Greiner, Lilli

5068 Background: 177 Lu-PSMA-617 (LuPSMA) is a groundbreaking therapy for mPC, but biomarkers are urgently needed to improve patient selection. Plasma-derived EVs are emerging blood-based markers of tumor burden. Here, we analyzed levels of EVs of 123 patients (pts) from 4 cohorts: a pre- and during LuPSMA treatment for a contemporary prospective Mayo Clinic cohort, pre-treatment plasma levels of EVs for a historical Tulane cohort, localized prostate cancer (LPC) after radical prostatectomy (RP), and male healthy donors (HD). Methods: Blood samples were collected from 51 pts pre-cycles (pre-C) 1-3 of LuPSMA at Mayo Clinic from 12/2024-12/2025 and 26 pts pre-C1 at Tulane Cancer Center between 12/2018-1/2025. Small and large EVs were incubated with fluorescently conjugated antibodies (PSMA, STEAP1, CEACAM5, and TROP2) and enumerated using nanoscale flow cytometry. EVs from blood samples of 10 HD and 36 LPC post-RP from 6/2020-8/2024 at Mayo Clinic as controls. AUC-ROC and logistic regression analyses were performed to identify marker signatures associated with PSA50 response. Results: Baseline demographics are shown in the table. Median levels of PSMA, STEAP1, CEACAM5, and TROP2 EV/milliliter (mL) were all significantly higher for pts with mPC (pooled Tulane and Mayo cohorts) compared to LPC post-RP (p<0.001) and HD (p<0.001). In the Mayo cohort, 10 of 51 pts (20%) had undetectable PSA pre-C1. There were no significant differences in median pre-C1 plasma EV levels for these 4 different types of EVs comparing pts with undetectable vs detectable PSA. For 41 Mayo pts with detectable PSA pre-C1, 20 achieved PSA50 response at time of data cutoff (49%), while 7 pts (17%) had persistent PSA rise on LuPSMA. For pts who achieved PSA50, there was a significant decline in both CEACAM5 and TROP2 EVs/mL pre-C1 compared to pre-C3 (19 x 10 6 vs 5.8 x 10 6 , p=0.003 and 16 x 10 6 vs 5.5 x 10 6 , p<0.001, respectively), while no significant changes occurred for the 7 pts with no PSA response. A composite score of pre-C1 CEACAM5 and TROP2 EVs (large- and small-sized EVs) demonstrated the strongest association with PSA50 response (OR=2.96, 95% CI: 1.27-6.44, p=0.038), achieving AUC of 0.73. PSMA and STEAP1 EVs did not show any significant changes from pre-C1 to pre-C3. Conclusions: Plasma-derived EVs are a promising biomarker to identify patients who will benefit from LuPSMA. Longer follow-up and enrolling a larger cohort are underway. Clinical trial information: NCT06200103 . Median values for pre-LuPSMA (Mayo and Tulane), post-RP for LPC, and HD. Mayo mPC (n=51) Tulane mPC (n=26) LPC (n=36) HD (n=10) Age (range) 73 (57-87) 71 (54-83) 66 (56-77) 55 (50-57) Prior lines (range) 1 (1-3) 4 (1-11) - - Gleason score 9 8 7 - PSA (range) 1.0 (0-182) 75 (0.8-503) - - PSMA EV/mL 3.7 x 10 6 14 x 10 6 2.0 x 10 6 1.4 x 10 6 STEAP1 EV/mL 30 x 10 6 45 x 10 6 12 x 10 6 12 x 10 6 CEACAM5 EV/mL 17 x 10 6 5.1 x 10 6 3.7 x 10 6 3.9 x 10 6 TROP2 EV/mL 13 x 10 6 5.2 x 10 6 4.5 x 10 6 3.6 x 10 6

Abstract Introduction Sepsis, a dysregulated immune response to infection, accounts for nearly 11 million deaths worldwide each year and remains a major cause of hospital mortality. The absence of precision diagnostics limits efforts to characterize immune dynamics and predict clinical outcomes. We evaluated whether liquid biopsies collected at ICU admission could provide insight into sepsis biology and enable precision diagnostics. Methods We applied cell-free RNA (cfRNA) liquid biopsies as a noninvasive method to highlight signaling networks, identify tissue-specific injury, and predict immune exhaustion. We collected serial samples throughout hospitalization from a cohort of 100 patients admitted with likely sepsis to a medical ICU. Comprehensive clinical data, including inpatient laboratory values and key outcome measures such as survival and ventilator-free days, were curated. Circulating cfRNA was isolated and purified from these samples and prepared for sequencing. We generated data from 254 samples at a minimum sequencing depth of 30x. We then designed a novel deep-learning computational method to enable deconvolution to better analyze and interpret these data. Results In our cohort, a total of 84 patients were adjudicated to have sepsis by the Sepsis-3 criteria, and 25 patients died within 90 days. Differential analysis was performed comparing circulating cfRNA in these septic patients to that of healthy controls, stratified by mortality along with specified subgroups including shock on admission, ventilator dependence, and preexisting immunosuppression. We identified multiple differentially expressed