Knutson, Keith L.

564 Background: HER2+ ILC is rare (~5% of ILC), with limited data on long-term adjuvant trastuzumab outcomes. We investigated late recurrence and immune landscapes of HER2+ ILC vs invasive ductal carcinoma (IDC) in the phase III NCCTG N9831 trial (NCT00005970) and a real-world cohort. Methods: We evaluated 3304 early-stage HER2+ breast cancer patients (pts) enrolled in N9831 (122 ILC, 3182 IDC). Recurrence-free survival (RFS) and overall survival (OS) were estimated (Kaplan-Meier) and compared using multivariable Cox regression adjusting for age, tumor size, grade, hormone receptor (HR), and nodal status. Findings were validated in a real-world cohort of HER2+ pts (Caris Life Sciences CODEai platform). Immune microenvironments were profiled by WTS deconvolution (Quantiseq). Real-world overall survival was calculated from trastuzumab start to last contact. Results: In N9831, ILC pts were older (median 54 vs 49 y), more ER+ (74.6% vs 51.4%), and had more N3 disease (25.4% vs 12.7%) compared to IDC. ILC pts derived a comparable magnitude of benefit from adjuvant trastuzumab compared to the overall population (HR 0.58 vs 0.67). However, long-term outcomes diverged: while the 5-year RFS was similar (77.1% vs 82.7%), ILC pts experienced a steep decline between years 5-15 (77.1% to 57.7%; ~20% decline) vs IDC (82.7% to 72.7%; ~10% decline) after multivariable Cox analysis (Table). In the real-world early/advanced cohort (n=1328; 26 ILC vs 1302 non-ILC [subset IDC n=595]) treated with trastuzumab-based therapy, ILC pts had worse mOS (33.7 vs 68.6 m, HR 2.01, 95%CI 1.00-4.07, p=0.04) compared to non-ILC. However, there was no statistically significant difference when compared specifically to IDC (HR 1.77, 95% CI 0.87-3.61, p=0.11). ILC had: i) numerically lower tumor mutation burden (TMB)-high (4.2% vs 9.5%, p=0.38); ii) higher infiltration of B cells, Tregs, and M2 macrophages (p<0.05); iii) upregulated MHC class II expression (HLA-DOA, p<0.05); and iv) downregulated proliferative and DNA repair pathways (E2F, G2M, MYC; FDR <0.25) compared to non-ILC. Conclusions: HER2+ ILC carries late recurrence risk despite initial trastuzumab benefit. The unique immune landscape (Treg/M2 macrophage enrichment and lower proliferative signatures) distinguishes HER2+ ILC from IDC, highlighting the need for tailored strategies. Long-term RFS and OS in HER2-positive ILC vs IDC (N9831 trial). Population N 5-year% (95% CI) 10-year% (95% CI) 15-year% (95% CI) ILC 122 RFS 77.13% (69.9, 85.1) OS 84.04% (77.7, 90.9) RFS 70.57% (62.7, 79.4) OS 76.0% (68.7, 84.2) RFS 57.67% (44.2, 75.1) OS 65.22% (56.6, 75.2) IDC 3182 RFS 82.71% (81.4, 84.1) OS 90.18% (89.1, 91.2) RFS 77.45% (75.9, 78.9) OS 81.81% (80.4, 83.2) RFS 72.68% (70.77, 74.6) OS 76.68% (75, 78.4)

536 Background: Folate receptor alpha (FRα) is overexpressed in ~80% of triple-negative breast cancers (TNBC). We previously demonstrated that patients (pts) with breast cancer naturally mount immune responses to FRα. In prior Phase I and II studies, a GM-CSF-adjuvanted FRα peptide vaccine (FRPV) was safe and induced durable FRα-specific T-cell immunity in 83–90% of pts with breast and ovarian cancer. Methods: Eligible pts were women ≥18 years with TNBC (ER/PR ≤10%) with 1+ of the following: stage ≥T1c disease, node-positivity, or residual disease after neoadjuvant chemotherapy. Pts were randomized 2:1 to receive FRPV or placebo (GM-CSF alone) every 28 days for cycles 2-7 and then every six months for 7 booster doses, with cycle 1 consisting of cyclophosphamide 50mg orally twice daily for days 1-7 and 15-21 (in both arms). Randomization was stratified by chemotherapy setting (adjuvant only vs neoadjuvant) and stage (I/II vs III). FRα expression was evaluated by immunohistochemistry. The study was designed to detect (at 30 months after the last pt enrolled) a disease-free survival (DFS) difference of 14% assuming a 3.5-year DFS rate of 70% in the control group and with 78% power. Results: 280 pts were enrolled (median age 52). The majority had stage I or II tumors (n = 219, 78%). There were numerically more node-positive tumors in the vaccine arm, 78 (41%) vs 27 (32%), p = 0.15. Median follow-up was 3.5 years, and 33 (FRPV: 25, placebo: 8) DFS events were observed; 3.5-year DFS rate was 88% with FRPV and 90% with placebo (HR 1.42, 95% CI 0.64–3.15; p = 0.39). Overall survival (OS) also did not differ by arm (HR 2.48, 95% CI 0.55–11.20; p = 0.22). Among node-positive pts (n = 105), DFS did not differ between arms (HR 0.95, 95% CI 0.34–2.66; p = 0.92). However, a numerical DFS event rate improvement (14% for vaccine vs. 26% for placebo) was observed in 61 patients with stage III disease (HR 0.51, 95% CI 0.16–1.68; p = 0.26). DFS did not differ by chemotherapy setting or by receipt of anthracycline, capecitabine, and/or an immune checkpoint inhibitor. FRPV was well tolerated, with predominantly grade 1–2 adverse events (AEs) and no vaccine-related grade ≥4 AEs. FRα expression was available from tumor in 230 pts, with 72% expressing > 0. Overall, DFS was not significantly associated with FRα expression (0 vs > 0; HR 0.55, 95% CI 0.21–1.44; p = 0.21), and no significant differences in DFS by FRα expression were observed between treatment arms. Conclusions: FRPV following oral cyclophosphamide was safe, with no vaccine-related high-grade AEs. While no significant improvement in DFS or OS was observed overall, a numeric trend toward improved DFS was observed in pts with stage III TNBC. This finding is hypothesis-generating and supports further evaluation of FRα-targeted vaccination in higher-risk TNBC. Immune correlative analyses are ongoing to better define vaccine immunogenicity and its relationship to clinical outcomes. Clinical trial information: W81XWH-15-1-0293.