Antibody-drug conjugates (ADCs) targeting cell surface proteins TROP2 or HER2 are effective in metastatic breast cancer, but the precise clinical contribution of epitope expression is uncertain. We prospectively monitored circulating tumor cells (CTCs) in 33 patients receiving ADC therapies using quantitative imaging. The expression of TROP2 and HER2 are heterogeneous across single CTCs from untreated patients, comparable to matched tumor biopsies, and display poor association with clinical response. Within three weeks of treatment initiation, declining CTC numbers correlate with a durable response (TROP2: median time to progression 391 versus 97 days, HR 4.15, P=0.0046; HER2: 322 versus 66 days, HR 9.12, P=0.0002). Neither TROP2 nor HER2 expression is reduced at progression, compared to matched pretreatment CTCs, and switching ADC epitope while maintaining a similar payload shows poor efficacy. Thus, epitope downregulation is not a common driver of acquired resistance to TROP2 or HER2 ADCs, and second-line ADC therapies may benefit from distinct payloads. SIGNIFICANCE:ADCs target tumor-associated antigens, followed by internalization and release of drug payloads. However, clinical studies of epithelial-targeting ADCs show efficacy despite low tumor epitope expression. Our finding that epitope downregulation does not commonly accompany acquired resistance suggests alternative drivers of clinical efficacy and the need for testing non-cross-resistant payloads to overcome resistance.

511 Background: Sacituzumab govitecan (SG) is a TROP-2 directed antibody-drug conjugate (ADC) approved for metastatic triple negative breast cancer (TNBC). Pembrolizumab (P), an anti-programmed death 1 monoclonal antibody, is approved for early-stage TNBC and metastatic PD-L1 positive TNBC. However, safety and efficacy of SG+P in early TNBC is not known. We published results of Arm A1 investigating neoadjuvant SG monotherapy in early TNBC (Spring et al. Annals of Onc 2024). Here we present results from Arm A2 of the NeoSTAR study investigating the combination of neoadjuvant SG + P in early-stage TNBC (NCT04230109). Methods: Patients (pts) with early TNBC (tumor size ≥2 cm or node positive) with no prior treatment were eligible. Pts received SG at starting dose of 10mg/kg on days 1,8 of a 21-day cycle for 4 cycles with P 200 mg given on day 1 of each cycle. After trial regimen, pts underwent imaging to determine residual radiographic disease per RECIST v1.1. A biopsy was performed if residual disease (RD) was suspected. Additional neoadjuvant chemotherapy (ANACT) was at discretion of the treating physician prior to definitive surgery. The primary objective was rate of pathologic complete response (pCR) with neoadjuvant SG/P. Secondary objectives included need for ANACT, radiographic response (RR), safety and tolerability (adverse events [AEs] per CTCAE v5.0) and event-free survival. A Simon two-stage design and standard descriptive statistics were utilized, including 95% binomial confidence intervals for all rates estimated. Results: From 5/19/23-8/13/24, 50 pts were enrolled (median age: 57 years, range 23-77). Clinical anatomic stage was II in 48 pts (96%) and III in 2 pts (4%). 64% of pts were node negative at diagnosis. 44 pts (88%) completed the trial regimen (5 pts had toxicity, 1 pt progressed on treatment). In interim analysis, 5/15 had pCR and so the remaining 35 were enrolled. The pCR rate per protocol (pts with pCR at surgery directly after SG/P without ANCT) was 16/50 (34%, 95% CI 19.5-46.7). The RR rate (complete CR or partial response PR) was 66% (95% CI 50-78%), 30% CR and 36% PR. Of 26 pts who received ANACT, 9 experienced pCR (2 biopsy-confirmed RD, 6 negative or non-diagnostic RD biopsy, 1 no biopsy). Overall, 25 (50%, 95% CI 35.5-64.5) pts had pCR at surgery. Of 5 pts with pathogenic BRCA mutations, 3 (60%) had pCR after SG/P, and 1 pt had pCR after ANACT. 20 pts (40%) had grade 3 or higher AEs. The most common AEs were nausea (28, 56%), alopecia (26, 52%), fatigue (23, 46%), and diarrhea (22, 44%). Dose reductions of SG occurred in 4 pts (8%). Updated survival and biomarker data will be presented at the meeting. Conclusions: In the first trial to investigate the SG/P combination in early TNBC, 34% of pts had pCR. Additional research is needed to determine the optimal duration and sequence of neoadjuvant SG/P and chemotherapy for pts with TNBC. Clinical trial information: NCT04230109 .