transcripts between septic patients and healthy controls, with significant alterations in signaling cascades associated with mitochondrial signaling, including alterations in oxidative phosphorylation and fatty acid metabolism. Using defined ligand-receptor interaction databases, we identified key pathways associated with immune and endothelial dysfunction in sepsis. Specifically, our analysis identified numerous alterations in CXCL8 (IL-8)-associated signaling cascades (engaging CXCR1, CXCR2, SDC1-3, and ACKR). Our analyses also highlighted key tissue factor pathway inhibitor (TFPI)-associated signaling via interactions with tissue factor and low-density lipoprotein receptor-related protein 1 (LRP1). These analyses are corroborated by our new deep-learning augmented approach to deconvolution, where we demonstrate the identification of rare tissue- and cell-specific signals that are associated with increased mortality in our cohort. Conclusion Liquid biopsy approaches such as cfRNA profiling can provide insight into mechanisms underlying sepsis and may uncover underlying drivers of endothelial dysfunction. These findings support ongoing efforts to develop liquid biopsy-based diagnostics and personalized therapeutic strategies for patients at high risk for sepsis and related mortality. This abstract is funded by: NIH

Abstract Background: Pediatric and AYA sarcomas are rare, biologically heterogeneous tumors with limited noninvasive biomarkers to guide diagnosis, personalized treatment, and disease monitoring. Standard-of-care evaluation relies on invasive biopsies that carry procedural risks and imaging modalities with limited sensitivity for detecting minimal residual disease and distinguishing tumor types. Cell-free DNA (cfDNA) methylation profiling offers a minimally invasive alternative capable of capturing tumor-derived epigenomic alterations and has consistently shown strong clinical utility in adult malignancies. However, genome-wide cfDNA methylation patterns across diverse pediatric and AYA sarcoma subtypes remain poorly defined. Methods: Plasma was collected from 93 pediatric and AYA patients (pediatric &lt;18 years; AYA 15-39 years) representing 27 Ewing sarcoma, 17 rhabdomyosarcoma, 11 liposarcoma, 10 chordoma, 8 synovial sarcoma, 7 chondrosarcoma, 6 osteosarcoma, and 6 fibrosarcoma cases, plus 19 matched healthy donors. cfDNA was isolated from 1 mL plasma, quantified by Qubit, and assessed by Bioanalyzer. Libraries were prepared using the NEBNext EM-seq v2 workflow with PCR cycles scaled to cfDNA input amount. Genome-wide sequencing (15× target depth) was followed by alignment, deduplication, and methylation calling. MultiQC was used to assess depth, duplication rate, conversion efficiency, and CpG coverage. Global methylation summaries were computed, and methylKit was used to generate per-CpG and 1-kb tiling matrices and initiate differential methylation analyses. Results: Ewing sarcoma patients had significantly higher plasma cfDNA concentrations than age- and sex-matched healthy donors (24.4 vs 4.6 ng/mL, p &lt; 0.0001), whereas cfDNA levels for other sarcoma subtypes overlapped with the healthy cohort. Low or undetectable cfDNA concentrations by Qubit were far more common among non-Ewing sarcoma samples (23 of 66) than Ewing sarcoma (1 of 27). All samples achieved sufficient CpG coverage for genome-wide methylation modeling. Global methylation summaries revealed no significant differences in mean β-values across sarcoma subtypes or versus healthy donors. An independent analysis of the Ewing subset identified 299 significant differentially methylated regions (DMRs), supporting the detectability of disease-specific methylation changes. Conclusions: These results demonstrate the feasibility of high-quality cfDNA methylome profiling across a large and diverse pediatric and AYA sarcoma cohort, including in plasma samples with cfDNA concentrations below the limit of detection. While global methylation levels appear broadly similar across subtypes, proof-of-concept DMR analysis in Ewing sarcoma suggests that subtype-specific epigenomic signatures could enable future noninvasive diagnostic tools for pediatric and AYA sarcomas. Citation Format: Lilli J. Greiner, Roman O. Kowalchuk, Jeff Szymanski, Pradeep S. Chauhan, Adam Amundson, Peter J. Schoettler, Wendy Allen-Rhoades, Stephanie F. Polites, Peter S. Rose, Kelly Bailey, Linda McAllister-Lucas, Anita Mahajan, Nadia Laack, Aadel Chaudhuri. Defining genome-wide cfDNA methylation landscapes in pediatric and adolescent and young adult (AYA) bone and soft-tissue sarcomas [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 7885.

