PURPOSE:The role of tumor-infiltrating lymphocytes (TIL) remains unclear in hormone receptor (HR)-positive/HER2-negative breast cancer, particularly in young patients, whose immune microenvironment could be altered by age-related host and tumor differences. EXPERIMENTAL DESIGN:Patients with stage I to III HR-positive/HER2-negative tumors were identified from a prospective cohort study of patients with breast cancer diagnosed at age ≤40 years. Multiplexed immunofluorescence and semiautomated quantitative software measured cytotoxic T, non-CD8 T, T regulatory, exhausted T, and PDL1+ cells in stroma and tumor. Univariate analyses assessed differences in clinicopathologic characteristics by high versus low immune infiltration, divided based on median. TIL subtypes were evaluated as a continuous variable per 10% increase in Cox regression analyses for invasive breast cancer-free survival, distant disease-free survival (DDFS), and overall survival, adjusted for clinicopathologic parameters. RESULTS:Among 390 patients, high immune infiltration was associated with increasing age, Black race, grade 3 tumors, and metaplastic or micropapillary histologic subtypes. Over a median follow-up of 8 years, higher stromal and intratumoral non-CD8 T-cell infiltration, T regulatory-cell infiltration, and PDL1 expression were associated with improved invasive breast cancer-free survival. Higher intratumoral non-CD8 T-cell infiltration, T regulatory-cell infiltration, and PDL1 expression were associated with improved DDFS; higher stromal PDL1 expression was also associated with improved DDFS. Higher intratumoral cytotoxic T-cell infiltration and PDL1 expression were associated with improved overall survival. CONCLUSIONS:Characterization of immune subpopulations could help refine the prognostic value of TILs in young patients with HR-positive breast cancer, who may benefit from risk stratification for treatment individualization. See related commentary by Salgado and Kok, p. 2133.

The intestinal microbiome shapes immune responses and is associated with patient outcomes in cancer following immunotherapy. We evaluated differences between the intestinal microbiome profiles of patients with early-stage invasive breast cancer (BC) and ductal carcinoma in situ (DCIS) by subtype using whole genome metagenomic sequencing. There were no significant differences in microbiome composition between DCIS and invasive BC as measured by alpha diversity (p = 0.20, ANOVA) or beta diversity (p = 0.52, PERMANOVA). Within invasive BC, patients with hormone receptor-positive (HR + )/HER2 + BC differed significantly in beta diversity relative to other subtypes (p < 0.05), with differences in six species (q < 0.25). Bacteroides ovatus was significantly more abundant in patients with stage III BC vs. stage I (p = 0.0003). Functional pathway analysis using HUMAnN3 revealed stage-specific enrichment of amino acid biosynthesis and nucleotide-related pathways. Altogether, these findings highlight potential microbial signatures associated with BC subtype and stage.

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 .

Previously published results demonstrated that the randomized phase 3 IMpassion031 trial met its primary objective: adding atezolizumab to neoadjuvant chemotherapy significantly improved pathologic complete response (pCR) rate in patients with stage II/III triple-negative breast cancer (TNBC). Here we report the prespecified final analysis of the secondary endpoints with 3 years' follow-up, together with exploratory analyses of circulating tumor (ct)DNA. Patients with previously untreated stage II/III TNBC enrolled in 75 academic and community sites in 13 countries were randomized 1:1 to receive neoadjuvant chemotherapy with either peri-operative atezolizumab (n = 165) or preoperative placebo (n = 168). Descriptive secondary endpoints included event-free, disease-free and overall survival. Long-term outcomes favored the atezolizumab group (event-free survival hazard ratio (HR), 0.76; 95% confidence interval (CI), 0.47-1.21; disease-free survival HR, 0.76; 95% CI, 0.44-1.30; overall survival HR, 0.56; 95% CI, 0.30-1.04). Among patients without pCR, 14 of 70 (20%) atezolizumab-treated and 33 of 99 (33%) placebo-treated patients received additional adjuvant therapy, frequently capecitabine. In exploratory biomarker analyses, patients with baseline ctDNA-negative status (6%) had excellent long-term outcomes. Most patients (87%) had cleared ctDNA at surgery. ctDNA-positive status at surgery identified a subset of non-pCR patients with poorest prognosis. Long-term safety was consistent with primary results. These data show that adding atezolizumab to chemotherapy for stage II/III TNBC is associated with favorable long-term outcomes, and ctDNA dynamics provide prognostic value beyond pCR. ClinicalTrials.gov identifier: NCT03197935 .

Supplementary Fig. S3. Summary of protein expression changes after 24 weeks of endocrine treatment (ET) and/or Palbociclib (palbo) treatment.

