Background: Prostate cancer is a complex and heterogeneous disease with multiple tumor foci, each potentially harboring distinct driver molecular aberrations. This complexity poses challenges to effective management. We took an innovative approach to gain a comprehensive understanding of the genetic underpinnings of each tumor focus and avoid overlooking more minor yet clinically significant foci. Instead of relying solely on a systematic sampling of dominant foci, we conducted molecular analysis on whole-mount radical prostatectomy specimens. Our study aimed to find distinct molecular subsets of prostate cancer and assess their correlation with clinical outcomes, focusing on Caucasians (CA) and African Americans (AA). Method: We randomly selected 2201 whole-mount radical prostatectomy cases, with 1207 (54.8%) from CA and 994 (45.1%) from AA patients evaluated for a 5-year biochemical recurrence-free survival rate (BCR). Of these 2201 cases, 834 (463 -56% were from CA and 371 -44% from AA patients) were subjected to molecular analysis using dual immunohistochemistry (IHC) for ERG and SPINK1, along with dual RNA in-situ hybridization (RNA-ISH) for ETV1 and ETV4 to evaluate tumor molecular heterogeneity on whole-mount specimens. The Chi-squared test examined racial disparities in aberrant oncogene expression. To assess BCR-free survival, we employed the Kaplan-Meier method and Cox-PH models for patients with distinct molecular subsets of prostate cancer. Additionally, Gleason Grade groups of prostate biopsies were summarized using a spaghetti plot and compared using linear mixed models. Results: Analysis of the 2201 cases revealed that AA with localized prostate cancer behaved differently with better 5-year BCR-free survival than CA after radical prostatectomy (AA: 0.82, 95% CI 0.80-0.85; CA: 0.71, 95% CI 0.68-0.75; p<.001). Molecular profiling of whole-mount specimens from 834 cases revealed that 16.4%, 58.4%, 21.7%, and 3.5% of patients with localized prostate cancer expressed none, one, two, and three of the four oncogenes, respectively. This finding identified new molecular subsets of prostate cancer with more than one driver mutation in a mutually exclusive manner within the multifocal disease. ERG and SPINK1 expression showed a negative correlation (p<.001). Notably, AA patients exhibited a lower incidence of ERG (38.8% vs. 60.3%) but a higher incidence of SPINK1 (63.3% vs. 35.6%) than CA patients. The incidences of ETV1 (9.4% vs. 9.3%) and ETV4 (4.6% vs. 3.9%) were not statistically significant between the two racial groups. However, significantly, ETV1 expression was associated with worse BCR-free survival in CA patients (hazard ratio [HR]=2.36, 95% CI 1.22-4.57, p=.02), while ETV4 expression was linked to worse BCR-free survival in AA patients (HR=2.65, 95% CI 1.15-6.09, p=.02). Moreover, ETV4 expression was associated with regional lymph node metastasis in AA patients (odds ratio [OR]=5.14, 95% CI 1.3-17.4, p=.01) but not in CA patients (OR=0.60, 95% CI 0.03-3.17, p=.63) at the time of radical prostatectomy. Additionally, in patients who underwent multiple biopsies before radical prostatectomy, the Gleason Grade group increased over time in AA patients (0.25 per year, p<.001) but remained unchanged in CA patients. ERG expression was associated with a lower Gleason Grade group (-0.20, p=.03), while ETV4 expression was linked to a higher Gleason Grade (0.54, p=.01). Conclusions: Our study reveals that AA with localized prostate cancer behaves differently and has better BCR-free survival than CA after radical prostatectomy, even after adjusting for known prognostic factors. Identification of new molecular subsets of prostate cancer with more than one ETS gene fusion within a multifocal prostate shows significant molecular heterogeneity between localized prostate cancer in CA and AA patients. Importantly, given the association of ETV1 and ETV4 expression with worse BCR-free survival in CA and AA, respectively, ETV1 and ETV4 emerge as potential prognostic markers, offering insights for clinical practice to predict prostate cancer recurrence after radical prostatectomy. Identification of new molecular subsets of prostate cancer with more than one ETS gene fusion and SPINK1 in a mutually exclusive pattern indicates the clonal origin of independent tumor foci, which is a rare and unique phenomenon in prostate cancer hitherto unidentified.

The authors have declared no competing interest.

This work was supported by the grant received from the Department of Defense to Nallasivam Palanisamy - fund number is provided in the manuscript

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This study is approved by the Henry Ford Health System Institutional review Board (IRB) # 10375. here is the approval letter: EXPEDITED CONTINUING REVIEW APPROVAL LETTER To: Nallasivam Palanisamy, Ph.D. Urology From: Jonathan K Ehrman, Ph.D. Date: November 09, 2023 Board Name: Henry Ford Health IRB IRB No.: 10375 Title: Comprehensive Molecular Profiling of Prostate Cancer in African American Population: Unraveling Molecular Heterogeneity Approval Period: November 09, 2023-November 08, 2024 On November 09, 2023, a Committee Member from the Expedited IRB Committee reviewed the Continuing Review submission referenced above through expedited procedures.

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