Gene mutations according to poverty level and RUCA codes

Description of rural-urban commuting area (RUCA) codes

Class II and III BRAF mutations are uncommon events, with limited data on their clinical and biological characteristics. We investigated clinical and molecular features of BRAF mutation classes in a total of 24,402 patients with mismatch repair proficient CRC (MMRp). Samples collected between 2006 and 2023 were profiled using next-generation sequencing and whole-transcriptome sequencing. We identified 1268 (5.2%), 132 (0.54%), and 323 (1.3%) patients with class I, II, and III BRAF-mutated CRC. Patients with class III mutations had significantly better median overall survival (OS) than those with class I mutations (23.6 vs 17.4 months; HR = 1.26, CI: 1.08-1.47, p = 0.004). Transcriptomic analyses revealed that the MAPK pathway score was significantly lower for class II and III BRAF mutations without concurrent RAS mutations than for those with RAS co-mutations. The cetuximab score, an RNA expression-based predictor of EGFR therapy response, was significantly better for class II and III BRAF mutations than for class I. The cetuximab score significantly improved for only class III BRAF mutations when those with concurrent RAS mutations were excluded. The results of this large multi-institutional analysis of BRAF mutation classes reveal the prognostic value of class II and III BRAF mutations, and their distinct clinical and molecular features.

COXPH model for patients with at least 6-mo overall survival

Overall survival by RUCA code and poverty

Overall survival by RUCA code and poverty

PURPOSE:Although estrogen receptor is well studied in breast cancer (BC), the role of androgen receptor (AR) in prognosis and therapy response is less understood. Here, we characterized the clinicopathologic and molecular features of AR gene expression in BC subtypes. METHODS:Ten thousand seven hundred twenty-eight BC samples were tested by next-generation DNA sequencing, whole-transcriptome sequencing, and immunohistochemistry at Caris Life Sciences (Phoenix, AZ). Tumors with AR-high and AR-low RNA expression were stratified by top and bottom quartiles, respectively. Treatment-associated survival was obtained from insurance claims and calculated from treatment start 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:AR-low was associated with basal-like tumors. AR-high tumors were associated with increased mutation rates in several genes-namely PIK3CA and CDH1-across all subtypes, while other associations such as RB1 and MAP3K1 were subtype-dependent. The immune landscape was differentially affected by AR expression in each subtype, but these differences did not correspond to differential responses to immune checkpoint blockade. Patients with AR-high tumors had a longer therapy response for most subtypes, but those with AR-high tumors that were human epidermal growth factor receptor 2-enriched and luminal B trended toward worse chemotherapy or hormone therapy response, respectively. CONCLUSION:Our data suggest a unique molecular profile of AR-high BC that is subtype-specific and generally associated with improved outcomes. Exploration of specific mutations and immune-oncology markers associated with AR-high may aid in molecularly selected clinical trial design for patients with advanced BC.

Statistically standardized estrogen receptor (ER) and progesterone receptor (PgR) differentiated prognosis. Here we examined statistically standardized human epidermal growth receptor 2 (HER2). CCTG MA.27 (NCT00066573) was an adjuvant phase III trial of exemestane versus anastrozole in postmenopausal women with ER + and/or PgR + tumors. We centrally quantitated machine-image immunohistochemical HER2, defined American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) dual-probe FISH HER2/CEP17 categories, determined ultra-low HER2 (IHC 0 with (0,10

