Citations

Abstract Purpose: B7-H3 (CD276) represents a promising therapeutic target tested in high-risk localized and treatment-refractory metastatic prostate cancer. To guide therapeutic development and treatment strategies, we examined prostate tumors and evaluated expression, molecular features, and overall survival (OS), accounting for tissue site, hormone sensitivity status, and race. Experimental Design: A total of 8,157 prostate cancer samples with paired DNA/RNA were analyzed based on annotations by tissue site, self-reported race, and disease state: hormone-sensitive prostate cancer (HSPC), castration-resistant prostate cancer (CRPC), or neuroendocrine prostate cancer (NEPC). Expression quartiles were B7-H3–high (>75th percentile) or B7-H3–low (<25th percentile). The OS was evaluated using Kaplan–Meier and Cox proportional hazards models. Results: B7-H3 expression was broadly maintained but varied by tumor site, hormone sensitivity status, and race. High expression aligned with AR-associated transcription factors (HOXB13 and FOXA1), AR-associated pathogenic dysregulations (AR-V7, SPOP, FOXA1, and TMPRSS2:ERG fusions), and actionable surface antigens (TROP2 and NECTIN-4). Weak correlations were found for lineage-plastic program regulators (EZH2, SOX2, and ASCL1) and NEPC-associated surface antigens (DLL3 and CEACAM5). High B7-H3 expression in primary tumors and HSPCs portended adverse OS [hazard ratio (HR), 1.342 and 1.30; confidence interval (CI), 1.19–1.512 and 1.15–1.46; q < 0.0001] although it was favorable in metastatic tumors (HR, 0.823; CI, 0.719–0.942; q = 0.0048). No significant differences in OS were observed among CRPCs and NEPCs, although OS varied by race, with the poorest survival in Asian/Pacific Islander patients with metastatic prostate cancer (HR, 3.72; CI, 1.49–9.29; q = 0.012). Conclusions: Maintained B7-H3 expression in various prostate cancer settings supports its viability as a target. The associations with AR-related molecular factors, surface antigens, and investigative targets for cell therapy or antibody–drug conjugates suggest potential dual-targeting strategies. See related article by Sharma et al., p. 3365

Abstract Annual breast cancer screening is a critical piece of women’s health. While mammography remains the gold standard, increased breast density leads to decreased sensitivity, underscoring the need for more sensitive and accessible screening methods. Liquid-based biopsies for early breast cancer detection are emerging but currently remain out of reach for clinical use. Recent data on nucleotide assessment from plasma in breast cancer have been mixed with 87% sensitivity for late-stage disease (stage 3-4) but only 20% sensitivity for early-stage disease (stage 0-2). Proteomics is an exciting area for early cancer screening where improvements in sample preparation and equipment have enabled major advances in early cancer detection. This is particularly true for a deep proteome assay as proteins can be identified which are 8-9 orders of magnitude less abundant than the most common plasma proteins. This study evaluates the presence of breast cancer using label-free shotgun mass spectrometry-based proteomics on less than 1 ml of plasma from 1,259 women classified as either healthy or newly diagnosed treatment naïve breast cancer patients with a focus on early-stage disease. We trained using a cohort of 845 women, comprising of 466 healthy and 379 with breast cancer and validated on 397 women (195 healthy and 202 breast cancer) using a protein-based machine learning classifier to distinguish these groups. All plasma samples were processed in a blinded manner coupled with semi-quantitative, label-free mass spectrometry (MS)-based analysis. The median number of proteins detected per patient across breast cancer and healthy individuals was 7,064 and 7,054, respectively. The validation performance achieved 92.3% specificity for healthy controls and an overall sensitivity of 92.6% with an AUC of 0.975 (95% CI: 0.961-0.987) for breast cancer patients. Sensitivity broken down by clinical stage (0-IV), molecular subtypes (HR+/HER2+, HR-/HER2+, HR+/HER2-, and HR-/HER2-) and pathological subtypes (LCIS, DCIS, ILC, and IDC) were ≥85% and indistinguishable across all stages and subtypes. Gene set enrichment analyses (GSEA) identified pathways including epithelial-to-mesenchymal transition (EMT) and PI3K-AKT signaling as enriched in the breast cancer samples, highlighting that our test can identify cancer-related proteins in early-stage patients. Overall, we have developed a highly sensitive blood-based assay that utilizes deep proteomic profiling to identify distinctive cancer specific signatures in women who are undergoing screening for breast cancer. This work enables us to develop a protein-based classifier from plasma for early detection of breast cancer and enhances screening strategies for women with dense breasts who are at average or high-risk based on family history, specific genetic mutations such as BRCA1/2, race, or other factors. Citation Format: Alec Horrmann, Yash Travadi, Jacob Carey, Kevin Mallery, Ella Boytim, Grant Schaap, Carissa Rungkittikhun, Kaylee Kamalanathan, Nathaniel R. Bristow, Catalina Galeano-Garces, Adam Groth, Alexa R. Hesch, Pooja Advani, Justin Hwang, Badrinath R. Konety, Justin M. Drake. Deep plasma proteomics for early-stage breast cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4073.

