Leon‐Ferre, Roberto A.

Abstract Background: Endocrine therapy (ET) with aromatase inhibitors (AIs) plus ovarian function suppression (OFS) is a standard endocrine strategy for premenopausal women with ER+/HER2- breast cancer. However, poor tolerability to AI+OFS limits its use, leaving tamoxifen as the only alternative. Prospective studies demonstrated that premenopausal women treated with neoadjuvant ET whose tumors exhibit Ki-67 ≤ 10% following 4 weeks of therapy achieve 5-year distant disease-free survival > 96%. Problematically, only 41% of patients reach this threshold with tamoxifen compared to 78% with AI+OFS (Nitz JCO 2022). (Z)-endoxifen (ENDX) is a potent selective estrogen receptor modulator that dually targets ERα and PKCβ1. At plasma concentrations of 3-5 ng/mL, it inhibits ERα; at ≥ 500 ng/mL, it targets PKCβ1, leading to AKT suppression. The EVANGELINE trial aims to examine endocrine sensitivity in terms of Week 4 Ki-67 in premenopausal women whose baseline Ki-67 > 10%, and objective response rate after 24 weeks of ENDX + goserelin in premenopausal women with baseline Ki-67≤ 10%. Methods: EVANGELINE (NCT05607004) is a multicenter, open-label, Phase 2 study comprised of three parts. Eligible patients are premenopausal women with Stage IIA/IIB ER+/HER2- breast cancer. • Part 1 (PK Run-in): assessed 40mg vs 80mg ENDX (+/- OFS) in 22 patients. • Part 2: patients with baseline Ki-67 >10% were randomized to 40 mg ENDX + goserelin or exemestane + goserelin for 6 months with the primary objective being 4-week ESD rate. Patients with a baseline Ki-67 ≤10% were assigned to a single-arm cohort of 40 mg ENDX monotherapy (no OFS) to evaluate the 24-week endocrine sensitivity disease rate. • Part 3 (replaces Part 2): all patients are treated with ENDX 40mg daily + goserelin every 28 days. A two stage Phase II design was chosen to assess outcomes as follows: Part 3a: Up to 45 patients with baseline Ki-67> 10% to assess Week 4 Ki-67≤ 10% rate is at least 65%. If 9 or more of these patients have a Week 4 Ki-67>10%, enrollment will be stopped due to futility. Otherwise, enrollment will continue. Part 3b: Up to 20 patients enrolled with baseline Ki-67≤ 10% to assess Week 24 objective response rate. Ki-67 levels are determined by central laboratory and images are centrally reviewed. Secondary objectives include safety, tolerability, surgical outcomes, and biomarker analysis using paired biopsies (baseline, week 4) and surgical specimens. Results: The PK run-in (completed Fall 2024) enrolled 22 patients and assessed ENDX doses (40 mg daily vs 80 mg daily (+/-OFS)). The Week 4 Ki-67 ≤ 10% rate with ENDX was 86% and did not differ according to dose. Early safety, PK, efficacy and compliance data supported the selection of 40 mg ENDX daily as the optimal dose. Enrollment in Part 2 began in May 2025. Part 3 will replace Part 2 with simplification of the trial design. Conclusions: EVANGELINE is the first trial to evaluate (Z)-endoxifen + OFS as neoadjuvant therapy in premenopausal ER+/HER2- breast cancer. By targeting both ER and PKC pathways, ENDX may offer a well-tolerated alternative to AI-based regimens and expand endocrine therapy options for this population. Clinical Trial Registration: NCT05607004 Citation Format: M. P. Goetz, V. J. Suman, C. Lopez, H. Erickson, L. Beaulieu, S. S. Hammer, L. Mina, R. Leon-Ferre, K. N. Hunt, M. Piltin, A. Degnim, J. N. Ingle, J. C. Boughey, B. A. Sarah, J. M. Reid, M. Schellenberg, J. R. Hawse, P. Advani, K. Giridhar, F. Batalini, D. Flora, J. M. Jones, N. A. Bagegni, J. Jakub, V. Abramson, V. Dabak, W. J. Irvin, E. Sima, K. Seema, S. C. Quay. A Phase 2 Trial of (Z)-endoxifen + Goserelin as Neoadjuvant Treatment for Premenopausal Women with ER+, HER2-, Breast Cancer (EVANGELINE) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS5-12-16.

