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1114 Background: While treatment and management of TNBC has improved, there is a need for novel prognostic biomarkers to better inform outcomes and guide therapeutic options. ILF2 is a poorly characterized protein with pleiotropic functions that is highly expressed in TNBC. Here we evaluated the associations of ILF2 with 1) genomic and transcriptomic data, 2) tumor microenvironment (TME), and 3) clinical outcomes in TNBC. Methods: 15,544 breast cancer (BC) samples, including 3,038 TNBC, were tested by NGS (592, NextSeq; WES, NovaSeq) and WTS (NovaSeq; Caris Life Sciences, Phoenix, AZ). ILF2 -high (H) and ILF2 -low(L) TNBC were defined by respective quartiles. Immune cell fractions were estimated by WTS deconvolution (Quantiseq). Real world overall survival (OS) was obtained from insurance claims and calculated from tissue collection to last contact using Kaplan-Meier estimates. Statistical significance was determined by chi-square, Fisher’s exact, and Mann-Whitney U test with p-values adjustments (q < .05). Results: ILF2 expression (median Log2(TPM+1) was higher (all q < .05) in key subgroups: ductal compared to lobular carcinoma (6.4 vs 6.0); primary compared to metastatic BC (6.4 vs 6.3); African American compared to White (6.4 vs 6.3); basal compared to luminal A, luminal B, HER2 PAM50 subtypes (6.9 vs 5.8, 6.3, 6.3); and TNBC compared to HR+HER2+, HR-HER2+, HR+HER2- subtypes (6.7 vs 6.3, 6.4, 6.2). Biopsied tissues from primary TNBC (pTNBC) and metastatic TNBC (mTNBC) patients were stratified into ILF2- H and ILF2- L groups. In both mTNBC and pTNBC, ILF2- H groups had 1) higher percentage of young patients (age < 50) (pTNBC: 35.5% vs 19.8%; mTNBC: 28.1% vs 17.3%; all q < .05); 2) higher mutation frequency of TP53 (pTNBC: 94.5% vs 79.6%; mTNBC: 92.4% vs 74.4%), but lower frequencies for PIK3CA ( pTNBC: 5.1% vs 23.4% , mTNBC: 8.8% vs 27.4%), CDH1 ( pTNBC: 0.8% vs 6.1%; mTNBC: 2.8% vs 12.2%; all q < .05); 3) higher infiltration of NK cells (pTNBC: 3% vs 2.6%; mTNBC: 2.8% vs 2.6%), but lower infiltration of M2 Mφ (pTNBC: 2.5% vs 3.3%; mTNBC: 2.6% vs 3.2%) and Tregs (pTNBC: 1.5% vs 1.9%; mTNBC: 1.4% vs 1.7%; all q < .05); 4) higher expression levels of immune checkpoint ( CD274, PDCD1LG2, CTLA4, LAG3, HAVCR2, FOXP3, IDO1, CD276 , FC: 1.2-3.1; all q < .05), stem cell genes (C D44, NANOG, POU5F1, KLF4, ALDH1A1 , FC: 1.4-2.4; all q < .05), and drug efflux genes ( ABCC3, ABCC11, ABCC2, ABCB1, ABCG2, ABCC1 , FC: 1.1-4.5; all q < .05) compared to ILF2- L group. In pTNBC, ILF2- H had significantly shorter OS vs ILF2- L group (22.3 vs 28.9 months, HR 1.2 [95% CI 1-1.5], p = .03), but no significant differences were observed between mTNBC ILF2 groups (HR 1.1 [95% CI 0.93-1.3], p = .2). Conclusions: ILF2 -H TNBC patients showed differential genomic and transcriptomic alterations that relate to therapy resistance, immune suppressive TME, and shorter OS. Further studies are warranted to validate the effects of ILF2 upregulation on therapeutic efficacy.