This cross-sectional study investigates the association between frailty and postoperative complications in oncologic breast surgery among older adults with nonmetastatic breast cancer.

Increasing incidence of breast cancer among young adults (YA), defined by the National Comprehensive Cancer Network (NCCN) as age < 40 years, has amplified concerns over financial toxicity (FT) in this demographic. This systematic review sought to clarify the association between YA status and FT. We queried bibliographic databases for full-text English language publications between 2010 and 2024 reporting FT using validated patient-reported outcome metrics among patients with stage I–III breast cancer. Two reviewers screened and extracted data between June and September 2024. Variables of interest included age and treatment-related adverse events (secondary outcome of interest). Of 4023 articles screened, 66 were included for review. Sixty-four were excluded for including in situ or metastatic disease, multiple cancer histologies, or FT as a parameter of a larger scale without validation for stand-alone use. While no studies compared FT using the NCCN YA threshold (i.e., < 40 versus ≥ 40 years), two studies demonstrated that younger age was associated with worse FT. One study compared patients age ≥ 65 to < 65 years using the Comprehensive Score for Financial Toxicity, and the other considered age as a continuous variable using the Psychological Sense of Economic Hardship Scale. The latter also reported treatment-related symptoms were associated with greater FT after adjusting for age. No study identified the multidimensional domain of FT as a primary outcome among YA versus non-YA women with non-metastatic invasive breast cancer. Two studies showed younger age was associated with greater FT. As FT has been shown to negatively impact patient outcomes, further investigations are needed to support policy efforts and to improve care for younger breast cancer survivors.

Background: Residual disease (RD) after neoadjuvant chemotherapy (NAC) for patients diagnosed with triple-negative breast cancer (TNBC) identifies a high risk population with increased rates of recurrence and poor prognosis. Adjuvant therapy (AT) with capecitabine is a standard management for patients with TNBC-RD based on the CREATE-X study. Alternative approaches, such as adjuvant capecitabine for unselected patients with TNBC (GEICAM 2003-11/CIBOMA 2004-01) have not shown benefit. Real-world utilization of adjuvant capecitabine for patients with TNBC-RD and the association of capecitabine with survival outcomes remains largely unknown. To address this gap, we investigated the utilization of capecitabine and other adjuvant therapies and association with outcome in a large cohort of patients with TNBC receiving NAC in the era prior to routine use of immunotherapy. Methods: Patients with stage I-III TNBC (including estrogen receptor and progesterone receptor <10%) who received NAC and underwent surgery between January 2016 and June 2019 were identified from databases from 3 comprehensive cancer centers. Treatment in the adjuvant setting was categorized as: capecitabine, other systemic therapy, or no AT. Propensity score methods were used to control potential confounding effects from baseline covariates (site, age at diagnosis, race, clinical stage, histology type, genetic status, residual cancer burden/RCB class, HR at diagnosis, HER2 at diagnosis, HR at surgery, HER2 at surgery, nodal disease at surgery, neoadjuvant anthracycline/taxane receipt). Survival outcomes were estimated using Kaplan-Meier and Cox proportional hazards models. Results: Among 977 patients with TNBC, the NAC regimen was primarily anthracycline-taxane (n=611; 62.5%), anthracycline-taxane-platinum (180; 18.4%), or taxane-platinum (39; 4.0%), while 122 (12.5%) patients received single-agent therapy or an alternate regimen. In the total population, 316/977 (32.3%) experienced pathologic complete response (pCR) while 661/977 (67.6%) had RD. Among patients with TNBC-RD, omission of AT was common: 45.1% (298/661) of patients with TNBC-RD did not receive any AT. Adjuvant therapy regimens among patients with TNBC-RD were diverse with 202/661 (30.6%) receiving capecitabine, 115/661 (17.4%) other chemotherapy, and 46/661 (7.0%) only targeted therapy. The primary objective was to evaluate survival outcomes among patients with TNBC-RD who received capecitabine versus no AT. At 3.5 years median follow-up, receipt of capecitabine was associated with significantly improved recurrence-free survival (RFS; hazard ratio/HR 0.70 95% confidence interval/CI 0.54-0.91, p=0.008), distant recurrence-free survival (DRFS; HR 0.71, 95% CI 0.54-0.93, p=0.01), and overall survival (OS; HR 0.66; 95% CI 0.49-0.90, p=0.009). As a sensitivity analysis, receipt of adjuvant capecitabine without any other chemotherapy (n=189) versus no AT demonstrated similar significant association with RFS, DRFS, and OS. Receipt of other adjuvant chemotherapy (n=109; with 67/109 single agent anthracycline, taxane, or platinum) versus no AT did not demonstrate significant association with RFS (HR 0.90, 95% CI 0.57-1.42, p=0.7), DRFS (HR 0.84, 95% CI 0.52-1.34, p=0.5), or OS (HR 0.63, 95% CI 0.37-1.08, p=0.08). Conclusion: In this large cohort of patients with TNBC-RD, omission of adjuvant therapy was common, with less than a third of patients receiving adjuvant capecitabine. However, receipt of adjuvant capecitabine was associated with significantly improved RFS, DRFS, and OS. These results highlight the importance of evaluating adjuvant capecitabine benefit in the immunotherapy era. Citation Format: Ava Strahan, Qingchun Jin, Akshara Singareeka Raghavendra, Danielle Brandes Zakon, Michael Grimm, Melissa E. Hughes, Mathew Cherian, Julie Vincuilla, Tonia Parker, Paolo Tarantino, Elizabeth A. Mittendorf, Tari A. King, Vicente Valero, Debasish Tripathy, Sara M. Tolaney, Nabihah Tayob, Nancy U. Lin, Daniel G. Stover, Ana C. Garrido-Castro, Carlos H. Barcenas. Real World Adjuvant Capecitabine Utilization and Patient Outcomes Among Patients With Triple-Negative Breast Cancer with Residual Disease after Neoadjuvant Chemotherapy [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 PS17-03.