Abstract Introduction: An Our Practice Advisory (OPA), formerly known as Best Practice Advisory (BPA), is an electronic health record (EHR) tool. OPAs identify key parameters to alert providers of potential interventions that could enhance quality of care. Such functionality is increasingly relevant given the rapid pace of new drug approvals. Within the Mayo Clinic system, we previously identified that amongst Mayo Clinic patients (pts) meeting the MonarchE criteria, 28% of pts did not undergo a documented discussion with their provider regarding the role of adjuvant abemaciclib (Premji, SABCS 2024). Further, a Flatiron study identified that only 40% of eligible pts received abemaciclib (Sandoval, Miami Breast 2025). To address this practice gap, in 2024 we began to implement EHR clinical decision support by creating an OPA trigger for pts eligible for an adjuvant CDK 4/6 inhibitor (CDK 4/6i). Here we report on ‘action taken’ versus not for the OPA and the most common reasons for re-prompts. Methods: The Mayo Breast Disease Group designed an OPA including pts who are within 14 months of a clinical or pathologic diagnosis of high-risk early-stage breast cancer. We leveraged structured data captured in the Epic staging forms (Epic Systems) to identify pts with clinical or pathologic T-stage, N-stage, grade, ± Ki-67/ multigene signature to identify ER+/HER2- pts eligible for adjuvant ribociclib or abemaciclib based on FDA approvals. The OPA was triggered by a patient encounter in oncology when endocrine therapy (but not CDK4/6i) was on the active medication list resulting in a hard stop. The OPA gathered limited details about whether adjuvant CDK4/6 inhibitors were discussed and whether the provider followed a link that resulted in prescribing one—considered an indicator of action taken. In this IRB-exempt quality improvement project, we describe the responses captured in the OPA trigger utilizing Epic tools. Results: The OPA went live on 1/10/2025. Over a 5-month period post OPA initiation, we identified 517 OPA alerts among 332 unique pts. Their median age was 62.5 years, and the majority were female (97%). The median time to resolve the OPA was 17.5 seconds (range 2-567 seconds). 12.6% (65/517) of the time there was ‘action taken’ by selecting the hyperlink to potentially start a treatment plan. Providers selected the following OPA pre-set responses: ‘re-prompt in one month’ 248 (48%), ‘patient not a candidate for therapy’ 48 (9%), ‘patient declines’ 69 (13%), ‘outside oncologist will prescribe’ 36 (7%), ‘not discussed’ 44 (9%), and unknown 72 (14%). The most common free text comments among the 517 OPA alerts (summarized) included: ‘revisit in future’ 108 (21%), ‘too early’ 65 (13%), ‘patient declines’ 66 (13%), ‘outside oncologist will start’ 31 (6%), ‘not a candidate’ 26 (5%), and ‘co-morbidities’ 18 (3%). Erroneous OPA prompting occurred in 14 (3%) who previously failed a CDK4/6i due to toxicity and in 18 (3%) for pts currently on a CDK4/6i. 30 (6%) OPA comments noted that they discussed or started therapy that day. Conclusion: The OPA offered clinical decision support and ensured that clinicians considered prescribing CDK 4/6i in all pts identified as eligible, and captured deferrals. Refinements are needed to ensure appropriate timing of OPA and minimize erroneous firing. The OPA remains active and captures the reason for deferral of adjuvant CDK 4/6i prescription. This data pairs real world decision making to better understand clinically meaningful application of improved invasive disease-free survival (iDFS). Citation Format: S. Premji, P. Advani, S. Chumsri, L. Mina, J. Taraba, J. Carroll, J. Hoppenworth, E. Cathcart-Rake, T. Haddad, B. Rudder, G. Choong, D. Idossa, C. O’Sullivan, S. Yadav, K. Ruddy, P. Peethambaram, T. Hobday, M. Goetz, R. Rao, B. Ernst, F. Batalini, R. Leon-Ferre, A. Tevaarwerk, K. Giridhar. Implementing an Our Practice Advisory (OPA) to provide clinical decision support for adjuvant CDK4/6 inhibitors in early-stage hormone receptor positive breast cancer: The Mayo Clinic Experience [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS5-11-10.

