Abstract 737: Androgen receptor-indifferent prostate cancer in radical prostatectomy cases detected by single-cell image analysis

Rational: A subset of castration-resistant prostate cancer (CRPC) displays an androgen receptor indifferent (ARi) phenotype. In these cases, the nuclear AR fails to promote the expression of transcripts in the AR cistrome, such as KLK3/PSA. Because of the aberrant functionality of AR, the ARi PC may not respond efficiently to hormonal therapies. As the ARi phenotype has only been described in CRPC, we hypothesized that it might exist in primary PC, potentially providing an opportunity for early detection of treatment resistance. Methods: 36 cases of pT3b prostate cancer with lymph node metastasis are displayed in 10 x 2mm cores per case on tissue microarrays. From each, we sampled 10 tumor regions including lymph node metastases, seminal vesicle and perineural invasion, extraprostatic extension, cribiform and non-cribifrom intraprostatic regions. TMA slides are stained with a multiplex panel of AR, PSA, Nkx3.1, PSMA, High molecular weight cytokeratin (HMW-CK, 34betaE12) and pan-cytokeratin (AE1/AE3) on the Leica Bond Rx and digitized using the Axioscan Z1. Manual annotations, cell and gland segmentation, and single cell protein expression measurements are conducted in QuPath. The clustering analysis is performed using CytoMap. Gene set enrichment analysis is used to determine the ARi phenotype in public RNA expression data sets. Significant differences are determined using T-statistics. Results: Using normal prostate glands as a reference, we identified the intensity of nuclear AR that is sufficient for PSA expression. We used this threshold to find cancer regions with less than 30% PSA-positive cells despite adequate AR expression levels in more than 50% PC cells, indicative of the ARi phenotype. Five of 36 cases contained regions with an ARi phenotype in at least one of the TMA cores. Out of all ARi regions, 23.9% are in lymph node metastasis and 21.7% in seminal vesicle PC. Within PC cases, percentages of AR positive cancer cells in lymph nodes don’t correlate with percentages at other PC locations, while percentages of AR-positive cells in seminal vesicle correlates with those in areas of extraprostatic extension or cribiform PC. Unsupervised k-means clustering using multiplex PC staining data reveals one of five clusters harboring most ARi TMA cores. The AR:PSA ratio is the most important variable for predicting the ARi phenotype, followed in importance by percentages of PSA, PSMA, Nkx3.1 and AR positive PC cells. Differential expression analysis of the TCGA-PRAD cohort cases confirms a subgroup of high AR and low KLK3/PSA cases with significant enrichment of the EMT hallmark gene expression signature, consistent with the ARi phenotype. Conclusion: Single cell analysis of AR and PSA identifies PC regions with ARi characteristics in primary PC and lymph node metastases, possibly predicting primary resistance to first line PC treatment. Citation Format: Beatrice S. Knudsen, Wei Zhang, Mason Hovinga, Erika Egal, Chance Walker, Yosep Chong, Galaxy Yang, Michael R. Freeman, Yi Qiao. Androgen receptor-indifferent prostate cancer in radical prostatectomy cases detected by single-cell image analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 737.