Background: Male breast cancer (BC) accounts for less than 1% of new BC cases annually. Androgen receptor (AR), a member of steroid and nuclear receptor superfamily is emerging as an important factor in pathobiology of BC. While the estrogen receptor (ER) is well-studied in BC, the role of the AR is less understood, particularly in male patients. Here, we aimed to characterize the molecular and immunological features of AR gene expression in male BC. Methods: 191 samples from male breast cancer patients were tested by NGS (592, NextSeq; WES, NovaSeq) and WTS (NovaSeq; Caris Life Sciences, Phoenix, AZ). MSI was tested by IHC and NGS. Tumor mutational burden (TMB) totaled somatic mutations per tumor (high>10 mt/MB). Immune cell fractions were calculated by deconvolution of WTS: Quantiseq. Tumors with AR-high(H) and AR-low(L) RNA expression were classified as above or below the 50th percentile, respectively. Real world overall survival (OS) and treatment-associated survival was obtained from insurance claims and calculated from tissue collection 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: AR-H male BC had lower frequency of TP53 mutations (20% AR-L vs 7% AR-H, p=0.02) compared to AR-L male BC tumors. AR-H had numerically higher frequency of PIK3CA (34.8% vs 27.2%) and CHEK2 (3.7% vs 1.1%), but lower frequency of BRCA2 (7.1% vs 13.6%) and PTEN (2.3% vs 6.9%) compared to AR-L, all p = 0.1-0.2. AR-H male BC had lower frequency of TMB-high (3.45% vs 12.22%) and PD-L1 positivity (5.08% vs 18.57%), all p<0.05. Analysis of inferred immune cells revealed that AR-H had higher infiltration of NK cells (3.58% vs 2.56%), dendritic cells (2.43% vs 1.98%), and B cells (5.93% vs 5.26%), all p<0.05. AR-H had higher T-cell inflamed score (19 vs -72) and MAPK activation score (-0.24 vs -1.6) but lower IFNg score (-0.38 vs -0.33), all p<0.05. AR-H had higher expression of immune checkpoint genes (CD274, FOXP3, HAVCR2, LAG3; FC: 1.3-1.5) and stem cell-related genes (CD34, CD44, POU5F1, KLF4, ALDH2; FC: 1.2-1.4) compared to AR-L male BC, all p<0.05. AR-H male BC had higher AR protein (IHC) expression (100% vs 86.8%, q<0.05) and numerically higher frequency of AR-fusion variant (3.2% vs 0%, p=0.08) compared to AR-L male BC. AR-H male BC had worse OS (mOS: 35.9 vs 92.4 month; HR 1.6, 95% CI 1.0-2.5, p = 0.043) compared to AR-L BC. When analyzed by TP53 mutation status, AR-H with TP53-wt had numerically better survival (mOS: 35.6 vs 25.7 months, HR 0.52, 95% CI 0.20-1.35, p=0.17) compared to TP53-mt. Similarly, AR-L with TP53-wt had numerically better survival (mOS: 48.3 vs 23.2 months, HR 0.58, 95% CI 0.22-1.33, p=0.18) compared to TP53-mt. Conclusions: Our analysis suggests a strong association between AR expression and TP53 mutations, TMB-H, and PD-L1 positivity, immune cell infiltration, immune checkpoint and stem cell-related gene. Also, T cell inflamed and IFNy score were inversely related. Further exploration of specific alterations and immune-oncology markers associated with AR expression may help in clinical trial design for male patients with BC. Citation Format: Priya Jayachandran, Sachin Kumar Deshmukh, Sharon Wu, Jennifer R. Ribeiro, Irene Kang, Joanne Xiu, Francesca Battaglin, Darcy V. Spicer, Daphne B. Stewart, Shivani Soni, Wu Zhang, Janice Lu, Karam Ashouri, Joshua Millstein, William Flood, Jose P. Leone, Dario Trapani, Maryam Lustberg, Stephanie L. Graff, George W. Sledge Jr., Heinz-Josef Lenz, Evanthia T. Roussos Torres. Comprehensive characterization of the androgen receptor in male breast cancer [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 P1-03-22.

