Zorko, Nicholas A.

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 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.

PURPOSE The continuous development of new imaging approaches, molecular phenotyping, genetic subtypes, prognosis assessments, and effective therapies across a range of disease states has created a need to redefine terminology and best practices for clinical trial conduct in patients with advanced prostate cancer. Methods We convened an international expert committee of diverse working groups, the Prostate Cancer Working Group 4 (PCWG4), between 2016 and 2025. Our objective was to formulate updated criteria based on emerging evidence and clinical trial data in a biomarker context to provide guidance for clinical trial design, eligibility, and end point assessments for patients with advanced prostate cancer. RESULTS PCWG4 redefines terminology around the disease state and previous therapies in a patient-centric context and terminology focused on androgen pathway modulation. We consider imaging, with a particular focus on positron emission tomography (PET)–defined disease. New recommendations are provided for disease state terminology, defining eligibility criteria, response and delay/prevent end points, intervals for reassessments including imaging, and patient-reported outcome determination. We provide recommendations in a biomarker-based context of use for the intended indication, reflective of patient benefit for specific interventions. We emphasize the need for development of validated PET imaging and molecular and phenotypic criteria as well as trial designs to appropriately risk stratify patients, predict and assess benefit, and measure post-treatment outcomes reliably in a trial framework. CONCLUSION PCWG4 updates recommendations on patient and tumor characterization, therapy development, and imaging criteria and extends guidance into earlier androgen pathway modulator–naïve/sensitive disease states to reflect an evolving, heterogeneous, and diverse patient population to optimize treatment benefits for all patients.

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 B7-H3 (CD276) is a transmembrane glycoprotein of the B7 immune checkpoint superfamily that has emerged as a promising therapeutic target. To better understand the applicability of B7-H3–directed therapies, we analyzed 156,791 samples comprising 50 cancer types to interrogate the clinical, genomic, transcriptomic, and immunologic correlates of B7-H3 mRNA expression. DNA (592-gene/whole-exome) and RNA (whole-transcriptome) sequencing was performed from samples submitted to Caris Life Sciences. B7-H3 high versus low expression was based on top and bottom quartiles for each cancer type. Patients’ overall survival was determined from insurance claims data. Pathway analysis was performed using gene set enrichment analyses. Immune cell fractions were inferred using quanTIseq. B7-H3 is expressed across several human malignancies including prostate, pancreatic, ovarian, and lung cancers. High B7-H3 expression is associated with differences in overall survival, possibly indicating a prognostic role of B7-H3 for some cancers. When examining molecular features across all cancer types, we did not identify recurrent associations between B7-H3 expression and genetic alterations in TP53, RB1, and KRAS. However, we find consistent enrichment of epithelial-to-mesenchymal transition, Wnt, TGFβ, and Notch signaling pathways. In addition, tumors with high B7-H3 expression are associated with greater proportions of M1 macrophages, but lower fractions of CD8+ T cells. We have begun to define the genomic, transcriptomic, clinical, and immunologic features associated with B7-H3 expression in 50 cancer types. We report novel clinical and molecular features of B7-H3–high tumors which may inform how current B7-H3 therapeutics should be deployed and prioritized. Significance: B7-H3–targeting therapeutics have shown promising results in initial clinical trials. In this pan-cancer analysis of B7-H3 mRNA expression, we found that B7-H3 exhibits robust expression in many common cancer types. These results may inform further development of B7-H3–targeting therapeutics and may guide clinical decisions for patients with limited treatment options.