Abstract Introduction Serial assessments of circulating tumor cells (CTCs) in metastatic breast cancer (MBC) provide independent prognostic significance for progression free survival and overall survival. A low CTC burden (<5 CTCs / 7.5mL blood) at baseline is favorable prognostically, and reduction of CTCs during treatment indicates response to treatment (Janni, CCR 2025). Despite increasing evidence supporting its clinical validity, real-time serial CTC enumeration has not been utilized broadly in clinical practice in the U.S. The ACT-MBC study is a prospective observational study to evaluate the impact of serial CTC testing on treatment decisions and response assessment (NCT05662345). Provider (physician or advanced practitioner) perception of CTC testing is measured with questionnaires at baseline (PQ1), and for each pt during the first restaging visit (PQ2), and at the end of the study (PQ3). We previously reported PQ1 results (Giridhar, SABCS 2024). Here, we report on results of PQ2. Methods ACT-MBC prospectively enrolled patients (pts) at Mayo Clinic Rochester and Mayo Clinic Health Systems sites (Mankato, Albert Lea and Eau Claire). Pts with MBC were enrolled at time of progressive disease during any line of therapy (TNBC) or ≥2nd line metastatic therapy for ER+/HER2- disease. Pts were tested for CTCs using the FDA-cleared CellSearch® assay which included enumeration and biomarker testing on CTCs for ER, HER2, and PD-L1. The treating clinician determined the regimen and timing of restaging scans. CTC testing was performed once per cycle (every 3-4 weeks) with results returned to the treating provider within 7 days. We descriptively summarized PQ2 responses from providers who had at least one on-treatment CTC result available by the first restaging visit. We report the proportion (90% CI) of providers who agreed or strongly agreed that assessing CTCs enhanced their ability to determine response to therapy and that the CTC results helped guide their decision to continue or change therapy (co-primary endpoints) while accounting for within provider correlation. Results As of June 25, 2025, 15 providers enrolled a median of 2 pts each (range: 1 to 15 pts) and completed PQ2 surveys on 40 pts. 7 providers (47%) practiced in the community setting and 4 (27%) were nurse practitioners. Most pts were female (98%), White (98%), and had ER+/HER2- MBC (80%). Baseline (C1) CTC results were elevated (≥5 CTCs) in 24 (60%) pts (CTC range 0-609). At C2 (n=39), CTC values remained undetected in 7 pts, decreased in 21 pts, increased in 8 pts, and remained unchanged in 3 pts. At PQ2 (n=40), based on clinician interpretation of radiographic findings, results showed 2.5% of pts with complete response, 20% with partial response, 25% with stable disease, 17.5% with mixed response, and 35% with progressive disease. Clinicians reported that CTC results were concordant with imaging results in 34 (85%) responses, discordant with imaging in 7.5% of responses, and unclear in the remaining 7.5% of responses. Most clinicians (82.5%) agreed or strongly agreed that assessing CTCs enhanced their ability to determine response to therapy (90% CI: 68.3%, 96.7%). Further, 70% of providers agreed or strongly agreed that the CTC results helped guide their decision to continue or change therapy (90% CI: 50.8%, 89.2%). 32.5% of the time, CTCs results influenced the timing of the first restaging scans. In 11/25 pts without progressing disease, CTC results influenced the timing of future scans. In the 7/8 pts with a mixed response at first restaging scans with decreasing CTCs, 4 remained on the same systemic therapy. Conclusions Oncologists in academic and community practice responded that serial CTC assessments enhance ability to determine response of therapy, guide decisions to continue or change treatments, and influence timing of radiographic assessments. Citation Format: K. V. Giridhar, D. Zahrieh, M. Hanna, A. Singh, E. Al-Hattab, P. Schumacher, J. Carroll, J. Hoppenworth, T. Haddad, A. Tevaarwerk, B. Rudder, G. M. Choong, S. Basu, R. A. Leon-Ferre, T. O'Brien, A. Lewis, F. de Snoo, L. Stork-Sloots, F. K. Kuhr, S. Lazaro, R. Prendergast, D. S. Spinner, M. P. Goetz. Assessing the impact of real-time serial circulating tumor cell results on the management of patients with metastatic breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-03-10.