Abstract Background: Liquid biopsy approaches for cancer detection often rely on tissue-specific methylation or fragmentation features to infer tumor origin. However, a pan-aerodigestive cancer detection strategy is clinically valuable given shared risk exposures, field cancerization, and frequent diagnostic ambiguity among lung, esophageal, and head & neck cancers. We hypothesized that advanced-stage disease exhibits convergent chromatin remodeling, producing a dominant malignancy-associated fragmentomic signal that supersedes subtler tissue-of-origin patterns. Methods: Whole-genome EM-seq (∼15×) was performed on plasma cfDNA from 120 patients with stage III/IV aerodigestive cancers (lung, esophageal, head & neck; squamous and adenocarcinoma) and 60 age-matched controls. Four fragmentomic modalities were assessed: (1) genome-wide fragmentation in 5-Mb bins (DELFI-like), (2) NMF spectral components, (3) 4-mer end motifs, and (4) promoter-centric nucleosome scores (Griffin). All preprocessing occurred strictly within each training fold of a repeated stratified 10-fold CV (100 repeats) to prevent data leakage. No test-sample information was used in training. Hyperparameters were tuned only within training partitions. Random Forest classifiers were trained under this fully nested scheme. Results: The multimodal classifier robustly detected advanced aerodigestive malignancies with a mean AUROC 0.86 and AUPRC 0.94. However, modality dissection revealed a clear performance hierarchy: nucleosome positioning (Griffin) alone achieved AUROC 0.85 and AUPRC 0.93, nearly matching the full integrated model, whereas end motifs performed poorly (AUROC 0.61). Biological interpretation demonstrated prominent nucleosome depletion centered at promoters of proliferation-associated genes, indicating a convergent epigenetic state of promoter opening across late-stage aerodigestive malignancies. Conclusions: Advanced aerodigestive cancers exhibit a dominant, convergent chromatin remodeling signature detectable in plasma cfDNA, driven primarily by altered nucleosome positioning. In late-stage disease, this shared malignancy-associated fragmentomic signal outweighs tissue-of-origin-dependent patterns. These findings highlight the utility of nucleosome-based fragmentomics not merely for detection, but for resolving diagnostic ambiguity in patients with indeterminate masses and for monitoring total tumor burden in high-risk aerodigestive malignancies. Citation Format: Axel Misael Hidalgo, Ayesha Hashmi, Jeff Szymanski, Pradeep Chauhan, Lilli Greiner, Faridi Qaium, Daniel J. Ma, David M. Routman, Katie M. Van Abel, Aadel A. Chaudhuri.. Convergent chromatin remodeling enables pan-aerodigestive detection of advanced malignancies in plasma cfDNA [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 3862.