Immune exclusion inhibits anti-tumor immunity and response to immunotherapy, but its mechanisms remain poorly defined. Here, we demonstrate that Trophoblast Cell-Surface Antigen 2 (TROP2), a key target of emerging anti-cancer Antibody Drug Conjugates (ADCs), controls barrier-mediated immune exclusion in Triple-Negative Breast Cancer (TNBC) through Claudin 7 association and tight junction regulation. TROP2 expression is inversely correlated with T cell infiltration and strongly associated with outcomes in TNBC. Loss-of-function and reconstitution experiments demonstrate TROP2 is sufficient to drive tumor progression in vivo in a CD8 T cell-dependent manner, while its loss deregulates expression and localization of multiple tight junction proteins, enabling T cell infiltration. Employing a humanized TROP2 syngeneic TNBC model, we show that TROP2 targeting via Sacituzumab govitecan (SG) induces an anti-PD1 response associated with broad immune cell activation, and TROP2 is highly associated with lack of response to anti-PD1 therapy in human breast cancer. Thus, TROP2 controls an immune exclusion program that can be targeted to enhance immunotherapy response. This work was supported by DOD/CDMRP Grant BC200924 and by R01CA260890 (to L.W. Ellisen, A. Bardia), by the Tracey Davis Memorial Breast Cancer Research Fund (to L.W. Ellisen), and by NIH/NCI K99CA286969 and The Terri Brodeur Breast Cancer Foundation Fellowship (to B. Wu). Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

In this issue of Cancer Cell, Zhang et al. use single-cell RNA sequencing to compare immune cell dynamics in triple-negative breast cancers treated with the PD-L1 inhibitor atezolizumab plus paclitaxel or nab-paclitaxel. They identify distinct T cell activation patterns and highlight mast cells' role in immune activation exclusively in the nab-paclitaxel combination.

Background: Treatment of HR+/HER2- MBC often involves an antiestrogen agent and CDK4/6i, and following disease progression, multiple therapies are approved in the second line. Management is increasingly guided by a precision-based approach, including the use of AKTi in tumors harboring an AKT1 or PIK3CA mutation or PTEN loss. However, little is known regarding molecular factors that mediate resistance to AKTi. Results from the TAKTIC trial demonstrated antitumor activity and tolerability of the AKTi ipatasertib with endocrine therapy (ET) +/- palbociclib post-CDK4/6i (Wander et al., 2023). We hypothesize that next-generation sequencing (NGS) of tumors among patients (pts) receiving ipatasertib could inform genomic predictors of response to AKTi. Methods: TAKTIC was a phase Ib open-label trial evaluating ipatasertib in combination with fulvestrant, an aromatase inhibitor, or fulvestrant + palbociclib, in participants with HR+/HER2- MBC who received ≥1 line of prior therapy for MBC and had exposure to CDK4/6i (NCT03959891). An exploratory objective of TAKTIC was to identify genomic biomarkers that correlate with response to an AKTi-based combination regimen. Blood samples for circulating tumor DNA analysis were drawn at routine timepoints and archival tumor tissue was obtained. Mutational profiling was performed using commercially available NGS-based assays (frequently via Guardant360). Progression free survival (PFS) was estimated using the Kaplan-Meier method, and survival analysis was implemented with the Breslow approximation for ties. Univariable and multivariable hazard ratio (HR) and 95% confidence interval (CI) analyses were estimated using a cox proportional hazards model. Results: TAKTIC accrued 77 pts (6/2019 – 2/2022), enrolling 35 on doublet therapy (AKTi + antiestrogen) and 42 on triplet therapy (AKTi, fulvestrant, and palbociclib). Baseline NGS results were available in 58 of 77 pts, and alterations in PI3K/AKT pathway genes were found in PIK3CA (43%), AKT1 (7%), and PTEN (10%). Mutations in ESR1 (29%) were seen at rates consistent with prior studies in MBC post-ET. A subgroup of 20 pts who received the triplet ipatasertib regimen and had baseline NGS data within 60 days of drug start were analyzed. Mutations in PIK3CA (n=7, 35%; n=5 polyclonal) and PTEN (n=4, 20%; n=1 polyclonal) were detected, as were alterations in ESR1 (n=5, 25%), FGFR1 (n=4, 20%), KRAS (n=4, 20%), and ERBB2 (n=3, 15%); no baseline AKT1 mutations were seen in this subgroup. Univariate gene analysis demonstrated that FGFR1 amplification was associated with shorter PFS (HR 5.42, 95% CI 1.3 – 22.1, P=.019), and ERBB2 alteration trended toward worse outcomes (HR 3.38, 95% CI 0.8 – 13.6, P=.086); no significant difference was seen between ESR1 mutant vs. ESR1 wild-type tumors (HR 1.6, 95% CI 0.6 – 4.6, P=.384). Multivariate gene analysis demonstrated that PIK3CA/AKT1/PTEN altered tumors (n=9) had greater response to the AKTi triplet combination compared to tumors without mutations in this pathway (n=11) (median PFS 505 vs. 114 days, HR 0.2, 95% CI 0.1 – 0.7, P=.015). Breast cancers harboring alterations that upregulate RAS pathway signaling (KRAS/NRAS, BRAF, FGFR1/2, ERBB2, EGFR; n=9) showed a trend toward inferior outcomes compared to non-altered disease (n=11) (median PFS 114 vs. 253 days, HR 2.0, 95% CI 0.8 – 5.3, P=.160). Additional analyses at the individual gene and pathway level will be presented at the meeting. Conclusions: Genomic insights using NGS suggest that MBC post-CDK4/6i is more susceptible to an AKTi-based treatment with ipatasertib in the presence of a PI3K/AKT/PTEN pathway mutation, whereas alterations in FGFR1 are associated with worse outcomes. This effort is one of very few studies prospectively evaluating mediators of AKTi response, an area of active interest given changes in the therapeutic landscape. The results presented here are hypothesis-generating; future work is underway to further expand upon these data. Citation Format: Maxwell Lloyd, Geoffrey G. Fell, Elizabeth Scott, Jennifer C. Keenan, Laura M. Spring, Jennifer Shin, Steven J. Isakoff, Lianne Ryan, Sarah Padden, Elizabeth Fisher, Amber Newton, Beverly Moy, Andreas Varkaris, Leif W. Ellisen, Douglas S. Micalizzi, Daniel Haber, Dejan Juric, Aditya Bardia, Seth A. Wander. Genomic predictors of response among patients with hormone receptor-positive (HR+)/HER2- metastatic breast cancer (MBC) receiving the AKT inhibitor (AKTi) ipatasertib combined w/ endocrine therapy & a CDK4/6 inhibitor (CDK4/6i) in TAKTIC trial [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 P2-01-17.

