1017 Background: Endocrine therapy (ET) plus Cyclin-Dependent Kinase 4/6 inhibitors (CDK4/6i) is the standard 1 st line treatment for patients (pts) with Hormone Receptor-positive, Human Epidermal growth factor Receptor 2-negative, advanced Breast Cancer (HR+/HER2- aBC). ET+CDK4/6i continuation beyond progressive disease (PD) detection, plus/minus locoregional therapies, is often used in clinical practice for selected pts with oligoprogressive disease or minimal PD. However, the effectiveness of this approach has never been investigated in large studies. Methods: In this analysis of the multicenter, real-world study PALMARES-2 (NCT06805812), we evaluated clinical characteristics and outcomes in HR+/HER2- aBC pts who continued 1 st line ET+CDK4/6i after detection of PD, as defined by physicians based on clinical-radiological assessment. The primary endpoint was real-world progression-free survival (rwPFS) beyond PD, defined as the time between the detection of the first PD event during 1 st line ET+CDK4/6i and the occurrence of subsequent PD leading to definitive ET+CDK4/6i discontinuation, or patient death. We used multivariable Cox regression models to explore the association of 15 covariates with rwPFS beyond PD. We also assessed real-world overall survival (rwOS), as defined as the time between 1 st line ET+CDK4/6i initiation and patient death. Results: Of 4,236 consecutive pts who initiated ET+CDK4/6i between January 2016 and September 2024 in 27 Italian Institutions, 2,434 pts experienced a PD event. Of these pts, 454 (18.7%) continued the same ET+CDK4/6i beyond PD and underwent radiotherapy (n = 306; 67%), surgery (n = 39; 9%), other locoregional treatments (n = 28; 6%) or no local therapies (n = 81; 18%). Pts continuing ET+CDK4/6i beyond PD were more likely to be younger and premenopausal, to have higher tumor estrogen receptor (ER) and/or progesterone receptor (PgR) expression, to have bone/lymph node metastases, and less likely to have liver metastases (p < 0.05 for all covariates). Median rwPFS beyond PD was 10.5 months (95% CI: 9.7-12). At multivariable analysis, higher tumor ER (p = 0.031)/PgR (p = 0.001) or lower Ki-67 (p < 0.001) expression, better ECOG Performance Status (p = 0.009) and the use of locoregional treatments (p < 0.001) were associated with longer rwPFS beyond PD. Pts continuing ET+CDK4/6i therapy beyond PD had better median rwOS than those discontinuing it after first PD detection (71.3 and 44.7 months, respectively; p < 0.001). Conclusions: This is the largest real-world study to show the effectiveness of 1 st line ET+CDK4/6i continuation beyond PD in HR+/HER2- aBC pts. Our findings support the use of this well-tolerated and effective approach in selected pts, such as those with less biologically aggressive tumors and/or amenable to locoregional therapies. Clinical trial information: NCT06805812 .

Supplementary Table S15: Results of the enrichment analyses looking for binding sites of specific TFs accumulating a greater or a smaller number of genetic variants than expected by change. For each TF and category (mutations significantly increasing or decreasing affinity) the observed and expected fraction of mutations overlapping the TF-bound sites are indicated, along with the difference between these two fractions, and the p-value of the corresponding χ2 test. Considering each TF and the mutations affecting the affinity to its target sites either positively or negatively (based on the p-value of the test) TFs could be either classified as showing significantly more or less mutations than expected, or not significant (ns).