Treatment guidelines recommend multimodal therapy for non-metastatic high-risk breast cancer in older adults. However, older patients may be less likely to receive this due to varying abilities to withstand intensive therapy. We aimed to quantify the incidence of, factors associated with, and reasons behind omission of multimodal therapy in older high-risk breast cancer patients. Women ≥ 70 years diagnosed with stage 2–3 HR−/HER2+ or triple-negative breast cancer were identified in the National Cancer Database, 2010–2020. Multimodal therapy was defined as surgery and systemic therapy; omission of multimodal therapy was defined as patients who did not receive one or both therapies. Chi-square tests were used to assess differences by therapy intensity. Multivariable logistic regression models adjusting for patient and disease-level characteristics were performed to determine the factors associated with therapy omission. Of 22,644 patients, 63.4

Background: Pathologic complete response (pCR) is prognostic in pts with early breast cancer (eBC). This study aims to assess individual prognostic factors for recurrence-free survival (RFS) and overall survival (OS) in pts with pCR vs residual disease (RD). Methods: This retrospective study included pts diagnosed at our institution with TNBC or HER2+ eBC who underwent surgery post-NAT and had available data for pCR/RD, RFS, OS. Clinical tumor size (cT), nodal status (cN), and subtype were evaluated as prognostic factors using Cox models and Kaplan-Meier method, in all pts and in pts with pCR (ypT0/is, ypN0) and RD. RFS and OS were defined as time from surgery to first recurrence or death from any cause, whichever occurred first, and as time from surgery to death from any cause, respectively (p<.05 significant). Results: 863 pts were identified with a median age of 50.2 yrs (21.0-85.4) who underwent surgery between 2016-2021. Median follow-up was 3.5 yrs (2.6-6.0). 387 pts (44.8%) had TNBC, 295 (34.2%) ER+HER2+ eBC, 181 (21.0%) ER-HER2+; 80 (9.3%) were stage I, 602 (69.8%) stage II, 181 (21.0%) stage III. Regarding NAT, 370 pts (42.9%) received chemotherapy-only, 473 (54.8%) received HER2-targeted regimens, 14 (1.6%) received immunotherapy-based regimens. Overall, 374 (43.3%) pts had a pCR, 489 (56.7%) RD. In TNBC, 125 pts (32.3%) had a pCR, 262 (67.7%) RD; in ER+HER2+ eBC, 119 pts (40.3%) had a pCR, 176 (59.7%) RD; in ER-HER2+ eBC, 130 pts (71.8%) had a pCR, 51 (28.2%) RD. 108 pts (12.5%) had disease recurrence, of whom 74 (68.5%) had TNBC, 22 (20.4%) ER+HER2+, 12 (11.1%) ER-HER2+ BC. Most common recurrence sites were brain (39, 36.1%), bone (37, 34.3%) and lung (33, 30.6%).Median survival measures were not reached. 3-yr RFS was 87% for the entire cohort (95% CI, 85-89), 80% (95% CI, 76-84) in TNBC and 93% (95% CI, 91-96) in HER2+ eBC. 3-yr RFS was 98% (95% CI, 97-100) in pts with pCR vs 79% (95% CI, 75-82) in pts with RD. In TNBC, 3-yr RFS was 97% (95% CI, 95-100) in pts with pCR vs 72% (95 CI, 66-77) in pts with RD; in HER2+ eBC, 3-yr RFS was 99% (95% CI, 97-100) in pts with pCR vs 87% (95% CI, 82-92) in pts with RD.Overall, higher cT (cT3-4 vs cT1-2, hazard ratio [HR]: 2.72; 95% CI, 1.88-3.94, p<.001), positive cN (cN+ vs cN0, HR: 2.43; 95% CI, 1.65-3.59, p<.001), TNBC subtype (TNBC vs ER+HER2+, HR: 2.57; 95% CI, 1.63-4.04, p<.001; ER-HER2+ vs ER+HER2+, HR: 0.94; 95% CI, 0.49-1.80, p=.8) and RD (pCR vs RD, HR: 0.11; 95% CI, 0.06-0.22, p<.001) were associated with poorer 3-yr RFS.In pts with pCR, 3-yr RFS was numerically higher in cT1-2 cases (99%) vs cT3-4 (95%) and in cN0 cases (99%) vs cN+ (97%), but differences were not significant (cT3-4 vs cT1-2, HR: 2.97; 95% CI, 0.84-10.52, p=.09; cN+ vs cN0, HR: 2.85; 95% CI, 0.74-11.01, p=.1); stratification by subtype revealed no associations.In pts with RD, higher cT (cT3-4 vs cT1-2, HR: 2.26; 95% CI, 1.54-3.33, p<.001), positive cN (cN+ vs cN0, HR: 2.31; 95% CI, 1.54-3.47, p<.001) and TNBC subtype (ER-HER2+ vs ER+HER2+, HR: 1.66; 95% CI, 0.79-3.49, p=.2; TNBC vs ER+HER2+, HR: 2.42; 95% CI, 1.51-3.86, p<.001) remained associated with poorer 3-yr RFS.In a multivariate model for RFS, no associations were found in pts with pCR. In pts with RD, higher cT (cT3-4 vs cT1-2, HR: 1.86; 95% CI, 1.24-2.80, p=.003), positive cN (cN+ vs cN0, HR: 2.13; 95% CI, 1.39-3.28, p=.001) and TNBC subtype (TNBC vs ER+HER2+, HR: 2.60, 95% CI, 1.63-4.17, p<.001) remained associated poorer 3-yr RFS. These results were consistent using ER<10% to define ER negativity. 3-yr OS will be presented at the meeting. Conclusions: The study confirms that pts experiencing pCR have better outcomes than those with RD. Overall, 3-yr RFS and 3-yr OS varied by cT, cN, and subtype at presentation. In pts with RD, cT, cN, and subtype remained independent prognostic factors. In pts with pCR, few events occurred, and no associations were found. Citation Format: Chiara Corti, Tianyu Li, Alyssa R. Martin, Melissa E. Hughes, Tonia Parker, Tyzaire S. Duporte, Giuseppe Curigliano, Tari A. King, Elizabeth A. Mittendorf, Nancy U. Lin, Nabihah Tayob, Sara M. Tolaney. Impact of Pathologic Response and Individual Prognosis After Neoadjuvant Treatment (NAT) in Patients (pts) with Early HER2+ and Triple-Negative Breast Cancer (TNBC) [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 P5-10-17.