274 Background: BRAF mutations (mts) are a heterogeneous group of molecular alterations seen in colorectal cancer (CRC). Class I BRAF mts (V600) are known to be associated with aggressive biology to CRC, but the knowledge of clinical characteristics of class II and III BRAF mts is limited. In this large cohort analysis, we evaluated the clinical and molecular features of class II and III BRAF mts and performed comparative analyses for their impact on survival outcomes. Methods: A total of 24402 MSS CRC samples were profiled by NGS (592-gene, NextSeq; WES, WTS NovaSeq) (Caris Life Sciences, Phoenix, AZ). BRAF mts were detected by NGS and classified using published literature (1). MAPK pathway activity score (MPAS) was calculated using RNA expression data. Real-world overall survival (OS) was obtained from insurance claims and calculated from tissue collection to last contact, while post-treatment survival from first treatment to last contact. KM estimates were calculated for defined patient cohorts. Significance was determined as P<0.05. Results: A total of 1270, 134, and 327 patients with class I, II, and III BRAF mts were identified. BRAF mts overall and class I BRAF mts were significantly less common among younger pts (age<50) compared to patients ≥ 50 (4.7% vs. 7.4 %; P< 0.001 and 3.2% vs. 5.7%, respectively, P<0.001). Class I BRAF mts were enriched with consensus molecular subtype 1 (CMS1) (class I, II, and III: 46% vs. 14% vs. 18% respectively) while class II and III mts had more CMS2 subtype compared to class I (2%, 30% and 29%, p<0.05). Higher MPAS scores were noted among patients with class I BRAF mts than those with class II and III mts (1.73 versus 0.38 vs 0.79). Class I BRAF mts and KRAS mts were nearly mutually exclusive (0.5%), while KRAS mts were relatively common among class II and III mts (12.8% and 27.4%, respectively). Median OS for patients with wild-type BRAF , class I, II, and III BRAF mts were significantly different (29.8, 17.3, 21.8, and 24.6 months, respectively, p<0.01). Similar OS was seen among patients who did not receive anti-EGFR therapy (28.3, 15.2, 20.7- and 21.9 months P<0.001). Patients with class III mts had significantly better OS compared to patients with class I mts (HR=1.28 CI: 1.09-1.48 p=0.002), and while no significant difference was noted for patients with class II vs class I mts, numerically more favorable outcomes were noted (mOS 21.8 vs 17.3 months P=0.133). Among treated with anti-EGFR, patients with class II and III BRAF mts had numerically better post-anti-EGFR survival compared to class I mts; however, this was not statistically significant (21.2 vs 14.2 vs. 10.4 respectively P=0.3). Conclusions: Class II and III BRAF mts represent a distinct biological subgroup of MSS CRC with distinct prognoses. Class II and III BRAF mts are associated with improved survival outcomes compared to patients with class I mts. 1. Sahin et al. JCO OP 2021.

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

PURPOSE:Regulator of chromosome condensation 1 (RCC1) and RCC2 have been shown to play important roles in the regulation of cell cycle, DNA damage response, and nucleocytoplasmic transport. MATERIALS AND METHODS:DNA (592-gene or whole exome) and RNA (whole transcriptome) sequencing was performed at Caris Life Sciences (Phoenix, AZ). Samples were stratified by RCC1 expression quartile thresholds (Q1: low, Q4: high) for small cell lung cancer (SCLC; n = 876), non-small cell lung cancer (NSCLC; n = 21,603), gastric cancer (GC; n = 1,908), pancreatic cancer (PC; n = 5,071), and colorectal cancer (CRC; n = 14,892). Statistical significance was determined using chi-square and Wilcoxon rank-sum tests and adjusted for multiple comparisons (*P < .05). Corresponding analyses were run for RCC2. RESULTS:Median RCC1 mRNA expression was highest in SCLC (14.3 transcript per million [TPM]), followed by GC (9.9), NSCLC (9.9), CRC (9.8), and PC (6.9). Similar to RCC1, the median RCC2 expressions were highest in SCLC (36.2 TPM). Tumor mutational burden-high rates were positively associated with increasing RCC1 expression quartiles (Q1-4) in NSCLC (31%-41%), GC (7%-22%), and CRC (5%-17%) and with increasing RCC2 expression in NSCLC and CRC only. Higher expression with RCC1 and RCC2 was associated with worse overall survival in NSCLC (hazard ratio [HR] for RCC1 and RCC2 were 1.3 and 1.3, respectively), PC (HR for RCC1 and RCC2 were 1.5 and 1.12, respectively), and CRC (HR for RCC1 and RCC2 were 1.3 and 1.03, respectively). CONCLUSION:RCC1 and RCC2 expression is a negative prognostic marker in NSCLC, PC, and CRC. Further studies to investigate RCC1 and RCC2 function at the molecular level may provide opportunities for novel targeted drug development.