1005 Background: Trastuzumab deruxtecan (T-DXd) is FDA-approved for HER2-low, but not HER2-0 metastatic triple negative (TNBC) and hormone positive breast cancer. Therefore, identifying HER2-low status is of great clinical importance. Prior studies have shown HER2-low status in TNBC is dynamic, but the correlation between the number of successive biopsies (Bxs) conducted and the likelihood of a HER2-low result is unknown. Methods: Patients (pts) were identified from an institutional database including all pts with TNBC treated in a single large academic center between 2017-2022. Only pts with TNBC at diagnosis were included. Bxs without known HER2 status were excluded. Pathological, clinical, and demographic data were extracted. HER2-low was defined as HER2 IHC 1+, or 2+ with non-amplified ISH. The type of Bx was categorized as core Bx, surgical Bx, or metastatic Bx based on the timing and method of Bx acquisition. For the early-metastatic matched analysis, the core Bx was considered the early Bx, unless the core Bx was missing and then the surgical bx was used instead. For cases with several metastatic Bxs the first metastatic Bx was used. Results: 529 consecutive pts with TNBC at diagnosis were included. The proportion of pts with HER2-low result increased as the number of successive Bxs increased (60%, 74%, 83%, 87% and 100% when 1 (192 pts), 2 (235 pts), 3 (52 pts), 4 (38 pts), and 5-9 (12 pts) Bxs were conducted, respectively). In women without a prior HER2-low result, about one third converted to HER2-low with each successive additional biopsy (e.g. 322/529 at 1 st biopsy, 44/131 on 2 nd biopsy, 8/25 at 3 rd biopsy, 3/8 at 4 th biopsy). HER2 status distribution did not significantly vary between the different types of Bx (58%, 63%, and 54% of pts had a HER2-low result in their core, surgical or metastatic Bx, respectively; p=0.2). Among 246 women with matched core-surgical biopsies, one quarter changed their HER2 status (55% from low to 0, 44% from 0 to low, and 1% from low to 3+). Core-surgical HER2 status conversion rates did not differ between women who had neoadjuvant therapy with residual disease and women who had surgery as their primary intervention. Among women with both matched early-metastatic (70 pts) or two matched metastatic Bxs (39 pts), nearly half (44%) converted their HER2 status (68%, 26% and 6% or 35%, 59% and 6% were converted from low to 0, 0 to low and low to 3+ in the matched early-metastatic or the two matched metastatic Bxs, respectively). Conclusions: Our findings show that HER2 status is dynamic in pts with TNBC and support the idea that HER2-low is a spectrum, not a specific entity. We further report the novel finding that for pts with TNBC without a prior HER2-low result, repeat Bxs at progression can increase the chance of obtaining a HER2-low result and provide clinically impactful information. Whether the dynamic HER2 result represents underlying biology or analytic variation remains to be determined.