Abstract Background: Tumor-infiltrating lymphocytes (TILs) in HER2+ and triple-negative breast cancer (BC) are prognostic and associated with therapy response; however, there are few data regarding distribution and significance of TIL values in ER+ HER2-negative (HER2-) MBC patients (pts) treated with CDK4/6 inhibitors (CDK4/6i). CDK4/6 inhibition may promote antitumor immunity by production of type III interferons and tumor antigen presentation, and by inhibition of regulatory T cells (Goel, Nature 2017). Further, the addition of immune checkpoint therapy to palbociclib numerically prolonged PFS in the 2nd line (2L) setting (Mayer, JCO 2024). We aimed to investigate the prognostic role of TILs derived from fresh metastatic tumor biopsies in pts with ER+HER2- MBC prior to starting CDK4/6i and endocrine therapy (ET) in the first line (1L) and 2L setting. Methods: PROMISE (NCT0281902) is a multicenter, prospective study that enrolled women commencing the CDK4/6i palbociclib (Pb) with letrozole (1L) or fulvestrant (2L) for HR+HER2- MBC. The primary goal was to identify novel genomic variants and pathways associated with PFS. Here, we performed a secondary analysis to quantitate stromal TILs using protocol mandated pretreatment tumor biopsies and determine their association with PFS. Using the Kaplan-Meier method, we reported median PFS (mPFS) and 3-year PFS percentage (%) (95% confidence intervals [CIs]) by TILs [≥20% vs <20%] separately within 1L and 2L. As sample TIL values were generally concentrated at zero (27/58 = 46.6%), coupled with wide spread of sample TIL values ≥20% [range: 20%- 80%], we applied a ≥20% threshold for PFS analysis (high vs low TIL group) as described by Luen, Salgado et al. Lancet Oncol 2017. Statistical significance was based on the Wilcoxon test of the null hypothesis that there was no difference in the PFS curves by TILs. PFS is reported at median follow-up of 21 months. Results: Pretreatment tumor biopsies from 58/68 (85.3 %) pts were evaluable (42 in 1L and 16 in 2L). Median [Q1, Q2] TIL values were higher in 1L (5% [0, 10]) vs 2L (0% [0, 15]). The most common biopsy sites were bone (44.8%), lymph node (17.2%), liver (17.2%) and breast (12%). Overall, TILs frequency (>20%) by the 4 most common biopsy sites were bone (2/20, 10%), lymph node (4/7, 57%), liver (2/6, 33%), and breast (1/7, 14%). In 1L, although PFS was not significantly different (P = 0.237), mPFS (44 vs 34 months) and 3-year PFS 75.0%, [95% CI: 50.3%, 100%] vs 46.4% [95% CI: 31.0%, 69.5%]) were longer in pts with high versus low TILS, respectively. In 2L, the 3-year PFS was numerically similar comparing high and low TILs groups, (25.0%, [95% CI: 4.6%, 100%] vs 22.5% [95% CI: 6.7%, 76.1%]) with no significant difference in PFS (P = 0.761). Conclusions: In pts with ER+HER2- MBC treated with Pb and ET, PFS was numerically longer comparing high vs low TIL values in 1L but not 2L settings. Future studies evaluating TILs in larger datasets are needed to validate these findings. Genomic analysis (DNA/RNA) and its association with TIL levels is underway and will be presented at the meeting. Funding: The Center for Individualized Medicine, Mayo Clinic; Mayo Clinic Breast Specialized Program of Research Excellence (SPORE P50CA116201); Mayo Clinic Cancer Center (CA15083-40A2); TEMPUS (https://www.tempus.com/) and Conquer Cancer Foundation of ASCO (Career Development Award 2018, Dr. O’Sullivan) Citation Format: C. C. O'Sullivan, A. M. Moyer, A. Nassar, D. M. Zahrieh, V. Suman, X. Tang, K. J. Thompson, R. C. Hentz, J. J. Harrington, A. Moreno-Aspitia, D. W. Northfelt, T. C. Haddad, S. Chumsri, P. P. Peethambaram, K. J. Ruddy, K. V. Giridhar, R. A. Leon-Ferre, R. Weinshilboum, L. Wang, K. R. Kalari, M. P. Goetz. Tumor-infiltrating lymphocytes in advanced ER-positive, HER2-negative breast cancer prior to treatment with palbociclib and endocrine therapy: a retrospective analysis of the PROMISE study [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS1-10-22.