1107 Background: Chronic stress-mediated β2-adrenergic receptor (β2-AR) signaling promotes tumor growth via immunosuppression in the tumor microenvironment (TME) in preclinical models. Blockade of β2-AR has shown higher survival benefit in patients with TNBC in observational studies compared to other breast cancer (BC) subtypes. However, the molecular and immunological features associated with ADRB2 (gene for β2-AR) gene expression in TNBC are unknown, prompting this investigation. Methods: 3,038 TNBC samples were analyzed via NGS (592-gene panel, NextSeq; WES/WTS, NovaSeq; Caris Life Sciences, Phoenix, AZ). Immune cell fractions were calculated by deconvolution of WTS: Quantiseq. TNBC ADRB2 -high(H) and ADRB2 -low(L) RNA expression were classified as above or below the 50 th percentile, respectively. Real-world overall survival (OS) was obtained from insurance claims and calculated from tissue collection to last contact using Kaplan-Meier estimates. Statistical significance was assessed using chi-square and Mann-Whitney U tests with multiple comparison adjustments (q < 0.05). Results: ADRB2 gene expression was lowest in TNBC (median (TPM: 1.6) compared to N = 453 HR-HER2+ (1.9), N = 629 HR+HER2+ (2.0) and N = 4,918 HR+HER2- (2.2) BC (all q < 0.05). African American or Black patients (N = 670) had lower expression of ADRB2 compared to European American or White (N = 1,412) TNBC patients (1.3 vs 1.7, q < 0.05). ADRB2 -H TNBC had higher mutation frequency of PIK3CA (21% vs 15.4%), CDH1 (7% vs 3.5%), NF1 (8% vs 4%), AKT1 (3.5% vs 2.1%), but lower frequency of TP53 (81.6% vs 87.5%), NOTCH1 (2.5% vs 4.5%) and NOTCH3 (4.4% vs 11.7%) compared to ADRB2 -L, all q < 0.05. ADRB2 -H had greater PD-L1 (22C3) positivity (39.1% vs 30.2%, q < 0.05), higher % of B cells (4.5 vs 3.4), M1 Mφ (3.4 vs 2.8), M2 Mφ (3.9 vs 2.2), Tregs (2.2 vs 1.3), NK cells (3.1 vs 2.6), DC (3.1 vs 2.9), CD8 + T cells (0.9 vs 0.2), all q < 0.05. ADRB2 -H TNBC had higher T-cell inflamed score (95 vs -80), IFN γ score (-0.23 vs -0.37), MAPK activation score (-0.46 vs -1.7), all q < 0.05; and higher expression of immune checkpoint genes ( CD274, PDCD1, PDCD1LG2, CTLA4, LAG3, HAVCR2, FOXP3, IDO1, TNFSF14, TIGIT, BTLA, CEACAM1, CD47, CD274 ; fold change: 1.6-3.7, all q < 0.05). ADRB2 -H tumors had higher expression of genes related to inflammatory response, IFNγ response, IL6-JAK-STAT3 signaling (normalized enrichment score (NES): 1.9 – 2.1), while ADRB2 -L had enrichment of MYC targets V1, MYC targets V2, E2F targets and G2M checkpoint (NES: 2.5 – 4.2), all FDR < 0.01. ADRB2 -H TNBC had better OS (mOS: 23.6 vs 18.6 months; HR 0.81, 95% CI 0.73-0.89, p < 0.0001) compared to ADRB2 -L. Conclusions: High ADRB2 expression in TNBC is associated with better survival and an immune enriched TME, elevated immune checkpoints and other targetable vulnerabilities. Future studies are needed to investigate ADRB2 as a potential stress biomarker and therapeutic target.

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.