GATA3 mutations are among the most common alterations in hormone receptor-positive (HR+) breast cancer (BC), yet these have no targeted therapies. MDM2 is an E3 ubiquitin ligase that targets p53 for degradation, and pre-clinical data suggests MDM2 inhibition may effectively treat GATA3mut HR+ BC. The GATA3 co-mutational landscape has been described only in primary BC tissue, and the mechanism of MDM2-driven efficacy is incompletely understood. Circulating tumor DNA (ctDNA) was assessed for GATA3 mutations via targeted sequencing. Associations with co-alterations and clinical/pathologic factors were estimated using Pearson's chi-squared test, two-sample Wilcoxon rank-sum, and multivariable logistic regression. Impact on survival was analyzed using multivariable Cox regression analysis. Tissue-based data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database was evaluated for expression and phosphorylation of GATA3 and associated proteins. Among 609 patients with HR + /HER2− MBC, ctDNA detected non-synonymous GATA3 variants ctDNA in 69 (11

PURPOSE:Antibody-drug conjugates (ADC) harboring topoisomerase I (TOP1) inhibitor payloads have improved survival for patients with metastatic breast cancer. However, knowledge of ADC resistance mechanisms and potential impact on the sequential use of ADCs is limited. In this study, we report the incidence and characterization of TOP1 mutations arising in the setting of ADC resistance in metastatic breast cancer. EXPERIMENTAL DESIGN:Patients with metastatic breast cancer treated with ADCs with available posttreatment plasma-based genotyping were included. TOP1 mutation incidence, mutant allele frequency, and functional characterization were assessed, and incidence was compared with that in patients with metastatic breast cancer not receiving ADC treatment and in The Cancer Genome Atlas. RESULTS:Plasma-based genotyping identified distinct TOP1 mutations (S57C, R364H, W401C, and G359E) in 12.9% of patients (4/31) at the time of disease progression on ADC, compared with 0.7% (3/420) in non-ADC-treated patients with metastatic breast cancer and 0.5% in The Cancer Genome Atlas. The appearance of mutations was associated with clinical cross-resistance, as median duration on the first ADC was 455 versus 52 days for the second ADC. The functional characterization of three novel TOP1-mutant proteins demonstrated that all exhibited reduced enzymatic activity, attenuated covalent DNA binding, and resistance to TOP1 inhibitor ADC payloads SN38 and deruxtecan. CONCLUSIONS:We describe the recurrent emergence of functionally altered, resistance-associated TOP1 mutations in vivo under selective pressure from ADCs and the potential impact on mediating cross-resistance to sequential ADCs. TOP1 mutation may represent a biomarker of resistance in this setting, and additional work is needed to optimize biomarkers and ADC payload design to improve outcomes for the sequential use of ADCs. See related commentary by Gwin and Hurvitz, p. 1824.

Triple-negative breast cancer (TNBC) is an aggressive and heterogenous breast cancer subtype. RASAL2 is a RAS GTPase-activating protein (GAP) that has been associated with platinum resistance in TNBC, but the underlying mechanism is unknown. Here, we show that RASAL2 is enriched following neoadjuvant chemotherapy in TNBC patients. This enrichment is specific to the tumour compartment compared to adjacent normal tissues, suggesting that RASAL2 upregulation is tumour-selective. Analyses based on 2D/3D cultures and patient-derived xenograft models reveal that RASAL2 confers cross-resistance to common DNA-damaging chemotherapies other than platinum. Mechanistically, we found that apoptotic signalling is significantly downregulated upon RASAL2 expression. This feature is characterised by substantial alterations in the expression of anti-versus pro-apoptotic factors, pointing to heterogeneous mechanisms. In particular, RASAL2 upregulates BCL2 via activation of the oncogenic transcription co-factor YAP. CREB1, a YAP-interacting protein, was identified as the common transcription factor that binds to the promoter regions of RASAL2 and BCL2, driving their collective expression. A subset of RASAL2 colocalises with BCL2 subcellularly. Both proteins decorate mitochondria, where the high levels of mitochondrial RASAL2-induced BCL2 expression render the organelles refractory to apoptosis. Accordingly, mitochondrial outer membrane permeabilisation assay using live mitochondria from RASAL2-high/chemoresistant tumour cells demonstrated attenuated release of death signal, cytochrome c, when exposed to pro-apoptotic factors BAX and tBID. Similarly, these cells were more resilient towards chemotherapy-induced mitochondrial depolarisation. Together, this work reveals a previously undocumented molecular link between RAS GAP and apoptosis regulation, providing a new mechanistic framework for targeting a subset of chemorefractory tumours.