Abstract Introduction: Metaplastic breast cancer (MpBC) represents a rare subtype, constituting <1% of all breast cancer cases. It is pathologically characterized by epithelial and mesenchymal cellular components exhibiting a basal molecular pattern, hormone receptor and HER2-negativity, and high grade. Clinically, MpBC exhibits an aggressive course with early disease progression and poor survival. Antibody-drug conjugates (ADCs) have been shown to penetrate the central nervous system (CNS); however, little is known about the response of ADCs in MpBC, particularly in patients with CNS disease. Here we aimed to describe features of CNS disease in a subset of patients (pts) who received an ADC for MpBC . Methods: We conducted a multi-institutional retrospective observational study of pts with advanced MpBC treated with ADCs at 4 NCI-designated cancer centers (abstract #A-345). This analysis includes a subset of this cohort with CNS metastases, with the aim of examining the incidence, characteristics, and treatment patterns. Clinical outcomes were evaluated for pts with information available, including CNS median progression-free survival (mPFS; defined from initiation of systemic therapy to time of further documented CNS progression or death) and median real world PFS (rwPFS; defined from time of treatment initiation to documented disease progression or death). Results: We identified 48 pts with MpBC who received an ADC. Among them, we identified 15 (31%) who developed CNS metastases. Of these, the majority (n=11, 73%) had triple-negative breast cancer, and 4 (27%) had HER2-positive breast cancer. The median age was 50 years (32-75 years). The median interval from the initial MpBC diagnosis to CNS metastasis was 13 months (0-54 months). Excluding those with de novo CNS disease, the median interval from time of metastatic diagnosis to development of CNS metastasis [mets] was 3 months (0-22 months). 4 (27%) pts had de-novo metastatic disease, of which 2 (50%) presented with CNS disease. 4 (27%) pts had screening CNS imaging prior to their diagnosis of CNS metastatic disease. At the time of CNS disease diagnosis, 9 (60%) pts had oligometastatic intraparenchymal CNS disease (defined as <5 CNS mets), 5 (33%) had diffuse (>5 CNS mets) CNS disease, and 1 (6%) had leptomeningeal and intraparenchymal disease. Most pts 14(93%) with CNS disease were symptomatic at time of diagnosis. One pt underwent resection, 9 (60%) underwent stereotactic radiosurgery (SRS), 6 (40%) underwent whole brain radiation therapy (WBRT), and 1 pt received intrathecal chemotherapy with methotrexate. The median number of lines in the metastatic setting prior to documented CNS disease was 1 (range 0-3). The majority 13 (87%) underwent a change in systemic therapy at time of CNS disease diagnosis, with 60% changing to an ADC at the time of CNS diagnosis or progression. Among pts with progression of CNS disease warranting change in systemic therapy, the median time from first CNS event to further CNS progression was 6.5 months. Median CNS PFS was 3.5 months (range 1 -74 months) and median rwPFS was 4.4 months. For those patients who used ADCs after diagnosis of metastatic CNS diagnosis (12/15), median rwPFS was 4 months. Conclusions: Within this CNS sub-cohort study, we found that 31% of pts with MpBC developed CNS metastasis. The majority of pts had neurological symptoms at diagnosis, but most pts did not undergo routing screening for CNS disease. Given the high rate of CNS mets within a short timeframe from diagnosis with poor outcomes, CNS screening should be considered. The extremely short median CNS PFS and low median rwPFS of this cohort highlight the guarded prognosis associated with MpBC. Given the small sample size of this cohort, larger studies are warranted to validate these findings. Citation Format: S. Premji, S. Jacob, A. LeVee, S. Marion, S. Fisch, L. Huppert, J. Mortimer, D. Schmolze, A. Nayak, A. DeMichele, P. Shah, R. Leon-Ferre, D. Idossa. Central nervous system disease in patients with metaplastic breast cancer receiving antibody drug conjugates: a multi-institutional study [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS1-09-07.