1051 Background: Hispanics or Latinos (HL) and African Americans or Black (AA) have a higher prevalence of advanced-stage breast cancer (BC) at diagnosis compared to Non-Hispanic Whites (NHW). To understand the role of immune system, we evaluated the tumor immune microenvironment (TIME) by race/ethnicity among HL, AA, and NHW BC patients. Methods: 15544BC samples were tested by NGS (592, NextSeq; WES, NovaSeq) and WTS (NovaSeq; Caris Life Sciences, Phoenix, AZ). Race/ethnicity data is self-reported. Immune cell were estimated using WTS deconvolution (Quantiseq). Gene expression profiles were analyzed for T-cell inflammation score (TIS) and interferon-gamma (IFN-gamma) score. Real-world overall survival (OS) was obtained from insurance claims and calculated from date of tumor biopsy to last contact using Kaplan-Meier estimates. Statistical significance was determined by chi-square and Mann-Whitney U test with p -values adjusted for multiple comparisons (q < .05). Results: 7170 NHW (35.3%, N = 2528) biopsied (bx) from primary breast cancer (pBC), 64.7% (N = 4642) metastatic bx (mBC), 1,508 AA (pBC 39.3% N = 592, mBC 60.7% N = 916), and 1,754 HL (pBC 44.1% N = 774, mBC 55.9% N = 980) cases were included. By subtype, there were 1,956 (60.4% NHW, 20.7% NHB, 18.9% HL) TNBC, 3425 HR+/HER2- (72.6% NHW, 11.9% NHB, 15.6% HL), and 694 HER2+ (64.6% NHW, 15.7% NHB, 19.7% HL). Across all cases, AA (20.5%) and HL (20.4%) had greater incidence (%) of PD-L1+ cases versus (vs) NHW (17.4%), all q < .05. TMB-High (³10 mut/Mb) was similar in NHW (11.5%), AA (10.8%), and HL (10.9%). AA tumors had lower median % cell infiltration of M2-like macrophages (M2 Mφ), B cells, and neutrophils vs NHW (Table). HL had a lower fraction of M2 Mφ and higher CD8+ T cells (Table). AA had lower TIS (-8 vs 1, p = .02) while HL had lower IFN-gamma (-0.38 vs. -0.35, q < .05) vs NHW. By subtype, AA had lower neutrophils (4% vs 4.3%) and increased DC fractions (3.1% vs 2.8%) in TNBC vs NHW, all q < .05; no significant changes seen in HL vs NHW. AA had worse mOS than NHW overall (31.8 vs 36.8 months (mo)), HR 1.1, 95% CI 1 – 1.2, p = < .01), in pBC (40.3 vs 49.9 mo, HR 1.3, 95% CI 1.1 – 1.5, p = < .01), but not mBC (27.4 vs 29.1 mo, HR 1, p = 0.2). HL had similar mOS vs NHW overall (37.4 vs 36.8 mo, HR 0.9, p = 0.9) and in mBC (29.1 vs 31 mo, HR 0.96, p = 0.4), but worse mOS in pBC (44.7 vs 50.0 mo, HR 1.1, 95% CI 1 – 1.3, p = .01). Conclusions: Our study shows worse mOS in AA and HL pBC cases vs NHW, possibly from a less inflamed TIME in AA and HL and lower fraction of neutrophils and M2 Mφ despite higher % of PD-L1+. Targeting Mφ and CD8+ T cells and converting cold to hot TIME may lessen race/ethnic disparities, especially in early-stage BC. Immune cell fraction of NHW, AA and HL BC. NHW (median %) AA (median %) HL (median %) q-valueNHW vs AA q-value NHW vs HL B cell 5.2 4.8 5.0 <.05 0.7 DC 2.6 2.7 2.6 0.08 0.4 M1 Mφ 2.5 2.4 2.4 0.4 0.9 M2 Mφ 4.6 3.7 4.2 <.05 <.05 Neutrophils 3.7 3.5 3.4 <.05 <.05 NK cell 2.9 2.9 2.9 0.7 0.6 CD8+ T cell 0.1 0.15 0.26 0.8 <.05 Treg 1.5 1.5 1.6 0.6 <.05