Hormone receptor positive (HR+) breast tumors are anomalous in their tendency for late recurrence. In the clinic, an mRNA ratio of HOXB13 to IL17RB (H/I) from the primary tumor is used to determine a patient’s risk for late recurrence and benefit of extended adjuvant hormonal therapy. Little is known about the tie between the H/I ratio and the biology leading to late recurrence. Herein we present data from primary breast tumors, in silico analyses and cell-based models that support our hypothesis that dysregulation of one-carbon metabolism and the SAM cycle is linked to the H/I signature and contributes to epithelial-mesenchymal plasticity (EMP), which has been shown to mediate the late recurring phenotype. We conducted proteomics analysis of 86 HR+ breast tumors and identified Choline Dehydrogenase (CHDH) as the most significant differentially expressed protein between HOXB13 high and low groups; with CHDH being associated with HOXB13 low tumors. Notably, RNA expression of CHDH is highly correlated with expression of IL17RB due to a shared promoter region. These links with both HOXB13 and IL17RB led us to hypothesize that CHDH contributes to the phenotype of late recurrence. To determine if signatures of differences in CHDH function could be seen in H/I low and high cell lines we used RNA expression and metabolomics data from the DepMap portal. Betaine, a standard readout metabolite for CHDH function, was significantly elevated in H/I low cell lines, consistent with our previous observations. We also observed that 1-methylnicotinamide, produced by Nicotinamide N-methyltransferase (NNMT), was identified as the metabolite most upregulated in H/I high cell lines. CHDH and NNMT both contribute to one-carbon metabolism in the SAM cycle, with opposing influences. Previous studies have demonstrated that dormancy and late recurrence in the context of HR+ breast cancer is driven by epithelial-mesenchymal plasticity (EMP). To test the association between SAM flux and differing epithelial and mesenchymal phenotypes, we created a CHDH:NNMT score and correlated this against epithelial-mesenchymal phenotypes in patient tumors from the TCGA Firehouse Legacy dataset. A significant association was found in all tumor types, with a conglomerate p-value across tumor types of <10-300. We also observed high SAM levels in tumors associated with markers of an epithelial-like state. We then demonstrated that shRNA knockdown of NNMT is sufficient to increase expression of epithelial proteins in cell lines displaying a mesenchymal phenotype. We will next test the ability of CHDH and NNMT to alter EMP in breast cancer cells and identify EMP associated metabolic pathways. In total, we have demonstrated that metabolic enzymes regulating SAM flux are highly associated with late-recurrence in HR+ breast tumors. Ongoing experiments will assess how metabolism controls epithelial-mesenchymal plasticity that may mediate the phenotype. Bryce Ordway, Isabella Vianna, Johannes Kreuzer, Wilhelm Haas, Dennis Sgroi, Leif W. Ellisen. Metabolic mechanisms underlying late recurrence in hormone receptor positive 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 7472.

6533 Background: A breast biopsy tissue biobank is a valuable resource for studying breast cancer biology and treatment response. However, underrepresentation of patient populations in biobanks limits the generalizability of findings. The aim of this study was to assess potential age and racial/ethnic disparities in the recruitment process for an institutional breast biopsy tissue bank. Methods: Ultrasound-guided (USG) research biopsy cores were collected immediately after routine clinical biopsy from January 2019 to October 2022 at a large academic center. Study eligibility included patients (pts) aged > 18 years undergoing USG clinical breast biopsy with a radiographically evident mass ≥ 0.6 cm in the longest dimension. Eligible pts, identified by a research associate on the day of the biopsy, were invited to participate at the discretion of the radiologist performing the biopsy. Those approached either consented or declined a research biopsy. Demographic and clinical data of eligible pts were extracted from the EMR. Results: 2449 pts underwent USG breast biopsy and 1309 were deemed eligible for a research biopsy. Of the eligible population, 564 (43%) consented to the study, 322 (25%) declined, and 423 (32%) were not approached. Consented pts were younger compared to those who declined or to those not approached (median age 49, 51, and 51 years, respectively; p = .01). Comparison of study groups by age and race categories are shown in the table. Pts > 70 were less likely to be approached compared to pts < 70 (p = .01). However, The likelihood of pts being approached to consent did not differ significantly with age (p=.09 ). Of the eligible pts, 951 (73%) were White, 327 (25%) were non-White (10% Asian, 8% Black, 1% Hispanic, and 7% other race), and race was unknown for 31 (2%) pts. White and non-White pts were equally likely to be approached (p = .3). However, approached non-White pts were significantly less likely to consent compared to White pts (p =.01). Within the non-White population, Black pts were more likely to decline a research biopsy, with only 50% of approached Black pts consenting compared to 66% of White pts, 61% of Asian pts and 55% of pts of other race. Conclusions: We found disparities based on age and race/ethnicity in participation in a breast biopsy tissue biobank. Older pts were less likely to be offered participation but equally likely to consent when invited, while Black pts were equally likely to be offered participation, but less likely to consent when invited, compared to other racial subgroups. Our findings support targeted interventions to increase participation across diverse subgroups of pts. [Table: see text]