Triple-negative breast cancer (TNBC) remains the most aggressive subtype of breast cancer, with a higher risk of recurrence in the early-stage setting compared to other subtypes. While TNBC is defined as breast cancer that lacks estrogen receptor and progesterone receptor expression and is without human epidermal growth factor receptor 2 (HER2) overexpression, it is increasingly recognized as a very heterogeneous disease. Utilization of neoadjuvant chemotherapy (NAC), with or without immunotherapy (depending on the clinical stage), has significantly improved clinical outcomes in early-stage TNBC (particularly in Stage II and III disease). Use of NAC offers the opportunity to de-escalate surgical treatment and evaluate treatment response, allowing for improved prognostication and further tailoring of post-operative systemic therapy. However, there continues to be a need for the personalization of systemic therapy strategies according to recurrence risk. More effective systemic therapies are still needed for patients who have poor response to NAC. Conversely, there remains a need for the identification of appropriate candidates for systemic therapy de-escalation, particularly given the potentially life-altering toxicities of current chemo-immunotherapy strategies. In this review the authors outline the current neoadjuvant paradigm for early-stage TNBC and emerging therapeutic strategies in this challenging disease state, along with questions that remain unanswered in the field.

THE theme of this year’s American Society of Clinical Oncology (ASCO) Annual Meeting was ‘Driving Knowledge to Action: Building a Better Future’. This year’s annual meeting delivered on this theme with instant practice changing and practice informing presentations. Several highly anticipated breast cancer randomized clinical trials that could result in new indications were reported.

1089 Background: T-DXd has become an important treatment option in mBC and other malignancies. Interstitial lung disease/pneumonitis (ILD) occurred in 10-14% of pts in the DESTINY-Breast trials (0-2% G5 ILD), and with potential lower incidence/severity in earlier line settings (Krop et al, ASCO 2023). We previously reported the Mayo Clinic Rochester, MN experience with T-DXd related ILD in mBC (Hoppenworth et al, ASCO 2024). Here, we expand the data to include patients treated at all locations of the Mayo Clinic Enterprise. Methods: We retrospectively identified pts with mBC who received ≥1 dose of T-DXd across the Mayo Clinic enterprise (Rochester, MN; Mayo Clinic Health System locations in MN/WI; Scottsdale, AZ; and Jacksonville, FL) between July 2022-December 2023. Demographic, mBC characteristics, and pulmonary clinical variables were abstracted from the clinical records. Data were summarized using descriptive statistics. Diagnosis of ILD was determined by treating clinicians, and severity was approximated to CTCAE V5 based on clinical documentation. Results: 252 pts with mBC received T-DXd during the study period. The majority were Caucasian (86%) and female (99%) with a median age of 63. 91 pts (36%) were current/formers smokers and 97 (39%) had prior pulmonary comorbidities. The majority had HER2 low (155, 62%) and HR positive (164, 65%) mBC. 35 (14%) developed any grade ILD [G1: 6 (17%), G2: 13 (37%), G3: 3 (8.6%), G4: 3 (8.6%), G5: 10 (29%)], with 15 (43%) presenting with ≥G3. 29 (83%) presented with at least 1 symptom (cough or SOB). Among those with previous pulmonary toxicity, 4 (33%) had previous pneumonitis in the ILD cohort. The median prior lines of all therapies (including endocrine therapy) were 5, with a median of 3 prior lines of chemotherapy in both the ILD (range 1-13) and non-ILD cohorts (range 1-13). Median onset to any grade ILD was 7 cycles. 