Background: Metaplastic Breast Cancer is rare and aggressive form of BC with majority having triple-negative receptor status. There are no standard therapeutic approaches for MBC and patients are treated similar to invasive ductal triple negative breast cancer (ID-TNBC) but with worse outcomes in comparison to ID-TNBC. Hence, there is an urgent need for development of new drug targets and therapies to improve outcomes in these patients. Here, we characterize the molecular and immune signature of metaplastic TNBC (M-TNBC). Methods: 455 BC samples (M-TNBC, n=91; ID-TNBC, n=364) were analyzed 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. Pathway enrichment was determined by GSEA (Broad Inst). Statistical significance was determined using chi-square and Mann-Whitney U test and p-value <0.05 was considered significant. Results: M-TNBC had a higher frequency of PIK3CA (50.0% vs 14.5%), PTEN (16.1% vs. 8.4%), EGFR, (2.2% vs 0%), PIK3R1 (14.4% vs. 5.5%), TERT (29.7% vs. 0.5%), but lower frequency of TP53 (70.6% vs. 89.7%) compared to ID-TNBC (all p<0.05). There was no difference in the frequency of TMB-high (3.3% vs 5.2%, p=0.58), dMMR/MSI-H (2.2% vs 1.1%, p = 0.34) and PD-L1 positivity (22c3) (50.0% vs 42.6%, p=0.38) between M-TNBC and ID-TNBC. M-TNBC had lower AR protein expression (11.0% vs 24.8%) and lower frequency of fusion variant-AR (0% vs 4.9%) compared to ID-TNBC (all p < 0.05). Analysis of inferred immune cell infiltrates showed that M-TNBC had increased infiltration of M2 macrophages (3.3% vs. 2.9%) and neutrophils (5.9% vs. 2.6%) but decreased infiltration of B cells (3.9% vs. 4.5%), T regulatory cells (Treg) (1.0% vs. 1.9%), Dendritic Cells (DC) (1.7% vs. 3.2%) and CD8 T cells (0% vs. 0.5%) (all p < 0.05). M-TNBC had decreased IFNγ score (-0.31 vs -0.24, p=0.05), but increased MAP kinase pathway activity score (0.9 vs 0.08, p<0.05). ID-TNBC had higher expression of immune checkpoint genes (FOXP3, IDO1; FC: 1.3-1.5), cell cycle genes (CDKN1B, E2F1, CCNE1; FC: 1.2-1.4), inhibition of apoptosis genes (BIRC3, BIRC6, BCL2; FC: 1.1-1.3), but lower expression of stem cell-related genes (CD44, ALDH1A2, KLF4, SOX2; FC: 1.2-2.3) (all p < 0.05). M-TNBC had gene set enrichment of epithelial to mesenchymal transition (EMT) pathway (NES: 1.5, FDR<0.25). Conclusion: These data indicate that M-TNBC is associated with an aggressive disease biology with higher frequency of PIK3CA, PTEN, PIK3R1, TERT, EGFR and gene set enrichment of EMT pathway. Higher expression of stem cell-related gene expression in M-TNBC indicates their association with therapy resistant phenotype. Also, M-TNBC had increased infiltration of M2 macrophages and neutrophils, decreased infiltration of B cells, Treg, DC and CD8 T cells and, lower IFNγ score and MAP kinase activity score suggesting differential tumor immune microenvironment compared to ID-TNBC However, these findings warrant further validation in larger studies. Citation Format: Pooja Advani, Sachin Kumar Deshmukh, Sharon Wu, Joanne Xiu, Jose P. Leone, Priya Jayachandran, Matthew Oberley, Maryam Lustberg, Stephanie L. Graff, George W. Sledge Jr, Asher Chanan-Khan. Molecular and Immune Landscape of Metaplastic Triple Negative Breast Cancer Compared with Invasive Ductal Triple Negative Breast Cancer [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 PS17-02.