570 Background: Major histocompatibility complex class I (MHC I) plays a critical role in immune surveillance by binding peptides derived from intracellular proteins and presenting them on the cell surface for recognition by CD8+ T cells. Loss or downregulation of MHC I expression has been identified as a key mechanism of immune evasion in cancers. Here, we evaluated MHC I expression and outcomes in all subtypes of breast cancer (BC). Methods: 9,038 BC samples were analyzed via NGS (592-gene panel, NextSeq; WES/WTS, NovaSeq; Caris Life Sciences, Phoenix, AZ), including triple-negative BC (TNBC) 3,038, HER2-positive (HER2+) 1,082, and hormone receptor-positive (HR+HER2-) 4,918. Immune cell fractions were estimated using WTS deconvolution (Quantiseq). MHC I ( HLA-A / HLA-B / HLA-C )-high (H) and -low (L) were classified by RNA expression above or below the 25th percentile. Real-world overall survival (OS) was derived from insurance claims and calculated from tissue collection to last contact using Kaplan-Meier. NanoString IO360 was performed in 114 samples from the FinXX trial (NCT00114816). Statistical significance was assessed using chi-square, Mann-Whitney U, ANOVA, and Cox regression with multiple comparison adjustments (q<.05). Results: TNBC had higher expression of HLA-A and HLA-B (median TPM: 169 and 191) compared to HER2+ (146.6 and 170, q<0.05) and HR+HER2- (141.7 and 157.5, q<0.05). However, there was no significant difference in HLA-C expression across 3 BC subtypes. In TNBC, MHC I-H tumors had higher frequencies of PD-L1 positivity (66.2% vs. 13.1%) as well as higher infiltration of B cells (4.5% vs. 3.2%), M1 macrophages (5% vs. 1.5%), M2 macrophages (4% vs. 2.1%), Tregs (2.8% vs. 0.8%), CD8 + T cells (1.8% vs. 0%), dendritic cells (3.2% vs. 2.8%), higher T-cell inflamed score (137 vs. -144), and IFN g score (0.02 vs. -0.49) compared to MHC I-L TNBC (all q<.05). MHC I-H TNBC was associated with significant improvement in median OS (30.1 vs. 15.2 months, HR 0.55, 95% CI 0.46-0.65, p<0.0001). However, this survival difference was not observed in patients with MHC I-H vs. MHC I-L in HER2+ (HR 1.04, 95% CI 0.74-1.47, p = 0.81) and HR+HER2- (HR 0.87, 95% CI 0.75-1.02, p= 0.09) BC subtypes. We further validated the MHC I expression in the FinXX trial. Similarly, patients with MHC I-H had significant improvement in recurrence-free survival (HR 0.27, 95%CI 0.11-0.66, p = 0.002) and OS (HR 0.23, 95% CI 0.09-0.57, p = 0.0005) compared to MHC I-L. Conclusions: Our findings demonstrate that higher MHC I expression is associated with higher immune infiltration and improved outcomes in TNBC but not in HER2+ or HR+HER2- BC subtypes. These results suggest that MHC I expression plays a critical role in the tumor microenvironment of TNBC. Future studies are needed to evaluate the prognostic value and potential therapeutic target of MHC I in TNBC. Support: Breast Cancer Research Foundation, Bankhead Coley, W81XWH-18-1-0564. Clinical trial information: NCT00114816 .

