Deshmukh, Sachin Kumar

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)

1024 Background: Invasive lobular carcinoma (ILC) is a distinct breast cancer (BC) subtype with dissemination patterns differing from invasive breast carcinoma of no special type (IBC-NST). While genomic differences between ILC and IBC-NST have been described, the extent to which molecular and immune profiles vary by metastatic organ and histology is poorly characterized. Methods: We conducted a retrospective analysis to evaluate organ- and histology-specific molecular features of metastatic BC (mBC). Patients with metastatic IBC-NST or pure ILC underwent NGS (592, NextSeq; WES/WTS, NovaSeq; Caris Life Sciences). Tumor mutational burden (high ≥10 mut/Mb), PD-L1 expression (22C3), and immune cell fractions (RNAseq deconvolution, quanTIseq) were assessed. Analyses were restricted to sites with ≥10 biopsies per histology. Comparisons used chi-square or Mann–Whitney U tests with multiple testing correction (q < 0.05). Results: A total of 2645 mBC biopsies (605 ILC; 2040 IBC-NST) were analyzed (Table). In ILC, ERBB2 mut frequency differed by metastatic site (q<0.01), with higher prevalence in liver (32.7%) and lower in gastrointestinal (GI) lesions (3.7%). In IBC-NST, ESR1 and GATA3 mut and FGFR1 amplification were enriched in liver metastases, whereas PD-L1 expression was highest in skin (20.7%) and lowest in liver (5.0%). No organ-specific differences were observed for PIK3CA, AKT, PTEN, BRCA1/2 or RB1 . Across metastatic sites, both ILC and IBC-NST showed differences in B cells, macrophages (M1/M2), neutrophils, NK cells, dendritic cells, CD8+ T cells, and Tregs (all q < 0.05); CD4+ T-cell differences were observed only in IBC-NST (q = 0.002). In organ-specific ILC vs IBC-NST comparisons, CDH1 mut were more frequent in ILC across sites, along with higher PIK3CA mut in skin (ILC 51.3% vs IBC-NST 34.4%) and higher ERBB2 mut in liver (32.7% vs 3.3%), whereas a higher TP53 mut frequency was observed in IBC-NST in skin (28.5% vs 52.3%) and lymph nodes (LND) (23.5% vs 55.5%) (all q < 0.01). Conclusions: mBC exhibits marked organ-specific molecular and immune heterogeneity that differs by histology. These findings support histology- and site-aware interpretation of metastatic biopsies and may inform biomarker assessment and treatment decisions in advanced disease. BC-NST ILC Skin LND Bone Breast Liver Perit. CNS q-value Skin LND Bone Breast GI Liver GYN Perit. CNS q-value N (specimens) 785 401 109 361 220 15 44 - 172 84 62 56 56 54 43 37 11 - ERBB2 mut (%) 3.2 3.1 3.2 2.3 3.3 7.1 2.5 1.0 7.1 7.4 16.4 15.4 3.7 32.7 2.4 5.7 11.1 <0.01 ESR1 mut (%) 9.4 5.2 8.3 16.0 28.2 7.1 2.4 0.0 7.8 7.2 8.9 9.8 16.7 26.0 4.9 20.0 0.0 0.9 FGFR1 amp (%) 10.1 6.6 5.6 12.8 16.5 0.0 9.7 0.02 4.4 3.4 6.7 6.1 2.4 5.6 7.7 18.5 0.0 1.0 IHC-PD-L1 (%) 20.7 - 5.3 16.0 5.0 11.1 10.3 0.02 8.9 - 5.0 5.7 6.1 0.0 0.0 0.0 14.3 1.0 TMB-High (%) 10.7 10.1 8.8 8.8 8.5 14.3 21.4 0.8 27.6 11.0 19.6 28.0 11.3 19.6 9.8 32.4 22.2 0.8 Perit: peritoneum; CNS: central nervous system; GYN: genital tract.