20 pts (57%) received steroids for ILD. 14 (40%) had a bronchoscopy, all had a CT chest, 16 (46%) were hospitalized, 7 (20%) were intubated and 1 (3%) had a lung biopsy. Pts with G5 ILD had a median of 3 lines of chemo and a median of 8 cycles prior to onset of ILD, compared to 3 lines of chemo and a median of 7 cycles for those with G1-4 ILD. G5 pts were all Caucasian females, 5 were former smokers, 5 were non-smokers, 1 had a history of pneumonitis. T-DXd was rechallenged in 4 pts (G1-2 ILD) without ILD recurrence. Conclusions: In this retrospective case series,14% of pts treated with T-DXd experienced any grade ILD (4% G5) which is in alignment with the rate observed in the pivotal DESTINY-Breast trials, though with higher rates of G5 toxicity. Among those with ILD, increased lines of therapy were not associated with increased risk. Further research is needed to correlate risk.

529 Background: Neoadjuvant chemotherapy (NAC) is commonly administered to patients (pts) with estrogen receptor alpha (ER) negative (immunohistochemistry [IHC] 0%) and ER-low (IHC 1-10%) breast cancer (BC). For pts without pathological complete response (pCR), ER expression may differ between baseline and residual BC following NAC. We previously demonstrated adjuvant endocrine therapy (ET) omission in ER-low early-stage BC was associated with significantly worse overall survival (OS); however, this effect was restricted to pts with residual BC following NAC and those with higher baseline ER levels (IHC 6-10%) (Choong et al., ASCO 2024). Here, we assessed how often ER was expressed in residual invasive BC for pts treated with NAC for ER-negative and ER-low BC. Methods: Weidentified pts with pre-treatment (tx) ER-negative and ER-low (regardless of HER2 expression) stage I-III BC treated with NAC who underwent BC surgery at Mayo Clinic Rochester between 2009 and 2023. ER IHC was performed in a CAP/CLIA laboratory. We evaluated ER expression in residual invasive BC for pts without pCR. The percent of pts with a post-tx change in ER status was estimated and reported with 95% Wilson confidence intervals. Results: 955 pts (838 [88%] pre-tx ER-negative; 117 [12%] pre-tx ER-low) met inclusion criteria, of whom 69% had HER2-negative and 31% HER2-positive BC. The median age at diagnosis was 52 (range: 24-86). 496 (52%) had residual BC. Residual BC was more common in HER2-negative versus (vs) positive tumors (56% vs 42%, p < 0.001) but did not differ significantly by ER status (ER-negative 51% vs ER-low 57%, p = 0.22). Of those with residual BC, 277/496 (56%) had ER re-testing. Rates of ER re-testing did not vary for pre-tx ER-negative vs -low (57% vs 51%, p = 0.37) but were significantly lower for HER2-positive vs HER2-negative tumors (39% vs 62%, p < 0.001). Among those with post-tx testing, 31/277 (11%, 95% CI: 8-15%) had an increase in ER expression from pretreatment levels (defined as ER IHC < 1% to either 1-10% or > 10%; or ER IHC 1-10% to > 10%). In these 31 pts, the original NAC was for either TNBC (21/31; 68%) or HER2+ BC (10/31; 32%). In pts with pre-tx ER-negative BC, 27/243 (11%, 95% CI: 8-16%) had ER expression in the residual BC including 14/243 (6%, 95% CI: 3-9%) with ER > 10% and 13/243 (5%, 95% CI: 3-9%) with ER 1-10%. For pts with baseline ER-low BC, 4/34 (12%, 95% CI: 5-27%) had ER > 10% in the residual BC. Among the 31 pts where ER increased following NAC, 21/31 (68%) received adjuvant ET, including 17/18 if ER was > 10% in the residual disease and 4/13 in those with ER 1-10%. Conclusions: In pts treated with NAC for ER-negative or ER-low BC not achieving pCR, we identified higher ER expression in the residual breast cancer in > 10% of pts. Given that omission of ET in ER-low BC with residual cancer following NAC is associated with worse survival, repeat biomarker testing should be considered in those without pCR, and ET individualized according to ER expression.