1071 Background: Guidelines define ER+ breast cancer (BC) as ≥1% tumor nuclei staining positive by IHC. Data on managing ER-low tumors (1–10% ER staining) is limited, with mixed evidence suggesting outcomes similar to ER- but a higher risk of death with adjuvant endocrine therapy (ET) omission. We aimed to examine the genomic landscape of ER-low MBC compared to ER+ and ER-. Methods: This retrospective study included consecutive patients (pts) with MBC who consented to clinicopathologic data collection and genomic profiling (OncoPanel) on tumor samples with matched ER IHC through the EMBRACE (Ending Metastatic Breast Cancer for Everyone) program. For multiple sequencing timepoints, the first was analyzed. SNVs, CNVs and TMB were compared among ER groups. Genes altered in > 3% of pts were analyzed for ER status association, with Benjamini-Hochberg adjusted p < 0.2 subjected to Holm-corrected pairwise testing. Results: Between 10/2000-12/2020, 1199 pts were identified:48 ER-low, 797 ER+, and 354 ER-. Median age at diagnosis was 63.8 (34.8-86.8), 64.4 (30.8-96.3), and 62.6 (30.3-92.7) years for ER-low, ER+, and ER- groups, respectively. De novo stage IV disease was observed in 8.3% (4/48) of ER-low, 27.0% (215/797) of ER+, and 17.2% (61/354) of ER- cases. Overall, 801/1199 (66.8%) had metastatic and 398/1199 (33.2%) had primary samples sequenced. 27/48 ER-low (56.3%), 451/797 ER+ (56.6%), and 73/354 ER- (20.6%) pts received ET before sequencing. CDK4/6i were administered in 8/48 ER-low (16.7%), 107/797 ER+ (13.4%), and 5/354 ER- (1.4%) pts prior to sequencing. The most clinically relevant genomic alterations are shown in the Table. TP53 mutations (mts) were more frequent in ER-low vs ER+ BC, and not significantly different between ER-low and ER- tumors. PIK3CA and CDH1 alterations were more frequent in ER-low than ER- BC, with no significant difference compared to ER+. AKT1 and RB1 alterations were significantly higher in ER-low vs ER+ BC. ESR1 mts were not significantly different between ER+ and ER-low BC. Median TMB was higher in ER-low vs ER+ cases, without significant differences between ER-low and ER- cases. Conclusions: ER-low BC has a distinct genomic profile, with high TP53 mts (similar to ER-) and frequent PI3K pathway alterations (typical of ER+). Ongoing analyses of clinicopathologic features and survival across ER-low, ER+, and ER- cohorts will be presented. Characteristic ER+ (N = 797) ER-low (N = 48) ER- (N = 354) ER-low vs ER+ (p value) ER-low vs ER-(p value) ER+ vs ER-(p value) TP53 24% 79% 83% 1.45 x 10^-14 0.689 1.85 x 10^-79 PIK3CA 39% 31% 13% 0.291 0.00292 1.96 x 10^-21 CDH1 19% 17% 3% 0.85 0.00193 6.66x10^-14 AKT1 2% 15% 4% 0.0155 0.0155 0.635 RB1 1% 12% 9% 0.00354 0.35 0.000116 PTEN 9% 10% 14% 0.828 0.828 0.828 ESR1 11% 6% 0% 0.612 0.00322 3.06x10^12 CCND1 (CNV) 17% 12% 3% 0.551 0.0258 2.83 x 10^-11 TMB, median (IQR) 6.844 (4.562) 8.365 (6.917) 7.604 (4.562) 0.018 0.212 0.001

Supplementary Fig. S2. Comprehensive analysis of protein expression changes before and after endocrine treatment

Estrogen Receptor (ER) expression is often lost in treatment-refractory, hormone-receptor (HR) positive metastatic breast cancers (MBC). The biological mechanisms allowing tumors to maintain viability—without the key driver of HR+ disease—are relatively unknown. To better define this subclass of MBCs, we analyzed transcriptome and targeted DNA-sequencing data of HR+/HER2- cases that lost ER at the time of recurrence. A cohort of patients with primary HR+/HER2- breast cancer and subsequent loss of ER in the metastatic setting was identified. Loss of ER was defined as ER<1% in at least one metastatic sample. Transcriptome-capture RNA-sequencing was performed along with differential pathway analysis, PAM50 assignments, and fusion discovery. A subset of cases was analyzed with DNA-sequencing. We identified 46 patients with ER loss and archival tissue. Of them, 21 patient-matched primary and metastatic pairs were interrogated with RNA-seq. The metastases were collected at a median time of 6 years [range 1-14] from the HR+/HER2- primary diagnosis. In contrast to prior studies of patient-matched tumors, metastases harboring ER loss were more often transcriptionally distinct from their primary tumor—with 15/21 metastases (71.4%) having a discordant PAM50 subtype. The most common discordances were tumors switching from LumA to HER2-Enriched or LumA to LumB (both in 5 of 21 cases, 23.8%). Notably, none of the HER2-E cases harbored ERBB2 amplification. Luminal to basal switching was observed in two patients, but case review suggested the metastases being from occult primaries. Differential pathway expression analysis showed upregulation in mTOR, G2M checkpoint, and E2F pathways, suggesting selection of bypass mitogenic signaling in the absence of ER. Druggable features driving this transcriptional remodeling were identified, including an acquired in-frame 3’ BRAF fusion and biallelic loss of NF1 in two LumA to LumB cases. Further integration with clinicopathological features and DNA-level alterations will be presented. Altogether, these results demonstrate significant transcriptomic shifts in HR+/HER2- cancers that lose ER in the metastatic setting. We find ER loss is associated with ER-independent transcriptional reprogramming and multimodal mechanisms of druggable mitogenic pathway activation. In rare cases, an apparent luminal to basal switch occurs—but these events likely stem from occult disease. Importantly, most ER loss cases do not become basal-like, but rather retain their luminal identity in the absence of ER and co-opt other mitogenic pathways to achieve a more proliferative, ER-independent state. We propose that triple-negative disease in the setting of ER loss is biologically unique from canonical basal-like tumors and could benefit from a distinct therapeutic approach. Nolan Priedigkeit, Stefania Morganti, Alinés Lebrón-Torres, Kalie Smith, Melissa E. Hughes, Busem Binoga Kurt, Yvonne Y. Li, Hersh Gupta, Heather A. Parsons, Daniel L. Abravanel, Rinath M. Jeselsohn, Elizabeth A. Mittendorf, Adrian V. Lee, Steffi Oesterreich, Sara M. Tolaney, Andrew D. Cherniack, Nancy U. Lin, Ana C. Garrido-Castro. Luminal reprogramming, druggable mitogenic pathway activation, and rare outgrowth of occult basal cancers define ER loss in HR+/HER2- MBC [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 1319.