382 Background: Androgen receptor signaling drives prostate cancer (PC) progression, and genes involved in androgen production, uptake, and conversion (APUC) are key therapeutic targets. We previously defined a 6-APUC gene set ( HSD3B1, HSD3B2, CYP3A43, CYP11A1, CYP11B1, and CYP17A1 ), that identified metastatic PC with favorable prognosis and predict docetaxel benefit. However, the expression landscape and clinical relevance of APUC genes have not been interrogated in localized PC. Methods: We analyzed the expression of 21 APUC genes in 55,329 radical prostatectomy tumor samples that underwent Decipher prostate genomic classifier (GC) testing (Veracyte Inc) between 2016-2024. Baseline clinical/pathologic factors were retrieved from Decipher GRID database (NCT02609269). Associations between gene expression quartiles with PathoGenomic Risk Factors (PGRF) including Very High Decipher score >0.85 (VHD), Grade Group (GG) 4-5, LNI and SVI were examined. Associations with distant metastatic (DM) outcomes and metastasis free survival (MFS) were evaluated using logistic regression in Mayo545 and Cox regression in Meta855 RP cohorts, respectively. Results: High expression of SRD5A2 was associated with favorable PGRF (lower odds ratio (OR), p<0.0001), lower rate of development of DM (OR 0.36, p<0.05), and longer MFS (Hazard ratio (HR) 0.51, p<0.05). HSD17B6 showed similar results for PGRF (lower OR, p<0.0001), DM (OR 0.58, p<0.05) and longer MFS (HR 0.39, p<0.05) despite being associated with GG4-5 and SVI+ in samples with VHD. In contrast, high expression of SRD5A3 and HSD17B10 had high OR for PGRFs (p<0.0001). High expression of SLCO2B1 , an androgen uptake gene, had the strongest association with GG4-5 and SVI/LNI (p<0.001 for both). We noted that SRD5A2 lacked correlation with SRD5A1 and SRD5A3 , which instead correlated with each other. Compared to all APUC genes, SRD5A2 had limited correlations with almost all APUC genes, including ones that exhibited robust cross correlation with each other . Similar to our prior finding, most APUC genes demonstrated negative correlation with AR expression and AR activity signatures. Key outcomes were validated based on transcriptomic data from 492 samples from TCGA database. Conclusions: Comprehensive analysis of 21 APUC genes in over 55,000 PC identified distinct, context-dependent associations with metastatic outcomes and clinical and molecular risk factors. Notably, high SRD5A2 expression was associated with lower odds of high-risk features and metastatic outcomes, whereas SRD5A1 and SRD5A3 linked to adverse outcomes, suggesting critical yet divergent roles among 5-alpha-reductase family members. These findings underscoring androgen-metabolic heterogeneity and highlighting SRD5A2 as a potential biomarker of indolent biology in localized prostate cancer.

