Our integrative proteogenomics (genome, proteome and phosphoproteome) of localized prostate cancer (PCa) in an equal-access Military Health System patient cohort (57 Black and 55 White) revealed significant ancestry-associated differences. Somatic and germline regulatory differences converged on androgen, metabolic, PI3K/AKT/mTOR, and DNA damage response (DDR) pathways, with ancestry-specific immune- and stromal-associated signals. Black patients displayed greater genomic variability, enhanced androgen response, fatty-acid metabolism, and epithelial-mesenchymal transition, while White patients showed prevalent DDRG alterations, activated oncogenic signaling (MYC, E2F, mTORC1), and cell cycle regulation. Phosphoproteomics highlighted distinct kinase activities and candidate druggable dependencies. Multiomics integration revealed three exploratory tumor subtypes whose distinct biological programs were reproducibly validated. Ancestry-associated eQTLs supported inherited regulation of the proteome independent of CNAs. Ancestry-specific CNA and protein panels improved progression risk prediction beyond PSA and pathology models. These findings provide a framework for ancestry-informed prognostic models and generate testable hypotheses for precision therapies to reduce outcome disparities. ### Competing Interest Statement The authors have declared no competing interest. Uniformed Services University of the Health Sciences, https://ror.org/04r3kq386

Black men experience higher incidence and mortality rates from prostate cancer, yet the molecular drivers of outcome disparities are not clearly defined. We performed a multi-omics analysis of localized PCa in the equal access Military Health System to identify ancestry-associated biological differences and prognostic markers. We analyzed tumors from 112 patients (57 Black, 55 White) using whole genome sequencing, quantitative proteomics, and phosphoproteomics. To identify ancestry-associated differences, we leveraged gene set enrichments from over-representation analyses and then used Multi-Omics Gene-Set Analysis (MOGSA) to determine the contribution of multi-omics features to these enrichments. Integrative clustering using iClusterBayes was applied to define molecular subtypes based on shared variation across multi-omics data. Germline regulation of the proteome was assessed via eQTL analyses. Prognostic significance was evaluated with Cox proportional hazards models (adjusted for age, PSA, and percent African ancestry); and ancestry-specific biomarker panels were validated using Kaplan–Meier and AUC analyses. Black patients displayed higher genomic variability, stronger androgen response, fatty-acid metabolism, and EMT. White patients were enriched for DNA repair gene deletions, MYC/E2F signaling, mTORC1 activity, and cell-cycle progression. Phosphoproteomics revealed ancestry-specific kinase dependencies, with CK2α and CHEK2 activity elevated in Black patients, and CDK1/2, PRKD1, and CAMK2D/B activity in White patients. eQTL analyses highlighted germline regulation of the proteome independent of CNAs. MOGSA revealed 56 pathways grouped into five ancestry-associated subtypes, contrasting White-enriched apoptosis and epigenetic pathways with Black-enriched structural and DNA repair responses. Multiomics integration with iClusterBayes defined three reproducible molecular subtypes, externally validated, providing a basis for risk stratification with clinical potential. Somatic and germline regulatory differences converged on steroid hormone signaling, metabolic reprogramming, PI3K/AKT/mTOR, and DNA damage response pathways, with ancestry-associated immune and stromal signals. Prognostic analyses identified ancestry-specific and shared alterations associated with PCa progression. Prognostic CNA and protein panels exceeded PSA and pathology models (AUC >0.8), with ancestry-specific and universal biomarkers validated across cohorts. This integrative proteogenomics study of localized PCa defines ancestry-associated pathways, identifies reproducible molecular subtypes, and highlights germline regulatory influences on the tumor proteome. Ancestry-aware CNA, protein, and phosphoproteomic biomarker panels improve risk prediction and highlight potential therapeutic vulnerabilities, including kinase and metabolic dependencies. These findings provide a framework for refining ancestry-informed risk stratification and testing hypotheses of precision treatments to reduce outcome disparities. Cara C. Schafer, Tamara S. Abulez, Xijun Zhang, Kun-Lin Ho, Jiji Jiang, Denise Young, Jesse Fox, Kelly A. Conrads, Brian L. Hood, Gauthaman Sukumar, Darryl Nousome, Praveen-Kumar Raj-Kumar, Mariano Russo, Ayesha A. Shafi, Xiaofeng A. Su, Albert Dobi, Amina Ali, Sally Elsamanoudi, Jennifer Cullen, William D. Figg, Gyorgy Petrovics, Clifton L. Dalgard, Matthew D. Wilkerson, Nicholas W. Bateman, Thomas P. Conrads, Isabell A. Sesterhenn, APOLLO Research Network, Leigh Ellis, Craig D. Shriver, Gregory T. Chesnut, Shyh-Han Tan. Integrated proteogenomics uncovers ancestry-specific and shared molecular drivers in localized prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr A057.