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.

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.

Background: Human epidermal growth factor receptor 2 (HER2) gene expression is an important predictive and prognostic biomarker in breast cancer (BC), which also guides treatment recommendations. The expression of HER2 is a continuum from null to positive and includes HER2-low and ultra-low as targets for anti-HER2 antibody–drug conjugates (ADC). However, HER2-low and ultra-low have not been studied in male BC. Here, we analyze whether there are any differences in molecular and immunological features between HER2-low, ultra-low and HER2-null/negative expression in males with BC. Methods: 199 male breast tumor samples were included in this study. HER2-null expression was defined as infiltrating cancer cells completely free of HER2 immunohistochemistry (IHC) staining. HER2 ultra-low expression was defined as ≤10% cancer cell showing incomplete and faint/weak membrane staining. HER2-low expression was defined as HER2 (IHC) 1+ or 2+ with negative chromogenic in situ hybridization (CISH) assay. HER2-positive expression was defined as HER2 IHC 3+ staining or 2+ with positive CISH assay. Mutations and gene expression were detected by next-generation sequencing (NextSeq; WES, NovaSeq) and Whole Transcriptome Sequencing (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ), respectively; tumor mutational burden (TMB) totaled somatic mutations per tumor (high>10 mt/MB). Immune cell fractions were calculated by deconvolution of WTS:Quantiseq. Statistical significance was determined using chi-square and Mann-Whitney U test and p-value <0.05 was considered significant. Results: Of 199 samples, there were 70 (35.2%) HER2-null tumors, 49 (24.6%) HER2 ultra-low tumors, 64 (32.2%) HER2-low tumors, and 16 (8.0%) HER2-positive. The proportion of HR+ was 81.4% in HER2-null, 93.8% in HER2 ultra-low, 87.5% in HER2-low and 81.2% in HER2-positive tumors. HER2 ultra-low male BC had higher frequency of PIK3CA (44.19% vs 23.64%) compared to HER2-null and KMT2D (7.5% vs 0%) compared to HER2-low, all p<0.05. HER2-low male BC had numerically lower frequency of TP53 (8.7% vs 16.6%, p=0.2) and ESR1 (1.6% vs 5.4%, p=0.2) compared to HER2-null. No significant differences were noted in TMB-high frequency (7.1% vs 5.0% vs 6.9%) and PD-L1 positivity (22C3) (8.11% vs 9.0% vs 10.6%) between HER2 ultra-low, HER2-low and HER2-null (all p=1.0). Analysis of inferred immune cells revealed that HER2 ultra-low and HER2-low male BC had higher infiltration of B cells (6.6% vs 5.8% vs 4.9%) but lower infiltration of neutrophil (2.4% vs 2.0% vs 3.8%) (all p<0.05). HER2-low had lower expression of immune checkpoint gene LAG3 (fold change (FC): 1.6), stem cell-related genes (KLF4, POU5F1; FC: 1.3-1.7) and drug-efflux gene ABCB5 (FC: 2.2) compared to HER2-null tumors (all p<0.05). HER2 ultra-low had lower expression of drug-efflux gene ABCG2 (FC: 1.5) compared to HER2-null tumors (p<0.05). Data adjusted for HR-subtype will be presented at the meeting. Conclusions: Our findings add valuable information to the current understanding of the HER2 spectrum in the male breast cancer, including frequency distribution and molecular characterization. With some exceptions, HER2-low, ultra-low and null breast cancer in men shared genomic features, suggesting that the disease biology may not be different across the spectrum of what historically has been considered HER2-negative disease. Interestingly, HER2 ultra-low, HER2-low and HER2-null had differential tumor immune microenvironment that warrant further exploration. Citation Format: Dario Trapani, Sachin Kumar Deshmukh, Sharon Wu, Joanne Xiu, Priya Jayachandran, Nancy U. Lin, Giuseppe Curigliano, Milan Radovich, Maryam Lustberg, Stephanie L. Graff, George W. Sledge Jr, Sara M. Tolaney, Jose P. Leone. Molecular and immunological characterization of HER2-low, HER2 ultra-low, and HER2-null male breast cancer [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 P3-01-25.