Abstract Background Clival metastases are a rare and clinically aggressive manifestation of advanced prostate cancer, associated with cranial nerve palsy and poor survival. The molecular features of prostate cancers giving rise to clivus metastases remain unknown. Patients and methods We performed a multi-center retrospective study across six institutions, identifying prostate cancer patients with radiographically confirmed clival metastases and available next-generation sequencing (NGS) data. Baseline characteristics and clinical outcomes were collected. Genomic alterations from tissue- and/or blood-based assays were aggregated at the patient level and compared with a publicly available metastatic castration-resistant prostate cancer (mCRPC) cohort (SU2C/PCF). Results Fifty-nine patients with clival metastases contributed 87 molecular assays. More than half of patients had Gleason grade group 5 cancer and presented with de novo metastatic (M1) disease. The median interval from initial prostate cancer diagnosis to clival metastasis was 71.4 months (95% CI, 42.0-101.7), while median overall survival following clival involvement was only 15.3 months (95% CI, 6.9-22.8). Compared with the SU2C/PCF mCRPC cohort, clival metastases showed significant enrichment of BRAF and CHEK2 alterations as well as homologous recombination repair (HRR) with relative depletion of AR-related, PI3K pathway, and G2–M pathway alterations. Conclusion Prostate cancers giving rise to clival metastases exhibit a distinct molecular profile enriched for DNA damage–repair and RAF kinase alterations, suggesting unique metastatic biology and potential therapeutic vulnerabilities.

Abstract Natural killer (NK) cells represent key effectors of antitumor immunity, yet emerging evidence highlights populations with distinct roles in cancer. Despite such expanded diversity within the NK cell repertoire, we lack an understanding of how this heterogeneity affects immune responses and downstream clinical outcomes. Using single-cell RNA sequencing, we systematically profiled NK cells across cancer and uncovered a dichotomous phenotypic and functional landscape of tumor-infiltrating NK cells shaped by opposing intrinsic signaling programs that drive the expression of IFNG or TGFB1. These divergent programs are associated with distinct transcription factor circuits that integrate cues within the tumor microenvironment and skew NK cells toward proinflammatory or suppressive functions. We found that the capacity for NK cells to engage in either functional direction is intrinsically linked to their phenotypic identity. Canonical NK cells recruited from circulation predominantly directed suppressive TGFB1 signals toward effector CD8+ T cells in tumors. Of note, these subsets exhibited higher TGFB1 expression than intratumoral myeloid cells across tumor types. In contrast, a tissue-resident (TR) adaptive subset exhibited exclusively proinflammatory IFNG-driven profiles and was associated with prolonged survival in both primary and metastatic tumor settings. Moreover, these TR adaptive NK cells, but not other subsets, were linked to response to immune checkpoint blockade. Collectively, our study reveals a previously unrecognized regulatory axis in NK cells that shapes NK cell diversity and augments broader antitumor immune responses.

e14555 Background: Natural killer (NK) cells are innate lymphocytes that engage in direct cytotoxic responses against malignant cells and promote pro-inflammatory tumor microenvironments. While studies have predominantly focused on conventional NK cell subsets, recent findings uncovered adaptive and tissue-resident (TR) subsets with unique functional properties. The influence of distinct NK cell subsets on tumor immunobiology and clinical outcomes remains unknown. Methods: Single-cell RNA-sequencing (scRNA-seq) of primary, therapy-naïve tumors from 276 patients across 14 cancer types, as well as 39 patients treated with immune checkpoint blockade (ICB), was accessed through publicly available datasets. We employed systematic computational analyses to phenotype and examine intra-tumoral NK cells. Gene signatures for NK cell subsets were applied to bulk RNA-sequencing from 212,105 tumor samples to delineate their associations with clinical outcomes using the Caris CODEai database. Results: Six main subsets of NK cells were identified in solid tumors, including conventional CD56 dim and CD56 bright NK cells, alongside adaptive, TR adaptive, TR CD56 bright , and TR CD56 bright /adaptive subsets. Across cancer, TR adaptive NK cells emerged as the most prognostic subset, with high infiltration conferring significantly longer overall survival (HR[95% CI] = 0.68[0.66-0.69], p < 0.001) than conventional CD56 dim and CD56 bright subsets. This was also observed in cohorts of head and neck, non-small cell lung, and esophageal cancers, particularly those treated with ICB. Notably, elevated TR adaptive NK cell infiltration was more prognostic than high tumor mutational burden in these settings. This was most apparent in ICB-treated head and neck cancers (HR = 0.48[0.41-0.57], p < 0.001 vs HR = 0.90[0.76-1.04], p = 0.16). Additionally, TR adaptive NK cells were strongly correlated to the infiltration of CD8 + T cells and B cells in tumors. Underlying these observations, our scRNA-seq analyses demonstrated that TR adaptive NK cells exhibit the highest immunomodulatory activity of all subsets, expressing high levels of pro-inflammatory cytokines ( CCL3 , CXCL13 , and IFNG ) and low levels of immunosuppressive cytokines ( TGFB1 ). Mechanistically, this activity was linked to the upregulation of transcription factors associated with NK cell activation, function, and persistence and downregulation of genes related to stress response and exhaustion. Interestingly, scRNA-seq from paired pre- and post-ICB-treated tumors revealed a unique expansion of cytotoxic GZMB + TR adaptive NK cells after treatment. Conclusions: TR adaptive NK cells represent a novel subset of NK cells that may drive anti-tumor immune responses, particularly in the context of ICB treatment. Features of this unique population may be harnessed to design more effective NK cell-based therapies against solid tumors.