We analyzed genomic data from the prostate cancer of African- and European American men to identify differences contributing to racial disparity of outcome. We also performed FISH-based studies of Chromodomain helicase DNA-binding protein 1 (CHD1) loss on prostate cancer tissue microarrays. We created CHD1-deficient prostate cancer cell lines for genomic, drug sensitivity and functional homologous recombination (HR) activity analysis. Subclonal deletion of CHD1 was nearly three times as frequent in prostate tumors of African American than in European American men and it associates with rapid disease progression. CHD1 deletion was not associated with HR deficiency associated mutational signatures or HR deficiency as detected by RAD51 foci formation. This was consistent with the moderate increase of olaparib and talazoparib sensitivity with several CHD1 deficient cell lines showing talazoparib sensitivity in the clinically relevant concentration range. CHD1 loss may contribute to worse disease outcome in African American men.

We analyzed genomic data derived from the prostate cancer of African and European American men in order to identify differences that may contribute to racial disparity of outcome and that could also define novel therapeutic strategies. In addition to analyzing patient derived next generation sequencing data, we performed FISH based confirmatory studies of Chromodomain helicase DNA-binding protein 1 (CHD1) loss on prostate cancer tissue microarrays. We created CRISPR edited, CHD1 deficient prostate cancer cell lines for genomic, drug sensitivity and functional homologous recombination (HR) activity analysis. We found that subclonal deletion of CHD1 is nearly three times as frequent in prostate tumors of African American men than in men of European ancestry and it associates with rapid disease progression. We further showed that CHD1 deletion is not associated with homologous recombination deficiency associated mutational signatures in prostate cancer. In prostate cancer cell line models CHD1 deletion did not induce HR deficiency as detected by RAD51 foci formation assay or mutational signatures, which was consistent with the moderate increase of olaparib sensitivity. CHD1 deficient prostate cancer cells, however, showed higher sensitivity to talazoparib. CHD1 loss may contribute to worse outcome of prostate cancer in African American men. A deeper understanding of the interaction between CHD1 loss and PARP inhibitor sensitivity will be needed to determine the optimal use of targeted agents such as talazoparib in the context of castration resistant prostate cancer.

Background: Immunobiological factors may, in part, contribute to racial disparities in prostate cancer (PCa), the second leading cause of cancer-related death for US men. African American (AA) men face a greater risk of developing PCa and having a poorer outcome of this disease, compared to Caucasian American (CA) men. The goal of this study is to identify immune-specific differences influencing PCa health disparity as well as PCa progression. Methods: Ex vivo tumor biopsies were obtained following radical prostatectomy (RP) from 51 treatment-naïve patients (26 AA, 25 CA) under approved IRB protocols. Total RNA from fresh frozen tissue was isolated and amplified by PCR using NanoString multiplexed target enrichment protocols. We evaluated differences in resulting transcripts by self-reported race, pathology, and other clinical and pathologic features. Relative abundances of immune cell subsets were determined using computational methods, including previously published deconvolution algorithms, to estimate cellular content. Results: We identified differentially expressed genes between AA and CA tumors that are characterized by genes regulating Natural Killer (NK) cell function and Wnt signaling cancer pathways. Among the entire cohort, high expression of two of these differentially expressed genes, DVL2 and MAP3K5, were associated with worse biochemical recurrence (BCR)-free and metastasis-free survival. No difference in immune cell abundance was observed by self-reported race. Strikingly, among the entire cohort, low abundance of intratumoral mast cells was also associated with worse PCa pathology and poorer BCR-free and metastasis-free survival. Conclusions: Using a race-matched military cohort, we found that AA and CA tumors differ in the expression levels of Wnt and NK signaling-related pathways, yet cellular estimations of immune abundance did not significantly differ. Remarkably, the level of mast cell abundance within tumors at time of RP may be protective and prolong BCR-free and metastasis-free survival of patients. These types of in-depth immune profiling assessments have the potential to guide precision medicine and treatment decisions in the clinic. Disclaimer: The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of Uniformed Services University of the Health Sciences (USUHS), The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., the Department of Defense (DoD), the Departments of the Army, Navy, or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Cara Schafer, Jiji Jiang, Sally Elsamanoudi, Darryl Nousome, Denise Young, Yingjie Song, Isabell Sesterhenn, Gregory Chesnut, Shyh-Han Tan. Immune cell profiling of human prostate tumors identifies a protective and pathological role for intratumoral mast cells in the development of biochemical recurrence and metastasis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B036.