Novel anti-HER2 antibody-drug conjugates (ADCs), such as trastuzumab deruxtecan (T-DXd), have shown efficacy in tumors with varying HER2 expression, including HER2-low and even tumors with minimal HER2 presence. This has sparked interest in the biology underlying the HER2 expression spectrum. Using molecular and multiplexed imaging, we revealed distinct immune and stromal features in treatment-naive, hormone receptor-positive (HR+) HER2-low versus HER2-0 tumors. HER2-0 tumors exhibit inflammatory and tissue remodeling gene signatures, with enrichment of APOE⁺ tumor-associated macrophages (TAMs) and DOCK4⁺ CD4 T cells. In contrast, HER2-low tumors are more immunosuppressed, with elevated cell cycle, metabolic, and estrogen signaling pathways, suggesting increased proliferative activity. These findings underscore key biological differences between HR+ HER2-low and HER2-0 breast cancers, and may inform more tailored therapeutic strategies. Statement of significance:This study revealed the distinct biological profiles of HR+ HER2-low and HER2-0 breast tumors. HER2-0 tumors exhibit inflammatory and tissue remodeling signatures, whereas HER2-low tumors have elevated cell cycle, metabolic, and estrogen signaling. These insights may help refine therapeutic approaches to improve outcomes for breast cancer patients.

Trial data demonstrate that omission of select locoregional therapies in women ≥ 70 years with early-stage HR+/HER2− breast cancer does not affect overall survival. However, the potential downstream effects of omitting sentinel lymph node biopsy (SLNB) on rates of adjuvant radiotherapy (RT) and endocrine therapy (ET) are unclear. We examined the association between SLNB omission, RT referral rates, RT receipt, and ET adherence in this population. Patients ≥ 70 years with unifocal cT1-2N0 HR+/HER2− disease undergoing upfront lumpectomy from January 2016 to January 2021 were identified from an institutional database. Radiotherapy referral, receipt, and ET adherence were examined by SLNB receipt. Multivariate logistic regression adjusting for patient and disease-level characteristics was used to assess factors associated with RT receipt and ET adherence. Of 670 patients, 460 (68.7