Triple negative breast cancer (TNBC) is historically defined as oestrogen receptor (ER) < 1% and HER2-negative. Increasing evidence supports that ER-low breast cancer (BC) (ER 1-10%) shares similar clinical behaviour and biology with classic TNBC. However, how ER cut-off definitions have been applied as eligibility criteria in clinical trials for TNBC over time remains unclear. In August 2024, the Clinicaltrials.gov database was systematically queried using the following string: "breast cancer" AND "interventional studies" AND "phase II-III-not applicable". We included trials started from January, 1, 2010 to August, 7, 2024 restricted to TNBC, using either ER<1% or any other ER cut-off up to 10%. Differences across study descriptors were assessed using Mann-Whitney U and χ2 tests. A total of 6747 studies were retrieved. Of 620 eligible trials, 474 (76.5%) restricted the inclusion to TNBC and 146 (23.5%) included ER-low tumours. The percentage of trials allowing ER-low was lower for phase III versus phase I and phase II (16.8%, 17.5%, and 27.2%, respectively; p = 0.018); in industry versus investigator-driven studies (11.6% vs 29.1%, p < 0.001), and in metastatic versus early-stage settings (18.0% vs 33.5%, p < 0.001). ER-low inclusion increased over time, particularly in investigator-driven trials (since 2015), while remaining limited in industry-sponsored studies. We evaluated the ER cut-off adopted in trials testing therapies for TNBC. Although more inclusive criteria have been applied in recent years, patients with ER-low disease remain largely excluded from registrational trials, particularly in the metastatic setting, potentially limiting access to effective therapies. Broadening inclusion criteria could improve treatment opportunities for this high-risk population.

Supplementary Figure 6. SIDP results are consistent across ER+ BC cell lines.

Supplementary Figure 12. Validation of DF and IF SIDP hits.

Supplementary Figure 14. TLR5 expression in ER+ cells from normal and cancer samples.

Clinicopathologic characteristics at breast cancer diagnosis and at mBC diagnosis.

Supplementary Figure 8. SIDP identifies regulatory regions with context-dependent activity in MCF7 cells.

Supplementary Figure S1. SIDP identifies Cis Regulatory regions in MCF7 with high reproducibility.

Supplementary Figure 13. The promoters of genes belonging to the TLR5 cascade are active in ER+ breast cancer patients.

BRCA1/2 pathogenic variants (PVs) may influence metastatic dissemination in breast cancer (BC), but existing data are often confounded by differences in subtype distribution among carriers. We assessed associations between germline BRCA1/2 status and metastatic sites in HER2-negative BC. Patients treated for HER2-negative metastatic BC (2007–2025) with known BRCA1/2 mutation status were identified. Metastatic sites at diagnosis and throughout the disease history were collected. CNS involvement included brain metastases and/or leptomeningeal involvement. Among 184 patients (33 BRCA1, 28 BRCA2, 123 noncarriers), 131 presented a HR + BC and 53 a triple negative BC (TNBC). In HR + BC, BRCA1/2 carriers presented less frequently bone metastases at first metastatic diagnosis (31.4