Abstract Introduction: Long intergenic noncoding RNAs (lincRNAs) and circular (circ) RNAs have been shown to play key roles in carcinogenesis and tumor progression. Urine represents a promising source of tumor-reflective signals shed directly from the urothelial microenvironment in bladder cancer (BC) patients. Here, we evaluate the urine cell-free (cf) RNA transcriptome in a cohort of 50 patients with localized BC. Methods: A total of 50 localized BC patients and 24 healthy adults were enrolled. Pre-operative urine (collected on the day of radical cystectomy) was processed to isolate cfRNA from urine supernatant. Libraries were prepared using an Illumina tagmentation-based RNA enrichment workflow and sequenced to 100M paired-end reads per sample on a NovaSeq X. FASTQ files were concatenated per sample and QC-checked with RNA-SeQC and Samtools. Reads were aligned to GRCh38/hg38 with STAR, and gene counts were generated using Feature Counts. Linear differential expression analysis (DEA) was performed with DESeq2. For discovery we used random halves of TCGA-BLCA and healthy urine cfRNA samples, alongside a second DEA with random halves of urine BC cfRNA vs. controls; overlapping upregulated genes were validated in the remaining samples. For circular (circ) RNAs, annotations from MiOncoCirc were used; junctions with ≥5 supporting reads were retained, and DEA was conducted with edgeR. Results: In this study, the median age was 68 years, and 80% (40/50) were male. Among the cohort, 66% (33/50) had muscle-invasive bladder cancer (MIBC), of whom 54% received neoadjuvant chemotherapy. Differential expression analysis of TCGA-BLCA tumors and BC urine cfRNA, identified 27 genes commonly upregulated, with 23 of the 27 shared genes strikingly being long intergenic noncoding RNAs (lincRNAs). These BC/urine-specific lincRNAs—including LINC00930, LINC01004, DLG5-AS1, APTR, PSORS1C3, and SERPINB9P1—have known roles in proliferation, EMT, metabolism, and immune signaling. Outside of lincRNAs, tumor-reflective coding transcripts in BC/urine were enriched for autophagy, driven by MAP1LC3B2.TCGA-BLCA survival analysis showed that LINC00930 and PSORS1C3 were associated with worse overall survival. Expanding beyond linear RNA sequences, analysis of urine circRNAs from BC patients revealed a strong cancer-specific dysregulation pattern, with 77 circRNAs significantly upregulated compared with healthy controls. Upregulated urine circRNAs arose from host genes involved in BC-relevant pathways, including invasion (DOCK1, ARHGAP5), autophagy/ER stress (VMP1, ERN1), and luminal differentiation (GRHL2, ESRP1). Conclusion: Cell-free RNA profiling reveals a distinct transcriptomic signature including differential lincRNA- and circRNA-driven autophagy, EMT, proliferation, metabolic, differentiation, and immune signaling programs in the urine of bladder cancer patients. Citation Format: Pradeep Chauhan, Li Lin, Irfan Alahi, Jessica Linford, Lilli J. Greiner, Ayesha Hashmi, Anushka Viswanathan, Nathan Colon, Faridi Qaium, Peter K. Harris, John Sheng, Jacob J. Orme, Eric H. Kim, Zachary L. Smith, Woodson Smelser, Christopher A. Maher, Aadel A. Chaudhuri. Urine cell-free RNA captures tumor-reflective long intergenic noncoding and circular RNA programs in bladder cancer [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 1133.