553 Background: Approximately one third of all TNBC diagnoses are stage 1. No validated biomarker is routinely utilized to guide treatment at this early stage. Methods: Samples from patients with stage I TNBC or ER-low (1-10%) breast cancer undergoing surgery at Dana-Farber/Brigham Cancer Center between 2016 and 2021 were identified. The 10-gene Core Immune Gene (CIG) signature and the 4-gene proliferation signature (both part of the TNBC-DX tool) were derived from extracted RNA. Central evaluation of sTILs was conducted at Dana-Farber, with 5% and 20% used as thresholds. All markers were tested for prediction of recurrence free survival (RFS) using the Kaplan-Meier method. Results: We identified 253 patients with stage I TNBC (n=218) or ER-low tumors (n=35) treated at Dana-Farber. Median age was 61 (31 – 85), most tumors were ductal (89%), high-grade (73%), 48% were >1 cm and 65% received chemotherapy. 5-year RFS in the overall cohort was 86.8%, with numerical variation by tumor size (T1a 100%, T1b 93.8%, T1c 81.7%, p=0.26). Gene signatures and sTILs were obtained for 117 and 123 patients, respectively (both for 110 patients), with their association with outcomes described in Table 1. Median follow-up was 3 years. A total of 20/117 patients (17.1%) had medium-high CIG score, with none experiencing RFS events prior to year 5. Similarly, no recurrence was observed prior to year 5 in 29 patients (24.8%) at the upper CIG quartile (vs 83-88% 5-year RFS in other quartiles). Worse outcomes were seen among patients in the upper quartile of proliferation (5-year RFS 83%, vs 88-100% in other quartiles). Overall, 33/123 patients (26.8%) had high sTILs (>20%) and experienced the highest 5-year RFS (97%, vs 78% if low sTILs). OS data will be presented. Conclusions: High expression of the 10-gene CIG immune signature or high sTILs are associated with numerically improved outcomes in patients with stage I TNBC that did not reach statistical significance, warranting further study as prognostic tools. 3- and 5-year recurrence free survival (RFS) according to gene signatures and sTILs. P values were obtained via Cox proportional hazard models. N 3-year RFS 5-year RFS CIG score - Low- Med-High 9720 91% (85%, 98%)100% (100%, 100%) 89% (80%, 98%)100% (100%, 100%) CIG score (quartiles)- ≤25%- 25-50%- 50-76%- >75% 49102929 94% (87%, 100%)83% (58%, 100%)87% (74%, 100%)100% (100%, 100%) 88% (74%, 100%)83% (58%, 100%)87% (74%, 100%)100% (100%, 100%) Proliferation score - Low- Med-High 5859 96% (89%, 100%)90% (82%, 99%) 90% (78%, 100%)90% (82%, 99%) Proliferation score (quartiles)- ≤25%- 25-50%- 50-76%- >75% 30292929 100% (100%, 100%)90% (77%, 100%)100% (100%, 100%)83% (68%, 100%) 88% (67%, 100%)90% (77%, 100%)100% (100%, 100%)83% (68%, 100%) sTILs- 1-5%- >5-20%- >20% 622833 94% (87%, 100%)91% (81%, 100%)97% (90%, 100%) 78% (63%, 98%)91% (81%, 100%)97% (90%, 100%)

Supplementary Fig. S6. Effect of hormone deprivation and NFKB pathway activation HR+ breast cancer cells.