1106 Background: TNBC, although an aggressive breast cancer (BC) subtype, is highly immunogenic and the only BC subtype where the ICI pembrolizumab is approved. However, predictive biomarkers for pembrolizumab benefit are limited. The chemokines CXCL9 and CXCL10 attract CD8 + T cells into the tumor microenvironment (TME) and are associated with chemotherapy benefit, but little is known about their role in prediciting pembrolizumab benefit in TNBC. We investigated the association of CXCL9 , CXCL10 and their cognate receptor CXCR3 with TME and ICI efficacy. Methods: 3,038 TNBC samples were analyzed via NGS (592-gene panel, NextSeq; WES/WTS, NovaSeq; Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) totaled somatic mutations per tumor (high > 10 mt/MB). Immune cell fractions were estimated using WTS deconvolution (Quantiseq). CXCL9 / CXCL10 / CXCR3 -high (H) and -low (L) tumors were classified by RNA expression above or below the 50th percentile. Real-world overall survival (OS) was derived from insurance claims and calculated from start of pembrolizumab to last contact using Kaplan-Meier. Statistical significance was assessed using chi-square and Mann-Whitney U with multiple comparison adjustments (q < .05). Results: TNBC expressed higher levels of CXCL9 and CXCL10 (median (TPM): 5.3 and 14.7) compared to N = 1,082 HER2+ (4.8 and 10.3, p < 0.05) and N = 4,918 HR+HER2- (2.7 and 7, q < .05) BC. CXCR3 expression was higher in TNBC compared to HR+HER2- (1.9 vs 1.7, q < .05), but no difference when compared to HER2+ (1.9 vs 2, q = 0.97) BC. CXCL9 / CXCL10 / CXCR3 -H TNBC had higher median OS post pembrolizumab [ CXCL9 -H vs -L: 26.5 vs 15.7 months (mo), HR: 0.65 (95% CI 0.5-0.84); CXCL10 -H vs -L: 26.0 vs 20.6 mo, HR 0.74 (0.57-0.95); CXCR3 -H vs -L: 32.6 vs 18.3 mo, HR 0.68 (0.52-0.88), all p < 0.05 ]. CXCL9 -H, CXCL10 -H and CXCR3 -H had higher PD-L1 positivity (22C3), TMB high, higher T cell inflamed score, TP53 mutations, elevated B and CD8 + T cells infiltration, but not neutrophils, and higher expression of immune checkpoint genes (Table). Conclusions: High CXCL9 / CXCL10 / CXCR3 expression is associated with longer survival in patients with TNBC post pembrolizumab, and characterized by an immune-enriched TME. Further investigation is needed to evaluate this chemokine axis in TNBC and its potential as a therapeutic target to enhance ICI efficacy. TME characteristics by CXCL9, CXCL10 and CXCR3 expression. CXCL9 CXCL10 CXCR3 High Low q-value High Low q-value High Low q-value PD-L1 % 54 14 <.05 54 14.7 <.05 49 19 <.05 TMB high % 14.3 8 <.05 12.6 9.7 <.05 12.4 9.8 <.05 B cell (median %) 4.4 3.5 <.05 4.3 3.6 <.05 5 3.4 <.05 CD8+T cell (median %) 1.2 0 <.05 1 0 <.05 1.3 0 <.05 Neutrophil (median %) 4.2 4.5 <.05 4.2 4.4 <.05 4.2 4.3 0.2 T cell inflamed score 100 -84 <.05 98 -84 <.05 108 -100 <.05 CTLA4 (median TPM) 3.2 0.7 <.05 3 0.8 <.05 3.4 0.7 <.05 LAG3 (median TPM) 6.6 2.1 <.05 6.9 2 <.05 6.8 2 <.05 TP53 mutation % 88 81 <.05 90.5 78.6 <.05 86 83 0.08