BACKGROUND:Tumour-infiltrating lymphocytes (TILs) are a robust prognostic marker in patients with triple-negative breast cancer. Artificial intelligence (AI)-derived computational tools assessing TILs could improve efficiency, but require independent validation against clinical outcomes. We aimed to compare the prognostic performance of AI-derived TIL scores with pathologist-scored TILs in a large, prospectively collected dataset pooled from randomised controlled trials. METHODS:CATALINA was an independent, external validation study using prospectively collected long-term clinical outcome data pooled from seven randomised clinical trials conducted at multiple sites. We independently evaluated two previously validated AI pipelines that generate five computationally assessed tumour-infiltrating lymphocyte (cTIL) scores by masked, independent deployment of locked models. cTIL scores were correlated with the mean of the pathologist-scored stromal TILs (sTILs) in 220 digitised haematoxylin and eosin whole slide images in a cohort of patients with early-stage triple-negative or HER-2 positive breast cancer, previously scored by trained pathologists in a TIL-reproducibility study. Prognostic performance was assessed in a separate cohort of patients with early triple-negative breast cancer pooled from seven prospective, randomised adjuvant trials. Multivariable Cox regression models adjusted for clinicopathological factors and study heterogeneity assessed associations of cTIL score and sTIL score with invasive disease-free survival, distant disease-free survival, and overall survival. 5-year discrimination was estimated using time-dependent area under the receiver operating characteristic curve (AUC). FINDINGS:Individual data were collated from 1759 patients, of whom 1356 had complete clinicopathological data, pathologist sTIL scores, and cTIL scores available. Modest correlation (r 0·375-0·473) was observed between cTIL scores and the mean pathologist sTIL score. Both sTIL and cTIL were independently associated with 5-year invasive disease-free survival, distant disease-free survival, and overall survival after adjustment for clinicopathological factors (hazard ratio for invasive disease-free survival was 0·73 [95% CI 0·66-0·82]; q<0·0001, distant disease-free survival was 0·70 [0·61-0·79]; q<0·0001, and overall survival was 0·72 [0·63-0·82]; q<0·0001 for sTIL scores and 0·80 [0·73-0·89]; q<0·0001, 0·77 [0·69-0·86]; q<0·0001, and 0·79 [0·70-0·88]; q=0·0002, respectively, for percentage_lymphocyte scores). In models adjusted for clinicopathological variables and sTIL score, cTIL score did not maintain a statistically significant prognostic association. Both sTIL and cTIL scores improved the 5-year AUC over clinicopathological variables alone, while cTIL score did not significantly further improve AUC when combined with clinicopathological variables and sTIL score. INTERPRETATION:Two cTIL models deployed entirely without retraining or modification provided statistically significant prognostic information and improved risk discrimination compared with clinicopathological variables alone in this large, platform-based, independent validation study. Although cTIL score did not incrementally improve prognostication compared with models combining clinicopathological variables with sTIL score, these findings support the application of cTILs as a reproducible prognostic biomarker, particularly in settings where routine or widespread pathologist assessment is unavailable. FUNDING:Breast Cancer Research Foundation (USA).

Supplementary Table S11: De novo coding variants. The table lists all the variants (SNVs and short INDELs) identified in the profiled sub-cohort of 58 SIDV metastatic samples. Chromosome and position on the chromosome (hg38 coordinates) are indicated for each variant, along with the reference, detected alternative allele and its frequency, the gene symbol and the ensemble transcripts, the type of alteration and the codon change, and the identifier of the sample in which the variant was identified.

Supplementary Figure 17. Activation of TLR5 via paracrine signaling.

Supplementary Table S2: sgRNAs sequences and metadata for the SIDP assays. S2.1: for each sgRNA targeting a region in the human genome, an identifier (using the corresponding hg38 genomic coordinates), the DNA sequence, the genomic coordinates (hg38) including the strand, along with efficiency and specificity scores as estimated by CRISPR-do, are provided. S2.2: for each positive control or non-targeting sgRNA, a custom identifier is shown along with the DNA sequence.

Advances in breast cancer (BC) therapy are limited by the absence of well-established biomarkers for DNA-damage targeted treatments. We evaluated the predictive and prognostic value of homologous recombination repair (HRR) deficiency (HRD) by RAD51 nuclear foci and stromal tumour-infiltrating lymphocytes (TILs) in early-stage BC patients with suspected germline susceptibility. Among 291 patients, HRD by RAD51 was found in 78.4% of tumours, and 69.8% had low TILs (<30%). In 178 patients treated with neoadjuvant chemotherapy, pathologic complete response (pCR) was higher in those with HRD vs HRR-proficient (HRP) tumours (52.3% vs 36.4%); RAD51 remained independently associated with pCR (p = 0.03). Overall survival (OS) favoured HRD, with 5-year OS of 89.2% vs 82.8% in HRP (p = 0.009), with stronger evidence in triple-negative TILs-low disease (p = 0.005). These findings support RAD51-based HRD assessment as a predictive and prognostic biomarker that may guide treatment decisions in early-stage BC.