5073 Background: B7-H3 ( CD276 ) is an immunomodulatory protein overexpressed in prostate cancer (PC), representing a promising therapeutic target. However, expression of B7-H3 across PC disease states (hormone sensitive [HSPC], castration resistant [CRPC], neuroendocrine [NEPC]) and across races is poorly understood. We analyzed PC samples from a large clinico-genomic database to comprehensively characterize B7-H3 expression and elucidate its therapeutic potential in PC patients. Methods: We analyzed 7,682 PC samples with paired DNA/RNA profiling from Caris Life Sciences. Using CD276 mRNA expression measured by transcripts per million (TPM), samples were stratified into B7-H3-high (>75% centile) or low (<25% centile) groups. Annotations of HSPCs and CRPCs were based on time of biopsy collection relative to first use of androgen deprivation therapies. NEPC was defined histologically (Epstein, AJSP 2014). Transcriptomic alterations were compared using Mann-Whitney U tests. Overall survival (OS) was obtained from insurance claims data and assessed by Kaplan-Meier and Cox proportional hazards analyses. Results: B7-H3 was expressed at similar levels across specimen sites (prostate, lymph node, bone, liver, lung). HSPCs and CRPCs had similar levels of B7-H3 expression, but was reduced in NEPCs (TPM = 5.07, 4.96, 4.48; q<0.0001). While B7-H3-high was associated with worse OS in HSPCs (HR 1.32, 95CI 1.14-1.53, p=0.0002), it was associated with better OS in CRPC (HR 0.82, 95CI 0.69-0.97, p=0.018). B7-H3 expression was comparable in white, African American (AA), and Asian patients. However, Asian patients with high B7-H3 had worse outcomes (HR 4.08, 95CI 2.10-7.93, p<0.0001) while AA patients had improved outcomes (HR 0.74, 95CI 0.57-0.97, p=0.027). In co-expression analyses, B7-H3 was positively correlated with AR transcriptional co-factors ( HOXB13 , FOXA1 ) (R=0.59, 0.47; q<0.0001) but had weaker correlations with lineage plastic factors ( EZH2 , SOX2 , ASCL1 ) (R=0.26, 0.11, -0.02). Further, B7-H3-high correlated with high AR-score and low-NEPC. Given emergent bi-specific therapeutics in PC, we examined co-expression of B7-H3 with other cell surface targets. TROP2 ( TACSTD2 ) and NECTIN-4 ( PVRL4 ) exhibited the greatest correlation with B7-H3 (R=0.42, 0.40; q<0.0001); whereas PD-L1 ( CD274 ), CTLA4 , DLL3 , and CEACAM5 had weaker correlations (R=0.18, 0.08, 0.14, 0.09). Conclusions: High B7-H3 expression worsens prognosis in HSPC but improved prognosis in CRPC. Outcomes differed by race, with B7-H3-high Asian patients exhibiting worse OS while AA patients had better OS. The positive correlation between B7-H3 and AR co-factors suggests that B7-H3 is AR-regulated, at least in CRPC. Lastly, bi-specific approaches may be valuable against B7-H3-high tumors, with co-targeting of TROP2 and/or NECTIN-4.