Introduction: ETS gene rearrangements involving ERG, ETV1 and ETV4, as well as PTEN deletions are among the most common genomic alterations in prostate cancer. Although these events are found to be mutually exclusive, the multifocality of prostate cancer suggests that they may co-occur in some tumors. The immunohistochemistry (IHC) detection of both ERG overexpression and PTEN protein loss are reliable substitutes for fluorescence in situ hybridization (FISH) assay to probe genomic alterations. The lack of specific antibodies that can discriminate between multiple ETS factors, however, has limited the evaluation for the co-occurrence of these genomic alterations in prostate cancer. Improving our ability to identify the multifocality or inter-focal heterogeneity of tumors can enhance our understanding of biological mechanisms of tumor initiation and progression, and help stratify patients for treatment. Methods: Using monoclonal antibodies (mAbs) that we developed against ETV1 and ETV4, we examined their expression in relation to ERG and PTEN expression by IHC using two TMAs constructed from primary prostate cancer specimens of independent patient cohorts: one was constructed from multiple 1 mm cores representing distinct tumor focus from multifocal tumors from 50 African American (AA) and 50 Caucasian American (CA) men; the other, from a single 2 mm core of individual tumors from a cohort of 152 AA and 304 CA men. Positive expression for each ETS factor and PTEN were evaluated for association with race and clinico-pathologic features that include PSA at diagnosis, Gleason score, pathologic T stage, biochemical recurrence, and metastasis. Univariate analysis of BCR free survival as a function of ERG, ETV1 and ETV4 and PTEN expression was performed for both patient cohorts. Results: ERG(+) expression alone or in combination with PTEN loss were significantly higher in CA men in both patient cohorts. When expression of ETS factors were evaluated together with PTEN status, positive expression of ETS factors and PTEN loss were significantly more prevalent among CA men in both patient cohorts ETV1 and ETV4 expression were detected at 5% or less in both patient cohorts. Cases harboring tumor foci that were positive for more than one marker evaluated frequently showed either intra-tumor or inter-tumor heterogeneity. Univariate analysis of PTEN-ETS protein expression status and BCR free survival showed that patients with loss of PTEN expression trends towards poorer BCR free survival regardless of ETS expression status. Conclusion: We showed that the IHC detection of multiple aberrantly expressed proteins such as ERG, ETV1, ETV4 and PTEN in prostate tumor specimens using highly specific mAbs can reveal the heterogeneity and multi-focality in prostate cancer, which could be used to enhance the stratification of patients for novel treatment strategies. Citation Format: Shyh-Han Tan, Cara Schafer, Denise Young, Jiji Jiang, Yingjie Song, Albert Dobi, Gyorgy Petrovics, Bettina F. Drake, Gregory T. Chesnut, Isabell A. Sesterhenn. Immunohistochemical detection of ERG, ETV1, ETV4, and PTEN in prostate tumor specimens reveals the heterogeneity of multi-focal prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A049.

Supplementary Figure S12 shows Mast vs TILs scores plotted by race for Gleason Sum, GG, and Dx Age.

Supplementary Table S1 shows the full list of differentially expressed genes in AA tumors along with log2 fold changes, p-values, and adjusted p-values.

Supplementary Table S3 shows Total TILs and individual cell type vs TILs scores evaluated as a continuous variable and by median and Youden index cutoffs for BCR-free and metastasis-free survival.

Supplementary Figure S1 shows differentially expressed gene pathways in AA tumors.