Background: Adding 2 yrs of adj abema to endocrine therapy (ET) has recently become standard for pts with ER+HER2–, node-positive EBC at high risk of recurrence. Characteristics of pts relapsing on adj abema have not been well characterized. Methods: Pts who received adj abema at a single institution and had recurred were identified. ER, progesterone receptor (PR) and HER2 expression pre- and post-abema were determined, and the duration of treatment (DoT) for adj ET, abema and the first treatment (tx) in the metastatic setting recorded. If NGS was performed at recurrence, genomic alterations related to ET and CDK4/6i resistance were described. Results: As of 04/2024, 163 pts received adj abema, starting between 2018-2024, with 15 (9.2%) experiencing recurrence. Among recurrent pts, median age was 48 yrs (31–86). All recurrences were distant, with the most common sites being liver (n = 6, 40.0%), bone (n = 5, 33.3%), lung (n = 2, 6.7%). 2 pts (13.3%) had germline (g)BRCA2 mutations (mts), 1 pt (6.7%) had a gCHEK2 mt. At diagnosis, stage distribution was: stage I, 2/15 (13.3%); stage II, 5/15 (33.3%); stage III, 8/15 (53.3%); nodal status was: cN0, 4/15 (26.7%); cN1, 9/15 (60.0%); cN2/N3, 2/15 (13.3%). ER IHC staining was ≥50% in 14 pts (93.3%) and >10% in all pts; PR was ≥50% in 6 pts (40.0%), >10% in 8 (53.3%), 1-9% in 5 (33.3%), and <1% in 2 (13.3%). Pts had HER2-low EBC in 7 cases (46.7%), HER2-0 in 7 (46.7%), HER2- (IHC unknown) in 1 (6.7%). 4 pts (26.7%) had upfront surgery, 11 (73.3%) neo-adj tx (chemotherapy [CT], 8/11 [72.7%]; ET, 3/11, [27.3%]). 9 pts (60.0%) had ductal histology, 1 (6.7%) and 5 pts (33.3%) had lobular and mixed, respectively. Pts had well, moderately, and poorly differentiated EBC in 2 (13.3%), 6 (40.0%), and 7 cases (46.7%), respectively. Among pts who received neo-adj CT, pathologic response was available in 6 cases: RCB-II, 1/6 (16.7%), RCB-III, 5/6 (83.3%). 11 pts (73.3%) had ≥4 metastatic lymph nodes. OncotypeDX was requested in 8 pts (53.3%), with RS 11-25 in 3 cases (37.5%) and 26-100 in 5 (62.5%). After surgery, 12 pts (80.0%) received adj ET, 3 (20.0%) CT+ET, no one olaparib. ET was as follows: aromatase inhibitor, 11/15 (73.3%), AI+OFS, 3/15 (20.0%), fulvestrant, 1/15 (6.7%). ER, PR and HER2 could be assessed pre- and post-abema in 12 (80.0%) pts. In this subgroup, 6 pts (50.0%) with strongly positive ER status (ER ≥ 95% in 5/6 cases, ER ≥ 20% in 1/6), had ER ≤ 10% and PR <1% at first recurrence. Of 7 HER2-0 cases, 4 (57.1%) remained HER2-0, and 3 (42.9%) became HER2-low; of 5 HER2-low cases, 3 (60.0%) remained HER2-low, and 2 (40.0%) became HER2-0. 7/15 pts (46.7%) recurred while on abema, 8/15 (53.3%) after stopping/completing abema. Median DoT was 8.0 mos (Q1-3: 3.8-21.2) for abema and 18.5 mos (Q1-3: 7.0-23.0) for ET. Median DoT for the first tx in metastatic setting was 3.0 mos (Q1-3: 1.6-5.0). At data cutoff, 2 pts were still on first-line tx. First-line tx received included clinical trials, 4/15 (26.7%; 3/4, ADC combination trials; 1/4, PARPi+immunotherapy); CT, 5/15 (33.3%); ET+CDK4/6i, 3/15 (20.0%); olaparib, 2/15 (13.3%); ET+capivasertib, 