4166 Background: GN is a front-line therapy for aPDAC. A Phase I/II study demonstrated that GCN has a higher overall response rate and median overall survival (mOS). Our previous study found no difference in outcomes among patients with DNA damage repair gene mutations; however, stromal gene expression correlated with mOS in patients receiving GCN. Here, we further evaluate differences in the outcomes with GCN and GN by site of biopsy. Methods: PDAC samples (n = 4,463) were analyzed by NGS (NextSeq/NovaSeq) or RNA (NovaSeq) (Caris Life Sciences, Phx, AZ). Expression of stromal and related genes ( ACTA2, ADIRF, HAS2, IL-6, MMP-2, MMP-9, SPARC, STAT3, TBGB1, TGFB2, TGFBR3, IDO1, HLA-DRB4, VEGFB) from different biopsy sites [high expression (H) >50% of RNA transcripts per million] was correlated with outcomes to GCN or GN. mOS was obtained from insurance claims and calculated from first treatment to last contact. The hazard ratio (HR) was calculated by the Cox proportional hazards model, and p-values were calculated using the log-rank test. Results: 4325 patients [primary biopsy (PT), n = 1,878; non-liver metastatic biopsy (N-LM), n = 818; Liver biopsy (LM), n = 1,629] received GN while 138 patients (PT, n = 45; N-LM, n = 28; LM, n = 65) received GCN. GCN was associated with longer mOS than GN [∆: 5.2 months (m), HR: 0.76, 95% CI 0.63-0.92, p = 0.01]. GCN was associated with longer mOS compared to GN in LM (∆: 5.6 m, HR: 0.66, 95% CI 0.50-0.87, p = 0.003), but it was not significant in PT (∆: 4.6 m, p = 0.13) and N-LM (∆: 4.4 m, p = 0.35). Median MMP2 (38.5 vs. 163.1 vs. 162.2), VEGFB (14.4 vs. 16.9 vs. 16.2) and TGFBR3 (11.4 vs. 15 vs. 16.4) expression were lower in LM compared to PT and N-LM while TGFB1 (41.6 vs. 34.5 vs. 39.8) and IL6 (1.61 vs. 1.19 vs. 1.41) expression were highest in LM (p < 0.05). In LM, IL6 -H (∆: -11 m, p = 0.037) and TGFB1 -H (∆: -5.9 m, p = 0.10) were associated with worse post-GCN survival compared to GN (p = 0.82 and p = 0.57). Whereas, in N-LM, TGFBR3 -H trended towards longer post-GCN survival (∆: 12 m, p = 0.18), while ADIRF -H trended towards shorter post-GCN survival (∆: -12 m, p = 0.056) compared to GN (p = 0.42 and p = 0.10). While in PT, MMP2 -H (∆: 11.3m, HR: 0.46, p = 0.16), TGFB1 -H (∆: 11.3 m, HR: 0.33, p = 0.09), HLA-DRB4 -H (∆: 12.9 m, HR: 0.44, p = 0.11) and VEGFB -H (∆: 10.7, HR: 0.34, p = 0.09) trended towards longer post-GCN survival compared to GN ( MMP2 -H, ∆: 3.4 m, p = 0.03, TGFB1 -H, p = 0.661, HLA-DRB4 -H, p = 0.67, VEGFB-H , ∆: 1.3 m, p = 0.03). Conclusions: GCN is associated with improved mOS compared to GN, especially in LM.Stromal gene expression in the liver differs from that in N-LM and the pancreas. While high stromal gene expression trends towards worse post-GCN survival in LM, it is associated with improved survival in N-LM and PT. Further validation is needed to understand the impact of stromal gene expression in different tumor sites on survival outcomes and signature development.