Elucidating the cellular immune components underlying aggressive prostate cancer, especially among African American (AA) men who are disproportionately affected by this disease compared with Caucasian American (CA) men, will support more inclusive precision medicine treatment strategies. We aimed to evaluate which immune-related genes and cell types are differentially expressed in AA tumors and how immunobiology impacts prostate cancer progression. We purified nucleic acid from tumor biopsies, obtained following radical prostatectomy, from 51 patients (AA = 26, CA = 25). Gene expression was measured using the NanoString platform from which we estimated immune cell abundances and assessed differences between groups based on clinicopathologic data. Product-limit estimates determined associations with biochemical recurrence (BCR)-free and metastasis-free survival. DVL2 and KLRC2 were significantly upregulated in CA tumors and were also associated with worse disease progression. No significant differences in immune cell abundances by race were observed. Highly significant reductions in abundances of mast cells versus tumor-infiltrating lymphocytes (TIL) were found in men with high-grade pathologies and in men who later developed metastases. Low ratios of mast cells versus TILs were associated with worse BCR-free survival and metastasis-free survival. Although estimated immune cell abundances were not different by race, we identified genes involved in metabolism and natural killer cell functions that were differentially expressed between AA and CA tumors. Among the entire cohort, depletion of mast cells within prostatectomy tumors was characteristic of advanced disease and susceptibility to disease progression. Significance: Our findings demonstrate that there are immune-related genes and pathways that differ by race. Impaired intratumoral cellular immune composition, especially for TIL-normalized mast cells, may be vital in predicting and contributing to prostate cancer disease progression.

Background: Aberrant ETV1 overexpression arising from gene rearrangements or mutations occur frequently in prostate cancer, round cell sarcomas, gastrointestinal stromal tumors, gliomas, and other malignancies. The absence of specific monoclonal antibodies (mAb) has limited its detection and our understanding of its oncogenic function.Methods: An ETV1 specific rabbit mAb (29E4) was raised using an immunogenic peptide. Key residues essential for its binding were probed by ELISA and its binding kinetics were measured by surface plasmon resonance imaging (SPRi). Its selective binding to ETV1 was assessed by immunoblots and immunofluorescence assays (IFA), and by both single and double-immuno-histochemistry (IHC) assays on prostate cancer tissue specimens.Results: Immunoblot results showed that the mAb is highly specific and lacked cross-reactivity with other ETS factors. A minimal epitope with two phenylalanine residues at its core was found to be required for effective mAb binding. SPRi measurements revealed an equilibrium dissociation constant in the picomolar range, confirming its high affinity. ETV1 (+) tumors were detected in prostate cancer tissue microarray cases evaluated. IHC staining of whole-mounted sections revealed glands with a mosaic staining pattern of cells that are partly ETV1 (+) and interspersed with ETV1 (-) cells. Duplex IHC, using ETV1 and ERG mAbs, detected collision tumors containing glands with distinct ETV1 (+) and ERG (+) cells.Conclusions: The selective detection of ETV1 by the 29E4 mAb in immunoblots, IFA, and IHC assays using human prostate tissue specimens reveals a potential utility for the diagnosis, the prognosis of prostate adenocarcinoma and other cancers, and the stratification of patients for treatment by ETV1 inhibitors.

Supplementary Figure S7 shows non-significant relative cell type scores by Dx PSA Group.

Supplementary Figure S11 shows BCR-free and Metastasis-free survival for cell types dichotomized by median cutoffs.

Supplementary Figure S10 shows BCR-free and Metastasis-free survival for top differentially expressed genes in AA tumors dichotomized by median cutoffs.

Supplementary Table 1. Table of primer sequences and PCR conditions used

Supplementary Figure S9 shows BCR-free and Metastasis-free survival for Total TILs scores dichotomized by median as well as Youden index cutoffs.

Supplementary Tables S1-S4 from Higher Expression of the Androgen-Regulated Gene PSA/HK3 mRNA in Prostate Cancer Tissues Predicts Biochemical Recurrence-Free Survival

Differentially expressed immune-related genes in AA and CA tumors and pathway overrepresentation analysis. A, Volcano plot showing cutoffs for top differentially expressed genes in 26 AA tumors compared with 25 CA tumors as baseline. Red symbol genes in plot represent differentially expressed genes, with unadjusted P values < 0.05 and log2FC values either above 1 or below −1. FDR correction was applied, and only genes at or below Benjamini–Yekutieli adjusted P value of 1 are listed. Genes listed in blue text are significant following FDR correction at adjusted P value < 0.05. B, Dot plot highlighting top 10 relevant pathways detected by over representation analysis of 120 genes with differential expression P value < 0.05 using the MSigDb-C2 database.