Abstract Introduction: Detectable circulating tumor DNA (ctDNA) is associated with inferior outcomes in Ewing sarcoma (ES); however, ctDNA clears in nearly all patients within 2 of the 6 chemotherapy cycles before radiotherapy (RT) or surgery. Given a 3-year EFS of 37% on AEWS 1221 (metastatic) and 5-year EFS of 78% on AEWS 1031 (localized), current ctDNA testing fails to predict most relapses. Extracellular vesicles (EVs) are microscopic particles studied as a biomarker in multiple cancer types. We hope to improve the sensitivity of ctDNA in ES and provide the first application of EVs in ES. Methods: cfDNA was isolated from 1mL plasma, quantified by Qubit, and sequenced as NEBNext Enzymatic Methyl-seq v2 libraries. We performed a systematic unsupervised clustering analysis of 27 ES plasma cfDNA profiles using 4 differentially methylated regions (DMR) subsets (63 strong DMRs, top 30, top 15, and 10 promoter-proximal loci), 3 clustering algorithms (k-means, Ward hierarchical, PAM), and k=2-5 clusters, testing associations with tumor burden and progression. EV analysis involved antibody detection against ES surface markers, including LINGO1, ENPP1, CDH11, and CD99. Concentrations of EVs were determined by standardized and calibrated high-resolution flow cytometry then analyzed with custom-built software to automatically determine and apply gates. Samples were de-identified, randomly coded, and run in triplicates. The senior research technologist responsible for EV analysis was blinded to patient information and clinical data. Results: EV analysis included 36 pre-RT samples from 16 metastatic and 20 localized ES patients from a prospectively maintained biobank, with 27 pre-RT samples in the cfDNA analysis and 19 healthy donor (HD) samples. 35 of 36 patients had chemotherapy before sample collection. cfDNA concentrations were markedly elevated in ES patients compared with HD (24.4 vs 4.6 ng/mL, p&lt;0.0001), and 74% of ES samples exceeded the HD 95th-percentile detectability threshold (7.0 ng/mL). Across clustering configurations, ES cfDNA profiles consistently exhibited reproducible methylation substructure. One configuration (PAM clustering, k = 5, 63 strong DMRs) showed a modest but permutation-supported association with primary tumor size (nominal p ≈ 0.04; empirical p ≈ 0.03-0.04). ES samples had increased EV concentration compared to HD (p&lt;0.001), with AUC 0.988 for distinguishing between ES and HD ENPP1 (p&lt;0.0001). The highest ENPP1 EV values were in patients with large primary tumors (&gt;8 cm vs. ≤ 8cm) and metastatic disease (p=0.066). Conclusions: cfDNA- and EV-based liquid biopsy provide encouraging signals for high ES detectability pre-RT. We also show the first study of EVs as a novel biomarker in ES, which may provide complementary information to ctDNA. Multimodal liquid biopsy in ES may identify candidate patients for systemic or local therapy escalation and facilitate personalized care. Citation Format: Roman O. Kowalchuk, Lilli J. Greiner, Yohan Kim, Rubia Noori, Jeffrey Szymanski, William S. Harmsen, Pradeep S. Chauhan, Adam C. Amundson, Andrea M. McMahon, Peter J. Schoettler, Wendy A. Allen-Rhoades, Stephanie F. Polites, Peter S. Rose, Kelly M. Bailey, Linda M. McAllister-Lucas, Anita Mahajan, Fabrice Lucien-Matteoni, Nadia N. Laack, Aadel A. Chaudhuri. Multimodal liquid biopsy of Ewing sarcoma reveals high sensitivity prior to radiotherapy [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 1149.

214 Background: 177 Lu-PSMA-617 (LuPSMA) has revolutionized care for patients (pts) with metastatic castrate-resistant prostate cancer (mCRPC), but only half achieves PSA decline ≥50%. Biomarkers are urgently needed to improve pt selection. EVs and ctDNA released by cancer cells into the blood are emerging biomarkers to estimate overall tumor burden and capture tumor heterogeneity in mCRPC. Here, we analyzed pre-treatment plasma levels of tumor EVs and ctDNA in a pilot cohort of 35 pts with mCRPC treated with LuPSMA. Methods: Blood samples were collected pre-cycle 1 of LuPSMA at Tulane Cancer Center between 12/2018-1/2025. Plasma-derived small and large EVs were incubated with fluorescently conjugated antibodies and enumerated using nanoscale flow cytometry. ctDNA fraction was estimated from plasma cfDNA methylation WGS (∼10×) with ichorCNA. We also analyzed EVs from blood samples of male healthy donors (HD) and pts with localized prostate cancer (LPC) collected 1 day to 8 months after radical prostatectomy (post-RP) from 6/2020-8/2024 at Mayo Clinic as controls. Results: Median age at collection for 10 HD was 55 yrs and for 36 pts with LPC post-RP