569 Background: Several studies established the prognostic role of both the amount and locations of tumor-infiltrating lymphocytes (TILs) in TNBC. Three distinct immunotypes were described based on the amount and locations of TILs: immune enriched (IN), immune excluded, and immune desert. Using single-cell spatial transcriptomic analysis in the Mayo Clinic TNBC cohort, our previous studies showed the central role of interferon (IFN) signaling in IN phenotype. Herein, we evaluated the association between IFN and outcomes in TNBC in 3 independent datasets. Methods: NanoString IO360 was performed in 114 samples from FinXX (NCT00114816) to generate 22-gene IFNα and 33-gene IFNγ signatures. RNA sequencing was performed in 388 samples from CALGB 40603 (NCT00861705). 3038 TNBC samples were tested by WTS (NovaSeq; Caris Life Sciences, Phoenix, AZ). Median values were used as cut-offs for high vs low IFNγ RNA expression and 18-gene IFNγ signature scores. Caris Life Science CODEai was used to evaluate real-world overall survival (OS) obtained from insurance claims and calculated from tissue collection to last contact using Kaplan-Meier estimates. Chi-square, Mann-Whitney U, ANOVA, and Cox regression were used. Results: A high 22-gene IFNα signature score was associated with significantly improved recurrence-free survival (RFS) in FinXX (hazard ratio [HR] 0.32, 95% confidence interval [CI] 0.14-0.74, p 0.007) and OS (HR 0.28, 95%CI 0.12-0.66, p 0.003). Similar findings were observed with 33-gene IFNγ signature with significant improvement in RFS (HR 0.21, 95%CI 0.09-0.51, p < 0.001) and OS (HR 0.18, 95%CI 0.08-0.44, p < 0.001). Furthermore, in CALGB 40603, both IFNα and IFNγ scores were positively associated with pathologic complete response (pCR: IFNα p 0.019 and IFNγ p 0.007) and residual cancer burden (RCB: IFNα p 0.044 and IFNγ p 0.013). Using the Caris data platform to further validate, we identified 2899 TNBC patients (pts) with genomic and clinical outcome data. High IFNγ expression was associated with significant improvement in OS (25.95 vs 17.43 months; HR 0.65, 95% CI 0.59 – 0.72, p < 0.001). Similarly, pts with high IFNγ signature scores had significant improvement in median OS (25.79 vs 16.22 months; HR 0.66, 95% CI 0.6 – 0.73, p < 0.001). Conclusions: This study underscores the pivotal role of IFN signaling in TNBC. High IFNα and IFNγ signatures were consistently associated with improved RFS, OS, higher pCR rates, and lower RCB across clinical trial cohorts and real-world data. These findings signify IFN signaling as a potential key biomarker and therapeutic target in TNBC. Support: U10CA180821, U10CA180882, U24CA196171; Breast Cancer Research Foundation, Mayo Clinic Breast Cancer SPORE (P50CA116201-17), Bankhead Coley, W81XWH-15-1-0292, P50CA015083, R35CA253187; https://acknowledgments.alliancefound.org . Genentech. Clinical trial information: NCT00114816 and NCT00861705 .