Abstract Background: Hispanic American (HA) men with prostate cancer (PC) are more likely to have more aggressive and advanced diseases and greater mortality rates compared to non-Hispanic white (NHW) men. Resistance to the current anticancer therapy is the major hurdle that leads to poor clinical outcomes. Hedgehog, Notch, and Wnt signaling pathways play a crucial role in development, progression, and resistance to the various anticancer therapies. Here, we characterized the enrichment of Hedgehog, Notch, and Wnt signaling pathways and molecular tumors differences between HA and NHW patients. Methods: Our data consist of 88 PC tumors samples (HA=34, NHW=54) obtained from treatment-naive metastatic hormone-sensitive prostate cancer (mHSPC) that were analyzed by next-generation sequencing (592, NextSeq; Whole Exome Sequencing, NovaSeq) and Whole Transcriptome Sequencing (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). Pathway enrichment was determined by Gene Set Enrichment Analysis (GSEA, Broad Institute) by measurable TPM (transcripts per kilobase million). The medical records were reviewed in a deidentified fashion for clinical features. Statistical significance was determined using chi-square and Mann-Whitney U (p&lt;0.05). Results: HA PC had enrichment of Hedgehog signaling (NES: 1.45, FDR=0.04), Notch signaling (NES: 1.41, FDR=0.07), and Wnt/β-catenin signaling (NES: 1.38, FDR=0.11) pathways compared to NHW tumors. HA tumors had higher expression of genes (HES1, Notch1, WNT1, WNT2; FC:1.1-2.5) associated with crosstalk between Hedgehog, Notch, and Wnt/β- catenin signaling pathways (all p&lt;0.05). Moreover, HA tumors had higher expression of stemness (ALDH1A2, ALDH1A1, PROM1; FC: 1.7-3.0), drug efflux (ABCC1, ABCB1; FC: 1.1- 1.5), epithelial to mesenchymal transition (EMT) (TWIST1, MMP2; FC: 1.1-1.5), cell cycle (CCND2; FC: 1.2), inhibition of apoptosis (BIRC2, XIAP; FC: 1.1) and epigenetic (EP300, TET1, TET2; FC: 1.3-1.4) related genes compared to NHW tumors (all p&lt;0.05). HA PC had higher frequency of Hedgehog and Wnt/β-catenin associated SPOP (16% vs 6.6%, p=0.2) and CTNNB1 (12% vs 2.2%, p=0.1) mutation compared to NHW PC. Conclusion: These results demonstrate that tumors from HA patients had enrichment of oncogenic Hedgehog, Notch, and Wnt/β-catenin signaling pathways, higher expression of stemness, drug efflux, EMT, cell cycle, inhibition of apoptosis and epigenetic-related genes, and mutations directly involved in activating the Hedgehog and Wnt/β-catenin signaling pathways. Together, these findings suggest that tumors from HA patients are enriched for gene signatures that promote cancer progression and therapy resistance. Additional research on collaboration and crosstalk between Hedgehog, Notch, and Wnt/β-catenin signaling pathways may help in designing better therapeutic strategies for HA men with mHSPC. Citation Format: Alejandro Recio-Boiles, Sachin Kumar Deshmukh, Ricardo J. Estrada-Mendizabal, Kenneth Barker, Sharon Wu, Joanne Xiu, Milan Radovich, Elisabeth I. Heath, Rana McKay, Juan Chipollini, Cynthia K. Miranti. Defining the role of Hedgehog, Notch, and Wnt signaling pathways in aggressive lethal prostate cancer of Hispanic men [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C100.