Optimal timing and dosing of adjuvant cyclin-dependent kinase (CDK) 4/6 inhibitor in early breast cancer is controversial. This prospective phase II clinical trial investigated tolerability and safety of two ribociclib dosing schedules. Patients with stage I–III hormone receptor-positive (HR+)/HER2– breast cancer on adjuvant endocrine therapy (ET) were randomized to two ribociclib dosing schedules: 400 mg continuous vs 600 mg intermittent, with initiation in early (prior ET < 2 years) vs delayed (prior ET ≥ 2 years) setting. Primary objective was to evaluate safety and tolerability of continuous vs intermittent schedule. Primary endpoint was proportion of patients who discontinued ribociclib before completion of all 12 cycles (measured at 12 months). Recurrence free survival (RFS) and circulating tumor DNA (ctDNA) detection were also evaluated. 81 patients were enrolled. Only six serious adverse events occurred, with no significant difference between treatment arms and no subject deaths. Twenty-five patients (31%) discontinued ribociclib before completion of 12 months, with no significant difference between treatment arms. Ribociclib discontinuation was higher in early vs delayed initiation (36% vs 21%). At median follow-up of 20 months, two patients in the intermittent arm (600 mg; Arm 2) experienced disease recurrence (2-year RFS 97%, 95%CI 88–99%), vs none in the continuous arm (400 mg; Arm 1) (2-year RFS 100%). ctDNA was only identified in the two subjects with recurrent disease at median of 7.5 months prior to radiological recurrence. Ribociclib is a safe and well-tolerated adjunct to adjuvant ET in early-stage breast cancer. Delayed initiation of ribociclib at 400 mg continuous dosing was feasible, better tolerated and associated with promising outcomes. ctDNA detection preceded clinical evidence of recurrence and may be considered as a surveillance tool in breast cancer.

Background: PI3K/AKT/PTEN signaling pathway alterations are present in approximately half of patients with hormone receptor positive (HR+), HER2 negative breast cancer (BC). The PI3K alpha-selective inhibitor (PI3Ki) alpelisib in combination with fulvestrant is approved for patients with PIK3CA-mutated HR+ advanced breast cancer, and the AKT inhibitor (AKTi) capivasertib in combination with fulvestrant is approved for patients with advanced/metastatic HR+/HER2- breast cancer (MBC) with alterations in PIK3CA, AKT1, or PTEN. Patients with PIK3CA mutant MBC are now candidates for both therapies, but there is minimal data to guide sequencing of these agents. Here we report our single-institution experience. Methods: All patients with HR+/HER2- MBC treated at the Mass General Cancer Center who received AKTi (either capivasertib or an experimental AKTi in a clinical trial/TAKTIC) were included. Patients were divided into three categories: those who received AKTi only (control), those who received PI3Ki first and then AKTi (PI3K > AKT) and those who received AKTi and later PI3Ki (AKT > PI3K). Patients could have intervening therapies between sequential PI3Ki/AKTi. Clinical information was abstracted by chart review. Progression-free survival (PFS) was evaluated as time from start of treatment to disease progression or death from any cause. Time on treatment was defined as the start of treatment to discontinuation. 95% confidence intervals were calculated for all mean and median estimates. Results: 56 patients were identified who received AKTi with/without PI3Ki for advanced/metastatic HR+/HER2- breast cancer from August 2019-June 2024. The median age at start of first PI3Ki/AKTi was 61.5 years. 42 patients (76.4%) experienced disease progression on their first AKTi/PI3Ki. 49 patients (89.1%) had known alterations in PIK3CA/AKT/PTEN pathway, and six patients (10.9%) had no known alterations and were treated on trial. Mutations of PIK3CA were present in 30 patients (54.5%), AKT in 8 patients (14.5%), and PTEN in 8 patients (14.5%). 39 patients (69.6%) received AKTi only, 10 patients (17.9%) were in the PI3K > AKT group, and 4 (7.1%) received AKT > PI3K, and the remaining 3 patients (5.4%) received 3 or more AKTi/PI3Ki. Overall median time on first treatment was 5.8 months (95% CI 4.6-11.0 months) and median time on second treatment was 5.4 months (95% CI 2.0-NR). Median progression-free survival (PFS) for patients who received AKTi alone was 5.5 months (95% CI 3.7-9.2 months). For patients who received PI3Ki before AKTi, the median PFS on second treatment (AKTi) was 4.6 months (95% CI 2.0-NR). An analysis of genomic predictors of response is in process and a multi-institution follow-up analysis is planned. Further updates to be presented at the meeting. Conclusions: In this single-institution study, we report median treatment times for patients treated with PI3Ki prior to AKTi and AKTi alone. Patients may still benefit from receiving second-line AKTi after PI3Ki. Biomarker data is needed to drive personalized selection of sequential use of agents targeting the same pathway. Citation Format: Rachel Abelman, Amanda Jung, Laura M. Spring, Geoffrey Fell, Neelima Vidula, Arielle Medford, Janice Kim, Steven J. Isakoff, Beverly Moy, Leif W. Ellisen, Dejan Juric, Aditya Bardia, Andreas Varkaris, Seth A. Wander. Sequencing PIK3CA and AKT Inhibitors in Hormone Receptor-Positive, HER2-Negative Metastatic Breast Cancer: A Retrospective Analysis [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 P3-10-10.