The tissue-level processes underpinning metastatic outgrowth remain unclear. We combined single-cell RNA sequencing, spatial transcriptomics, and AI-supported 3D imaging in human breast cancer with functional investigations in mice to uncover a 3D morphogenetic process essential for macrometastatic expansion. Macrometastases pervasively activate a metastatic trabecular morphogenesis (MTM) gene-expression program that redeploys developmental branching morphogenesis to build macrometastases as a 3D trabecular lattice of epithelial cords. MTMHIGH cells pre-exist in primary tumors destined to metastasize, whereas MTMLOW primaries are non-metastatic and display a compact, expansile growth architecture. Chromatin immunoprecipitation sequencing (ChIP-seq) on metastatic organoids identifies ETV1/4/5 as master regulators of MTM and branching cancer morphogenesis, required for metastatic outgrowth but dispensable for primary tumor take, bulk growth, and initial metastatic dissemination. Spatial and functional analyses reveal stromal fibroblast growth factor (FGF)→fibroblast growth factor receptor (FGFR) signaling as an actionable MTM dependency. Thus, we link metastatic outgrowth to a 3D developmental morphogenetic process, exposing therapeutic vulnerabilities specific to the lethal macrometastatic stage.

Supplementary Table S5: SIDP results in MCF7 grown in white media (-E2). S5.1-5: the tables follow the same structure of S3.1-5.

Importance Young BRCA carriers may undergo breast-conserving surgery (BCS) or mastectomy with or without contralateral risk-reducing mastectomy at the time of an index breast cancer diagnosis. A variety of factors may influence surgical decision-making. Objective To explore surgical patterns of care and factors associated with uptake of bilateral mastectomy among affected BRCA1 or BRCA2 ( BRCA1/2 ) carriers diagnosed with early-onset breast cancer. Design, Setting, and Participants The BRCA BCY Collaboration is an international, hospital-based retrospective cohort study conducted at 109 centers across 5 continents. Women aged 40 years or younger with germline pathogenic or likely pathogenic variants in BRCA1/2 who were diagnosed between January 1, 2000, and December 31, 2020, with invasive stage I to III breast cancer were included. Data were collected between January 2022 and June 2024, and analyses were performed between May 21 and 28, 2025. Main Outcome and Measures The main outcome was the evaluation of surgical management as primary treatment for a first diagnosis of invasive breast cancer, defined as BCS, unilateral mastectomy, or bilateral mastectomy. Results Of 5660 eligible BRCA1/2 carriers, 4715 women (median [IQR] age, 35 [31-38] years) had unilateral stage I to III breast cancer with surgical treatment details available. In the cohort, 1805 patients (38.3%) underwent BCS, 1752 (37.2%) underwent unilateral mastectomy, and 1158 (24.6%) underwent bilateral mastectomy. The proportion of BRCA1/2 carriers undergoing BCS decreased over the study period (55.7% [49 of 88] in 2000 vs 27.0% [82 of 304] in 2020), whereas the use of bilateral mastectomy significantly increased (8.0% [7 of 88] in 2000 vs 44.4% [135 of 304] in 2020). Bilateral mastectomy was more common in women who had genetic testing performed before (248 of 435 [57.0%]) or within 6 months (729 of 1892 [38.5%]) of a breast cancer diagnosis compared with those tested after diagnosis (111 of 2167 [5.1%]). On multivariable analysis, compared with those tested after diagnosis, earlier genetic testing was the factor most strongly associated with bilateral mastectomy receipt (testing prior to diagnosis: adjusted odds ratio, 20.72 [95% CI, 15.27-28.13]; testing at diagnosis: adjusted odds ratio, 6.83 [95% CI, 5.39-8.66]). In subgroup analyses, including 2327 patients who had genetic testing performed before or within 6 months of diagnosis, 977 (42.0%) underwent bilateral mastectomy, with the lowest rates reported in Asia and Africa (130 of 499 [26.1%]) and the highest rates reported in North America (233 of 351 [66.4%]). Conclusions and Relevance The findings of this cohort study of young BRCA1/2 carriers suggest that geographic region and timing of genetic testing were the factors most strongly associated with surgical management.

Supplementary Table S6: SIDP results in T47D grown in white media (-E2) media. S6.1-3: the tables follow the same structure of S3.1-3.