Abstract Background: Breast cancer (BC) in males represents a rare clinical entity, accounting about 1% of all breast malignancies diagnosed every year. Emerging evidence suggest that a gender medicine approach is critical in the research and treatment of cancer, based on the possibility that sex hormones, molecular differences, immune system, and other factors might play a crucial role in disease management. Here, we characterized molecular and immune differences between male BC (MaBC) and female BC (FeBC) across BC subtypes. Methods: 10,728 BC samples (male, n=137; female, n=10591) were analysed by next-generation sequencing (592, NextSeq; WES, NovaSeq), Whole Transcriptome Sequencing (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) totaled somatic mutations per tumor (high ≥10 mt/MB). Microsatellite-instability (MSI) was tested by IHC and NGS. Immune cell fractions were calculated by deconvolution of WTS: Quantiseq. Real world treatment-associated survival was extracted from insurance claims and calculated from sample collection to last contact using Kaplan-Meier estimates. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q &lt; 0.05). Results: MaBC represented higher HR+/HER2+ (6.61% vs 4.76%), HR+/HER2- (80.17% vs 60.66%) and lower HR-/HER2+ (1.65% vs 3.5%), TNBC (11.57% vs 31.08%) molecular subtypes compared to FeBC. Compared to HR+/HER2+ FeBC, MaBC had higher frequency of CHEK2, RAD51B, TSC1 mutation (Table 1) and RARα (100% vs 13.61%), DAXX (10% vs 0.16%) copy number alteration (all p &lt; 0.05). HR+/HER2- MaBC had higher frequency of BRCA2, FOXA1, CREBBP, FLCN mutation (Table 1), and FGF3 (21.62% vs 14.31%), MRE11 (1.75% vs 0.23%), MEN1 (1.75% vs 0.13%) copy number alteration, but lower frequency of TP53, CHD1, ESR1 and AKT1 mutation (Table 1) (all p &lt; 0.05). HR-/HER2+ MaBC had higher frequency of BRCA1 (p &lt; 0.05) mutation (Table 1). Male TNBC had higher frequency of CDKN2A, KMT2D, STK11, ASXL1, MYC, NF2, PPM1D, WT1, EPHA2, AMER1, ARHGAP35 mutation (Table 1) and BCL6 (4.35% vs 0.05%) copy number alteration, but lower frequency of TP53 mutation (all p &lt; 0.05) (Table 1). MaBC had higher AR protein expression (82.5% vs 60%) but lower frequency of fusion variant-AR (0.7% vs 3.9%) (all p &lt; 0.05) compared to FeBC. There was no difference in TMB high (7.1% vs 8.4%, p = 0.5) and dMMR/MSI-H (1.8% vs 0.8%, p = 0.1). Analysis of inferred immune cell infiltrates showed that MaBC had increased immune cell infiltration of B cells (5.8% vs 5.3%) and M2 Mφ (4.9% vs 4.3%), but decreased infiltration of DC (2% vs 2.6%) (all p &lt; 0.05). MaBC had increased expression of MHC class II gene HLA-DQB2 (FC: 1.3), but decreased expression of immune-related genes CD274 (FC: 1.2), IDO (FC: 1.4), IL1A (FC: 1.3) and IL12A (FC: 1.5) (all p &lt; 0.05). Correlative analyses with survival will be presented at the meeting. Conclusions: These data indicate that MaBC has a differential mutational frequency, copy number alteration, immune gene expression and immune cell infiltration and overall survival compared to their FeBC counterparts. A better understanding of these sex-based differences with additional research may help inform disease outcomes, provide a rationale for tailored therapeutic approaches and design future treatments. Table 1. Mutation frequency in male and female breast cancer Citation Format: Dario Trapani, Sachin Deshmukh, Sharon Wu, Joanne Xiu, Phil Walker, Pooja Advani, Nancy Lin, Giuseppe Curigliano, Stephanie Graff, Chadi Nabhan, George Sledge Jr, Sara Tolaney, Jose Leone. Molecular and immunological landscape of sex-based differences in breast cancer: a distinct disease in men [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS13-09.