1/15 (6.7%). Among pts with available NGS (n = 10) at recurrence, nearly all of them had genomic alterations in the P53 pathway (n = 9, 90%): n = 5 TP53 oncogenic mts, n = 1 TP53 homozygous deletions, and n = 3 MDM2 high amplifications (amp) (copy number >20). One ESR1 mt and no RB1 mt was detected. Other alterations include PI3K pathway (n = 3; n = 2 PIK3CA mt, n = 1 PTEN mt), RTK alterations (n = 4; n = 2 FGFR1 amp, n = 1 ERBB2 mt, n = 1 FGFR2 amp), and CCND1 amp (n = 3). Conclusions: In this series of 15 pts relapsing on adj abema plus ET for high-risk ER+ EBC, loss of ER expression occurred in 50.0% of evaluable cases. Median DoT for the first tx line (metastatic) was 3 mos. Post-abema genomic alterations include P53 in 90% of cases. Citation Format: Chiara Corti, Alyssa R. Martin, Patrick T. Kurnia, Jorge Gomez Tejeda Zanudo, Melissa E. Hughes, Tonia Parker, Paolo Tarantino, Giuseppe Curigliano, Tari A. King, Elizabeth A. Mittendorf, Nancy U. Lin, Sara M. Tolaney. Retrospective study evaluating the characteristics of patients (pts) with early-stage estrogen receptor-positive (ER+) HER2- breast cancer (EBC) who relapsed on or after adjuvant (adj) abemaciclib (abema) [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 P1-09-07.

PURPOSE:Prognostic staging after neoadjuvant chemotherapy (NACT) is not included in American Joint Commission on Cancer (AJCC) staging. This study addressed this deficiency by including responses to therapy with standardized staging variables in a validated prognostic staging system for patients treated with NACT. METHODS:The National Cancer Database was queried to identify 140,605 patients treated with NACT between 2010 and 2018. Three response categories (no response, partial response, and complete response [pCR]) were created on the basis of comparison of clinical and post-NACT pathologic staging. Univariate and multivariate analyses of clinical stage, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 (HER2), and grade were analyzed for each category. Predictive models for each response category were validated using the bootstrap technique. Calibration plots compared predicted and observed 3-year survival probabilities in the training and validation data sets. RESULTS:Each validated model demonstrated statistically significant survival differences in the postneoadjuvant prognostic stage assignment. Of all patients with a pCR, 94.2% were assigned to postneoadjuvant ypStage I compared with 35.5% of patients with no response. Advancing clinical stage had a progressive but small impact on overall survival (OS) with pCR (high-grade, triple-negative breast cancer [TNBC]: cStage I, 97% v cStage IIIB/IIIC, 91%; grade 2 luminal A: 97% v 91%) but was associated with a profound decrease in OS with no response for TNBC or HER2+ disease (high-grade TNBC 89% v 50%) and less profound for grade 2 luminal A disease with no response (97% v 81%). CONCLUSION:We present a novel, validated prognostic staging system that predicts OS according to the response to NACT. These data will provide AJCC stage assignments for a growing proportion of patients treated with NACT.