDNA+ occurred most frequently on the first test and ≤6 months from EODT. Pts, predominantly those with TNBC, had a high rate of radiographic recurrence at time of ctDNA+, consistent with early recurrence typical of TNBC. These findings have implications for future trial design, emphasizing the importance of early ctDNA testing, and careful selection of criteria defining risk of recurrence. Updated survival data and on-treatment ctDNA dynamics will be presented. Citation Format: Nicholas Turner, Isabel Pimentel, David Cescon, Hiroji Iwata, Wolfgang Janni, Aleksandra Lacko, Sibylle Loibl, Hope Rugo, Gianfilippo Bertelli, Hiroshi Ishiguro, Sumainizah Sukor, Tytti Ahola, Christian Kurzeder, Erin Hofstatter, Laura Austin, Fotini Kouri, Magdalena Zajac, Cheynna Crowley, Yufei Wang, Jon Chung, Angela Silvestro, Melinda Telli. Circulating tumor DNA surveillance in ZEST, a randomized, phase 3, double-blind study of niraparib or placebo in patients w/ triple-negative breast cancer or HER2+ BRCA-mutated breast cancer with molecular residual disease after definitive therapy [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 GS3-01.

PURPOSE To report the results of OPAL (ClinicalTrials.gov identifier: NCT03574779 ) cohort A, a single-arm substudy of niraparib plus dostarlimab and bevacizumab for the treatment of advanced, platinum-resistant ovarian cancer (PROC). METHODS Participants with PROC who received 1-2 previous lines of therapy were treated with niraparib (200 or 300 mg once daily), dostarlimab (500 mg once every 3 weeks for four 21-day cycles, followed by 1,000 mg once every 6 weeks), and bevacizumab (15 mg/kg once every 3 weeks). The primary end point was investigator-assessed objective response rate (ORR) per RECIST v1.1. Safety was also assessed. Exploratory biomarker end points included evaluation of changes in the tumor molecular profile and microenvironment using baseline and on-treatment tumor samples. RESULTS Of 41 enrolled participants (median age, 66.0 years [range, 37-83 years]), 9.8% had tumors that were BRCA-mutated, 19.5% were homologous recombination (HR)–deficient, and 17.1% were HR repair (HRR)–mutated. As of the cutoff date, all participants discontinued treatment. The ORR was 17.1% (80% CI, 9.8 to 27.0), including one complete response (2.4%); the disease control rate was 73.2% (80% CI, 62.3 to 82.2). Two participants withdrew before first postbaseline scan because of adverse events (AEs). Grade ≥3 treatment-emergent AEs were reported in 92.7% of participants, with the most common being hypertension (26.8%). Response was not correlated with BRCA, HRR, HR deficiency (HRD), or PD-L1 status. Changes suggesting immune activation were observed in on-treatment samples after triplet therapy. CONCLUSION Results demonstrated modest activity of niraparib, dostarlimab, and bevacizumab in participants with PROC, many of whom had prognostic factors for poor treatment response. Most participants with response were bevacizumab-naïve. No association was found with HRD, BRCA, or PD-L1 status. AEs were consistent with previous monotherapy reports, except that hypertension was reported more frequently.

Supplementary Data from The Pan-Tumor Landscape of Targetable Kinase Fusions in Circulating Tumor DNA

Gene sets enrichment and associations

Positive percent agreement (PPA) between patient-matched cancer tissue and liquid biopsies.

Gene sets defined and used in this study and Signature strength per sample

Distribution of the ratio of variant allele frequency to ctDNA fraction of mutations and rearrangements in the AR gene.

AR variants of unknown significance and concordance of detection between tissue/liquid.

Distribution of estimated tumor fractions in samples with or without detected copy number amplifications.

Frequencies of genomic alterations between three datasets.

Differentially expressed genes

RNA-seq under drug treatment

Ratio of detection rates in liquid versus tissue.

Supplemental Figure 1. Distribution of EGFR extracellular domain (ECD) mutations in ctDNA samples from patients with CRC. Supplemental Figure 2. Response to crizotinib in a patient with small bowel adenocarcinoma and GOPC-ROS1 fusion. Supplemental Figure 3. Detection of copy number amplification is associated with increased ctDNA fraction. Supplemental Table 1. List of 62 genes sequenced in this study using the FoundationACT ctDNA assay. Supplemental Table 2. Genomic alterations detected in temporally matched ctDNA and tissue samples.

PDF file - 74K, Effect of CDK2 on DNA replication fork dynamics. Replicating DNA was sequentially labeled with CIdU (red) and IdU (green), and the length of double-labeled DNA fibers were measured via confocal microscopy. (A) Distribution of fork rates in unperturbed HCT116 cells and the CDK2-/- derivative cell line. (B) Average fork rates in HCT116 parental and CDK2-/- cells. ** denotes significance (One-tailed paired Student's t-test, p<009). (C) Proportion of labeled fibers that represent replication origins and terminations. Colored arrows represent orientation of replicons.

Alterations in PI3K, WNT, RAS and FGFR components in liquid biopsy.

RNA-seq experiment conditions and QC stats

Variant allele fractions of DDR genes.

Supplementary Figure 1: (A) Survival of mice with DAOY-derived orthotopic tumors at 25 mg/kg MBZ (n=4) or mock-treated (n=3). One mouse from the mock treatment group did not develop symptoms of a growing brain tumor, and was euthanized after surviving for more than six months. This mouse was not found to have a brain tumor, and was therefore omitted from this analysis. (B) RNA was harvested from untreated and treated tumors and assessed by qRT-PCR for expression of GLI1, PTCH1, and PTCH2.