1098 Background: Leucine-rich repeat-containing protein 15 ( LRRC15) has emerged as a potential biomarker and therapeutic target for various cancers due to its high expression in cancer-associated fibroblasts (CAFs) and role in tumor progression. High LRRC15 expression is associated with poor prognosis in TNBC. This study aims to define the multiomic profile of LRRC15 in TNBC. Methods: 3,038 TNBC samples were analyzed via Next-Generation Sequencing (592, NextSeq; Whole Exome Sequencing, NovaSeq) and Whole Transcriptome Sequencing (NovaSeq; Caris Life Sciences, AZ). Immune cell fractions were estimated using WTS deconvolution (Quantiseq). Stromal cell abundance in the tumor microenvironment (TME) was estimated from RNA expression profiles using MCP Counter. LRRC15 -high (H) and -low (L) tumors were classified by RNA expression above or below the 25th percentile. Real-world overall survival (OS) and treatment-related survival were derived from insurance claims and calculated from tissue collection or treatment initiation to last contact using Kaplan-Meier. Statistical significance was assessed using chi-square and Mann-Whitney U tests with multiple comparison adjustments (q < .05). Results: LRRC15 -H TNBC tumors had higher frequency of PIK3CA (25.9% vs 16.8), PIK3R1 (6.2% vs 1.6%), PTEN (11.3% vs 5.8%), but lower frequency of RB1 (7.8% vs 12.1%) and KMT2D (2% vs 4.4%) compared to LRRC15 -L, all q < 0.05. LRRC15 -H had higher PD-L1 positivity (32.3% vs 24.5%, q < 0.05). Analysis of immune cells showed LRRC15 -H TNBC had higher infiltration of B cells (4.2% vs 3.5%), M1 macrophages (4.3% vs 2%), M2 macrophages (4% vs 2.5%), Tregs (1.9% vs 1.1%), neutrophils (4.9% vs 3.9%), CD8 + T cells (0.4% vs 0.1%), but lower dendritic cells (2.5% vs 3%), all q < 0.05. LRRC15 -H tumors had greater abundance of CAFs (575.6 vs 93.78, 6.14 fold change (FC)) and endothelial cells (7.3 vs 3.7, 1.97 FC), all q < 0.05. LRRC15 -H had higher T-cell inflamed score (71.5 vs -77) and IFNg score (-0.14 vs -1.72), all q < 0.05. LRRC15 -H tumors had higher expression of immune checkpoint genes ( CD274, PDCD1, PDCD1LG2, CTLA4, LAG3, HAVCR2, FOXP3, IDO1, TNFSF14, TIGIT, BTLA, CEACAM1, CD47, CD80, CD86, CD160, CD274 ; FC 1.2-2.5, q < 0.05). LRRC15 -H was associated with better OS (mOS: 24.7 vs 13.6 months; HR 0.61, 95% CI 0.53-0.7, p < 0.001). Post-pembrolizumab survival was longer for LRRC15 -H patients (mOS: 27.2 vs 19.4 months; HR 0.61, 95% CI 0.42-0.89, p = 0.01). Conclusions: LRRC15 -H TNBC exhibited better outcomes with pembrolizumab, likely due to higher immune cell fractions and increased CAFs. These findings highlight TNBC heterogeneity and position LRRC15 as a potential biomarker for tumor stratification, a possible adverse prognostic biomarker and a positive predictive biomarker. Ongoing phase I trials targeting LRRC15 show promise. Combining LRRC15 -targeted therapies with immunotherapy may improve TNBC outcomes, warranting further validation in breast cancer models.

Abstract Purpose: Around 10% to 15% of prostate cancers harbor recurrent aberrations in the Forkhead Box A1 gene, FOXA1, whereby the alteration type and the effect on the forkhead (FKH) domain affect protein function. We developed a FOXA1 classification system to inform clinical management. Experimental Design: A total of 5,014 prostate cancer samples were examined using whole-exome and -transcriptome sequencing from the Caris Life Sciences database. We denoted class 1 FOXA1 alterations as missense and in-frame insertions/deletions with subclasses oriented with respect to the FKH domain. These were in the first part of the FKH domain [class 1A: amino acids (AA) 168–246], within the Wing2 region of FKH (class 1B: AA 247–269), or outside FKH (class 1C: AA 1–167, 270+). Two hotspot missense mutations at R219 were denoted class 2. Class 3 included predicted truncating mutations with subclasses partitioned based on the FKH domain (class 3A: AA 1–269 and class 3B: AA 270+). Class 4 represented FOXA1 amplifications. Real-world overall survival and therapy outcomes were determined from insurance claims. Results: FOXA1 alterations did not influence survival when considered in aggregate but had distinct prognostic effects when stratified by class. In primary prostate samples, class 1A alterations were associated with overall improved survival (HR, 0.57; P = 0.03); a similar trend was seen in metastatic biopsies with class 1B (HR, 0.84; P = 0.09). Conversely, in primary specimens, class 1C exhibited worse survival upon second-generation androgen receptor signaling inhibitor treatment (HR, 1.93; P &lt; 0.001). Class 2 mutations (R219C/S) were enriched in neuroendocrine prostate cancers and were associated with overall poor survival (HR, 2.05; P &lt; 0.001) and worse outcomes to first-line androgen-deprivation therapies (HR, 2.5; P &lt; 0.001). Class 3A alterations indicated improved survival (HR, 0.70; P = 0.01), whereas class 3B alterations portended poor outcomes (HR, 1.50; P &lt; 0.001). Amplifications (class 4) indicated poor outcomes in metastatic samples (HR, 1.48; P = 0.02). Molecularly, different FOXA1 alteration classes harbored distinct mutational and immunologic features as well as unique transcriptional programs. Finally, relative to European Americans, African Americans had increased class 1C alterations, whereas Asian/Pacific Islander patients had increased class 1B alterations. Conclusions: FOXA1 alterations should not be interpreted in aggregate, as different classes are associated with divergent molecular features and clinical outcomes. Our revised classification schema facilitates clinical decision-making for patients with prostate cancer and uncovers important racial differences.