TPS1137 Background: Antibody-drug conjugates (ADCs) have demonstrated substantial improvement in progression free survival (PFS) and overall survival (OS) in phase III clinical trials in patients with metastatic triple negative breast cancer (mTNBC) and hormone receptor positive/HER2-negative (HR+/HER2-) metastatic breast cancer (MBC), offering an effective new treatment strategy. Several outstanding questions impact the use of these drugs clinically, and prospective real-world data is needed. First, it is important to understand the safety and efficacy of these agents in a real-world population with diverse patient characteristics. Second, it is critical to understand the safety and efficacy of these ADCs in sequence. Third, it is essential to identify biomarkers that can help clarify mechanisms of response and resistance to ADCs, which may inform future sequencing and treatment strategies. Methods: This is a multicenter prospective registry study of patients with HER2-negative MBC who are treated with sequential ADCs per standard of care (SOC) with the goal to understand the safety and efficacy of sequential ADCs in a real-world setting (NCT06774027). A total of 100 participants with HER2-negative MBC will be enrolled in this study, either prior to starting their first ADC per SOC (cohort 1 = HR+/HER2-; cohort 2 = mTNBC) or prior to starting their second ADC per SOC (cohort 3 = HR+/HER2-; cohort 4 = mTNBC). The dual primary endpoints are real-world progression free survival (rwPFS) of ADC1 and rwPFS of ADC2. Secondary endpoints include overall response rate (ORR), duration of response (DOR), best overall response (BOR), disease control rate (DCR), and real-world overall survival (rwOS), and safety for each ADC. Exploratory endpoints include translational correlates of response/resistance to ADCs (e.g., circulating tumor DNA, circulating tumor cells, and tissue spatial correlates) and patient-reported outcomes (PROs). rwPFS and rwOS will be estimated by the Kaplan-Meier method. Statistics will be descriptive. Enrollment to start in the first quarter of 2025. Clinical trial information: NCT06774027 .

e13015 Background: Metaplastic breast cancer (MpBC) is a rare, histologically distinct form of breast cancer. Around 90% of MpBC is negative for estrogen receptor (ER), progesterone receptor (PR), and HER2. Treatment strategies for MpBC are extrapolated from triple-negative breast cancer (TNBC) data. MpBC, however, has low representation in guideline-establishing clinical trials. MpBC is associated with inferior chemotherapy response and worse prognosis compared to non-metaplastic (non-Mp) TNBC. There is scarce literature on MpBC response to antibody-drug conjugates (ADCs). We compare clinical outcomes for metastatic MpBC TNBC vs metastatic non-Mp TNBC treated with sacituzumab govitecan (SG), a TROP2-based ADC approved for metastatic TNBC, and assess for targetable genomic and pathological characteristics in MpBC. Methods: Patients (pts) with metastatic TNBC treated at the Mass General Hospital Cancer Center, between 2000 and 2025, were assessed for MpBC and receipt of SG. MpBC cases were interrogated for biomarkers qualifying for targeted therapy: HER2-low status (IHC 1+/2+), combined positive score (CPS), and targetable genomic variants based on next-generation sequencing of plasma and/or tissue ordered by the treating provider. Multivariable Cox regression analysis assessed the association of MpBC under SG treatment with progression-free survival (PFS) and overall survival (OS), adjusting for number of prior therapies and SG combined vs monotherapy. Results: Eighty-five pts with metastatic TNBC were treated with SG, 13 (15%) of whom had MpBC. Two MpBC and 23 non-Mp TNBC pts received SG with a PARP inhibitor (clinical trial), while the remaining pts received SG alone. Both cohorts received a median of 2 prior lines of therapy (range 0-8). Median age at diagnosis was 47 years in MpBC and 48 years in non-Mp TNBC. Median PFS in MpBC was 1.9 months vs 5.6 months for non-Mp TNBC (p = 0.3).Median OS in MpBC was 18.5 months vs 16.7 months in non-Mp TNBC (p = 0.9). Genomic and pathologic annotation of MpBC pts revealed that 8/13 cases were HER2-low (candidacy for trastuzumab deruxtecan), 2/13 cases had a CPS ≥ 10, 3/13 cases had a TMB ≥ 10 mut/Mb (indications for pembrolizumab), 5/13 cases had pathogenic PIK3CA variants, and 2/13 had pathogenic PTEN variants. There were no tumor-agnostic targetable genomic variants. Conclusions: Pts with MpBC treated with SG had a numerically worse PFS compared to non-Mp TNBC. While the difference is not statistically significant, the sample size of MpBC cases was small, and these findings merit evaluation in a larger cohort. Over half of MpBC cases were HER2-low and/or had pathogenic variants, which may broaden treatment options to other ADCs and/or trials. Optimizing MpBC management will require multi-institutional collaboration to assess larger numbers of pts with interrogation for biological rationale and response to targeted therapy.