Prevalence of common mutations in tumor subtypes

A subset of cancers present with unclear or potentially incorrect primary histopathologic diagnoses, including cancers of unknown primary (CUP). We aimed to develop and validate an artificial intelligence (AI) tool, Genomic Probability Score AI (GPSai™), which predicts tumor tissue of origin in CUP and flags potential misdiagnoses for additional workup during routine molecular testing. The GPSai model was trained on whole exome and whole transcriptome data from 201,612 cases submitted for tumor profiling at Caris Life Sciences. Retrospective (N = 21,549) and prospective (N = 76,271) validations were performed. The clinical impact was evaluated over 8 months of live testing and through physician surveys. GPSai demonstrated 95.0% accuracy in non-CUP cases and reported on tumor tissue of origin in 84.0% of CUP and 96.3% of non-CUP cases. During the initial 8 months of implementation, GPSai changed the diagnosis on 704 patients (0.88% of all profiled cases), which were supported by orthogonal evidence including imaging, IHC, mutational signatures, hallmark fusions, or viral reads. Diagnosis changes prompted changes in targeted therapy eligibility based on level 1 clinical evidence in 86.1% of cases (n = 606/704). A majority (89.7%; n = 87/97) of physician responses indicated acceptance of the GPSai results, and 53.6% (n = 52/97) of responses stated that the results prompted a change in treatment plan. GPSai accurately identifies tumor tissue of origin and has the potential for clinical impact in a small but meaningful subset of patients with CUP or pathologically ambiguous tumors. Our results support the integration of this AI tool into routine molecular testing to improve diagnostic accuracy and guide subsequent therapeutic decisions. SIGNIFICANCE:Our findings show that GPSai, a deep learning-based tool, can support the identification of primary tumor sites with high accuracy in conjunction with orthogonal evidence. Its integration into routine tumor profiling furthermore allows simultaneous biomarker identification. Analysis of real-world implementation of GPSai shows that it enhances diagnostic accuracy, including resolution of CUP cases, and prompts clinically relevant therapeutic recommendation changes without requiring additional specimen.

HSD3B1 encodes an enzyme that catalyzes the conversion of adrenal precursors into potent sex steroids. A common germline variant (c.1100C) enhances this effect and is linked to breast cancer (BC) progression. As the HSD3B1 genotypes contribute to differences in local and adrenal steroid production, their transcriptional and phenotypic effects on cancers influenced by hormonal signaling such as BC and endometrial cancer (EC)—particularly in relation to menopausal status—remain unclear. We analyzed BC and EC sequenced from patients that received diagnostic tests in oncology clinics, and we determined the germline HSD3B1 c.1100 genotype (AA, AC, CC) from tumor DNA sequencing by using variant allele frequency, with inferred menopausal status assumed by age at molecular profiling. Whole-transcriptome RNA sequencing and gene set enrichment analysis showed that adrenal-permissive homozygous (CC) tumors in premenopausal ER + BC were enriched for hormone-related pathways, including Estrogen Response Early (NES ≈ +1.8). In premenopausal triple-negative BC, adrenal-restrictive homozygous (AA) tumors exhibited the elevated expression of immune and epithelial genes and the increased prevalence of MED12 alterations (AA 0.25% vs. CC 8%, p < 0.01). In endometrioid EC, CC tumors demonstrated the suppression of immune and proliferative pathways. Postmenopausal cases had higher progesterone receptor IHC positivity (AA 75% vs. CC 83%, p < 0.05) and numerically more frequent ESR1 copy number gains (AA 2.0% vs. CC 4.0%). Results highlight context-specific associations between germline HSD3B1 genotypes and tumor biology in BC and EC.