Abstract Purpose: Germline alterations in homologous recombination repair (gHRR) genes affect the pathogenesis, treatment options, and survival of patients with cancer. However, distinct gHRR gene alterations may differentially affect treatment response and oncogenic signaling. In this study, we interrogated genomic and transcriptomic data and assessed clinical outcomes of patients with gHRR mutations across four BRCA-associated cancers (breast, ovarian, pancreatic, and prostate cancers) to identify therapeutic vulnerabilities. Experimental Design: We assessed 24,309 patients undergoing matched tumor/normal next-generation DNA and RNA sequencing. Annotated gHRR gene variants [germline BRCA1, germline BRCA2, germline PALB2, germline ATM (gATM), and germline CHEK2] were analyzed. HRs were used to assess survival outcomes comparing germline versus sporadic groups. Somatic alterations and their frequencies were compared across gHRR-altered groups. Differential gene expression and gene set enrichment analysis were used to compare transcriptomic profiles. Results: Somatic TP53 mutations were depleted in gATM carriers (P &lt; 0.05) across all four BRCA-associated cancers by up to 2.5-fold. Tumors with germline BRCA1/2 mutations were associated with improved survival in patients with ovarian cancer and had consistent enrichment of TP53 mutations in all four cancers. gATM mutations displayed elevated p53 transcriptional activity in all four cancers, with significance reached in breast and prostate cancers (P &lt; 0.01). In breast, ovarian, and prostate cancers, gATM tumors demonstrated significantly increased inflammatory pathways (P &lt; 0.001). Finally, using gene dependency data, we found that cell lines that were highly dependent on ATM were co-dependent on canonical p53 function. Conclusions: gATM-associated cancers seem to require intact p53 activity and this synthetic essentiality may be used to guide targeted therapies that perturb canonical TP53 function.

202 Background: While several PARP inhibitors are approved in combination with hormonal therapies for metastatic castrate resistant prostate cancer (CRPC), their use in castrate sensitive prostate cancer (CSPC) is understudied. We previously reported that olaparib monotherapy (without ADT) shows significant clinical activity in BRCA2 -altered patients with biochemically recurrent (BCR) prostate cancer. Here, we explored prognostic and predictive PARP-related mRNA biomarkers derived from primary prostate tumors. Methods: We characterized the expression of 16 PARP transcripts using a cohort of 52,266 radical prostatectomy (RP) specimens (2016-2024) undergoing Decipher prostate genomic classifier testing (Veracyte Inc). Transcriptome-wide mRNA expression data as well as baseline clinical/pathologic factors were retrieved from the Decipher GRID database (NCT02609269). Associations between high PARP expression (top 25%), baseline factors, and gene signatures were examined using logistic regression. We assessed the association between PARP expression and distant metastases in 3 retrospective cohorts (n= 545, 855, and 325). Finally, we evaluated PARP expression in our prior phase II trial (NCT03047135, n=39) of olaparib monotherapy in the setting of BCR following RP. Results: Higher mRNA levels of PARP1 , 2 , 8 and 14 showed strong associations (all P <0.001) with known adverse prognostic factors such as very-high Decipher score (>0.85), high Gleason grade (GG5) and non-organ confined disease (EPE, SVI and LNI), while higher PARP6 and 7 expression were associated with more favorable disease characteristics. High PARP1 expression was positively correlated with DNA-repair deficiency (r=0.77) and tumor immune microenvironment (r=0.62) signatures. In 3 independent retrospective cohorts of intermediate-high risk PCa patients, higher PARP1 expression was strongly associated with development of distant metastases after RP (HR 1.70, 1.81, and 1.67 respectively; all P <0.05). Finally, in our phase II trial of patients with BCR who received olaparib monotherapy, PARP1 (but not PARP2 ) expression from RP was 1.5-fold higher in samples with BRCA2 mutations (median 0.416 vs 0.276, P =0.01) and was associated with overall improved PSA progression-free survival (continuous variable, HR 0.55, P =0.01) and longer time to next systemic treatment (continuous variable, HR 0.66, P =0.02). Conclusions: High PARP1 mRNA expression, measured at RP, is associated with aggressive disease biology and greater risk of metastasis. However, high PARP1 (but not PARP2 ) levels are associated with improved response to PARP inhibition in the setting of BCR prostate cancer. Future trials testing PARP (including PARP1-selective) inhibitors in the CSPC space should consider measuring PARP1 mRNA levels in addition to BRCA1/2 mutation status. Clinical trial information: NCT02609269 , NCT03047135 .

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.