BACKGROUND:CDK4/6 inhibitors (CDK4/6i) are used for management of hormone receptor-positive (HR+) metastatic breast cancer (MBC), and activation of the RAS/MAPK and PI3K/AKT signalling pathways has been implicated in resistance to these agents. Pathogenic NF1 mutations (pNF1m) dysregulate RAS signalling, but NF1 has not been linked to CDK4/6i resistance. We analysed multi-institutional data, real-world evidence, and preclinical models to characterise the impact of pNF1m on CDK4/6i sensitivity. METHODS:A retrospective cohort of patients with pNF1m tumours were identified from 4 institutions between 2/2015-5/2023 and evaluated for progression-free survival and intrinsic/acquired resistance on CDK4/6i. Real-world clinical-genomic data from GuardantINFORM between 6/2014 and 3/2023 was analysed for associations between pNF1m and time-to-next-treatment or overall survival following CDK4/6i, adjusted using propensity score weighting. We used CRISPR/Cas9 to delete NF1 in MCF7 and T47D breast cancer cells in vitro. NF1-knockout (NF1-KO) and -wild-type (WT) cells were analysed with respect to CDK4/6i sensitivity, MAPK and PI3K pathway activation, and sensitivity to MAPK and PI3K pathway inhibitors. In parallel, we assessed treatment response in a patient-derived organoid (PDO) harbouring NF1 loss, established from an HR+/HER2- breast tumor following progression on a CDK4/6i. FINDINGS:Among 1962 multicentre patients, we identified 38 with HR+/HER2- MBC, pNF1m, and exposure to CDK4/6i. NF1-associated intrinsic or acquired resistance to CDK4/6i was observed in a majority of tumours, and in those with baseline pNF1m on first-line CDK4/6i, a median progression-free survival of 6.2 months was much less than expected in routine practice. Real-world weighted analysis of 1161 patients comparing 28 pNF1m to 1133 NF1 non-altered tumours demonstrated shorter time-to-next-treatment on CDK4/6i regimens (4.2 vs. 12.4 months, hazard ratio 3.14, 95% confidence interval 2.01-4.93) and overall survival (15.8 vs. 45.2 months, hazard ratio 2.04, 95% confidence interval 1.09-3.82). NF1-deleted cells exhibited reduced sensitivity to CDK4/6i with or without oestrogen suppression, which was accompanied by induction of both MAPK and PI3K pathways, the latter of which was exacerbated by CDK4/6i. Blockade of RAS or AKT, but not MEK or ERK, reversed CDK4/6i resistance mediated by NF1 loss in cell lines and the PDO. INTERPRETATION:NF1 mutations are associated with shorter therapy duration on CDK4/6i in MBC. A causal link between NF1 loss and CDK4/6i resistance was supported by experiments in HR + breast cancer cells. NF1 deletion was accompanied by activation of ERK and AKT, and blockade of RAS or AKT combined with CDK4/6i was effective in NF1-deleted cells and an NF1-mutant PDO. FUNDING:Breast Cancer Research Foundation DRC-20-001, National Cancer Institute R01CA273246, National Institute of Health P30 CA142543, Susan G. Komen Breast Cancer Foundation SAB1800010, Department of Defence BC 210406, Mary Kay Ash Foundation International Postdoctoral Scholars in Cancer Research Fellowship.

We discuss a recent manuscript providing recommendations to improve use and access for liquid biopsy in oncology.

Supplementary Figures S1-S7. Supplementary Tables S1-S4

Drugs targeting the DNA damage response (DDR) are widely used in cancer therapy, but resistance to these drugs remains a major clinical challenge. Here, we show that SYCP2, a meiotic protein in the synaptonemal complex, is aberrantly and commonly expressed in breast and ovarian cancers and associated with broad resistance to DDR drugs. Mechanistically, SYCP2 enhances the repair of DNA double-strand breaks (DSBs) through transcription-coupled homologous recombination (TC-HR). SYCP2 promotes R-loop formation at DSBs and facilitates RAD51 recruitment independently of BRCA1. SYCP2 loss impairs RAD51 localization, reduces TC-HR, and renders tumors sensitive to PARP and topoisomerase I (TOP1) inhibitors. Furthermore, our studies of two clinical cohorts find that SYCP2 overexpression correlates with breast cancer resistance to antibody-conjugated TOP1 inhibitor and ovarian cancer resistance to platinum treatment. Collectively, our data suggest that SYCP2 confers cancer cell resistance to DNA-damaging agents by stimulating R-loop-mediated DSB repair, offering opportunities to improve DDR therapy.