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease and characterized by poor outcomes with a lack of targeted therapies. A comprehensive analysis of the molecular and immune landscape of invasive ductal TNBC can help improve our understanding of TNBC biology and identify novel targets and/or pathways for better management of this disease. Here, we characterized the molecular and immune signature of invasive ductal (ID) TNBC. Methods: 13,036 BC samples (ID TNBC, n=392; ID non-TNBC, n=927) were analyzed by NGS (592, NextSeq; WES, NovaSeq), WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). A total of 10 pro-apoptotic (BAX, BAK1, BID, BAD, BIK, BCL2L11, BMF, HRK, PMAIP1, BBC3) and 6 anti-apoptotic (BCL2, BCL2L1, BCL2L2, MCL1, BCL2A1, BCL2L10) BCL2 family genes were analyzed. Immune cell fractions were calculated by deconvolution of WTS: Quantiseq. Real world overall survival (OS) and treatment-associated survival was extracted from insurance claims and calculated from tissue collection or treatment start to last contact using Kaplan-Meier estimates. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q &lt; 0.05). Results: ID TNBC had a higher frequency of TP53, RB1, NF1, PIK3R1, NOTCH1, CREBBP, BRCA1, FANCI, and HRAS mutations (Table 1), NOTCH2 (4.3% vs 0.47%), EGFR (3.4% vs 0%), NFIB (4% vs 0.6%), MYB (3.5% vs 0.5%), CCNE1 (4.4% vs 1.1%) and AKT2 (2.4% vs 0.3%) copy number alterations, and NOTCH2 (1.6% VS 0.2%) fusion (all p &lt; 0.05) compared to invasive ductal non-TNBC. Analysis of inferred immune cell infiltrates showed that ID TNBC had increased infiltration of Tregs (2% vs 1.7%), DC (3.2% vs 2.4%), CD8 T cells (0.5% vs 0.1%), and M1 macrophages (M) (3.4% vs 2.9%), but decreased infiltration of B cells (4.5% vs 6%) and M2M (2.9% vs 4.5%) (all p &lt; 0.05). ID TNBC had increased T cell inflamed score (23 vs -9, p &lt; 0.05), IFNy score (-0.24 vs -0.34, p &lt; 0.05), MHC class I genes (HLA-A, HLA-B, TAP1, TAP2, FC: 1.1-1.5, all p &lt; 0.05), immune checkpoint genes (CD274, PDCD1, CTLA4, PD-L2, FOXP3, IDO, FC: 1.1-2, all p &lt; 0.05) and PD-L1 protein expression (SP142: 49.8% vs. 28.4%; 22c3: 41.7% vs. 16.8%, all p &lt; 0.05). ID TNBC had differential expression of BCL2 family genes (upregulation: BAK1, BID, MCL1, BCL2A1, BCL2L10, FC: 1.1-2.0; downregulation: BAD, BIK, BBC3, BCL2, BCL2L1, BCL2L2, FC: 1.2-2.8, all p &lt; 0.05) compared to invasive ductal non-TNBC. ID TNBC was associated with worse OS compared to ID non-TNBC (mOS: 24.5 vs 54.6 months; HR: 0.5, 95% CI 0.47-0.57; p &lt; 0.00001) but trend towards better survival with pembrolizumab (mOS: 29.4 vs 8.8 month; HR: 0.67, 95% CI 0.28-1.5; p=0.3) treatment. No significant OS difference was noted for by high vs. low pro and anti-apoptotic BCL2 genes except for anti-apoptotic BCL2A1 (higher expression associated with better OS (mOS: 5.7 months, HR: 0.78, 95% CI 0.6-0.99; p=0.047). Conclusion: These data indicate that ID TNBC is associated with distinct mutational profile compared to non-TNBC, has increased T cell inflamed score, IFNy score, MHC class I and immune checkpoint gene expression, and differential immune cell infiltration. Interestingly, TNBC was associated with increased M1 and decreased M2M. There is evidence to suggest that transcriptomically defined M1 high tumors are clinically aggressive and have worse OS in TNBC. BCL2 family genes are not prognostic for TNBC outcomes except BCL2A1, which needs further prospective validation. Table 1: Mutation frequency in invasive ductal TNBC and invasive ductal non-TNBC Citation Format: Pooja Advani, Sachin Deshmukh, Sharon Wu, Jacob Andring, Joanne Xiu, Alex Farrell, Jose Leone, Priya Jayachandran, Stephanie Graff, Matthew Oberley, George Sledge Jr, Asher Chanan-Khan. Comprehensive Molecular and Immunological Characterization of Invasive Ductal Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-01.