was 65 yrs. Median PSMA+ EV level was 2.0 x 10 6 /mL for post-RP and 1.4 x 10 6 /mL for HD. For 35 pts with mCRPC, 25 pts had complete data for analysis. Median PSMA+ EV level was 1.4 x 10 7 /mL, significantly higher than post-RP (p<0.001) and HD (p<0.001). At LuPSMA start, median age was 69 yrs (range 54-83), PSA was 82 ng/mL (range 0.8-503), median time from CRPC diagnosis to start was 50 months (range 6.9-285), median time from plasma collection to treatment initiation was 0.5 months (range 0-7.3). Median number of prior lines of life-extending therapy was 4 (range 1-11), including 100% androgen receptor pathway inhibitor and 75% docetaxel. Pts received median 4 cycles of LuPSMA. Median ctDNA fraction was 14.0% (range 0-51.1). Median time to reach nadir PSA for pts with any decline was 2.6 months. 15 pts (60%) achieved PSA40 (defined ≥40% decline in PSA from start to nadir) (range -99% to -42%) compared to 10 pts (40%) who did not (range -16% to rise), which predicted better median overall survival (OS) after LuPSMA start (40.7 v 6.0 months; HR 0.23, 95% CI, 0.07-0.71; p=0.0001). Pts were classified as LuPSMA responders using PSA40. Responders had a lower ratio of large to small PSMA+ EVs (0.30 v 0.53, p=0.0042) with an area under the receiver operating characteristic curve (AUC) of 0.83 (95% CI, 0.68-0.99), and a lower median ctDNA fraction (6.1% v 26.2%, p=0.0044) with an AUC of 0.83 (95% CI, 0.68-1.00). Incorporating both PSMA+ EV and ctDNA fraction together predicted PSA40 response with an AUC of 0.93 (95% CI, 0.84-1.00, p=0.0003). Conclusions: Using pre-LuPSMA plasma samples from a heavily pre-treated historical cohort, we integrated non-invasive liquid biopsy methods to successfully predict response to LuPSMA using PSA decline. Prospective validation is underway.

Abstract Purpose: Metastatic castration-resistant prostate cancer (mCRPC) resistant to androgen receptor signaling inhibitors (ARSI) is often lethal. Liquid biopsy biomarkers for this deadly form of disease remain under investigation, and underpinning mechanisms remain ill-understood. Experimental Design: We applied targeted cell-free DNA (cfDNA) sequencing to 126 patients with mCRPC from three academic cancer centers and separately performed genome-wide cfDNA methylation sequencing on 43 plasma samples collected prior to the initiation of first-line ARSI treatment. To analyze the genome-wide sequencing data, we performed nucleosome positioning and differential methylated region analysis. We additionally analyzed single-cell and bulk RNA sequencing data from 14 and 80 patients with mCRPC, respectively, to develop and validate a stem-like signature, which we inferred from cfDNA. Results: Targeted cfDNA sequencing detected AR/enhancer alterations prior to first-line ARSIs that correlated with significantly worse progression-free survival (P = 0.01; HR = 2.12) and overall survival (P = 0.02; HR = 2.48). Plasma methylome analysis revealed that AR/enhancer lethal mCRPC patients have significantly higher promoter-level hypomethylation than AR/enhancer wild-type mCRPC patients (P &lt; 0.0001). Moreover, gene ontology and CytoTRACE analysis of nucleosomally more accessible transcription factors in cfDNA revealed enrichment for stemness-associated transcription factors in patients with lethal mCRPC. The resulting stemness signature was then validated in a completely held-out cohort of 80 patients with mCRPC profiled by tumor RNA sequencing. Conclusions: We analyzed a total of 220 patients with mCRPC, validated the importance of cell-free AR/enhancer alterations as a prognostic biomarker in lethal mCRPC, and showed that the underlying mechanism for lethality involves reprogramming developmental states toward increased stemness. See related commentary by Nawfal et al., p. 7

Abstract Background: Metastatic castration-resistant prostate cancer (mCRPC) resistant to androgen receptor (AR)-targeted agents is often lethal. Unfortunately, biomarkers for this deadly disease remain under investigation, and underpinning mechanisms are ill-understood. Here, we applied epigenomic approaches to identify cell-free (cf) DNA features associated with these lethal AR-resistant mCRPC patients. Methods: Plasma from 99 mCRPC patients was collected from two independent institutions, either prior to first-line AR-targeted therapy (n = 63) or during treatment (n = 36). We applied EnhanceAR-Seq to detect alterations in the AR locus including the upstream enhancer. We then applied genome-wide cfDNA epigenomics to 43 pre-treatment plasma samples and performed both fragmentomics and methylation sequencing to delineate the biology of AR/enhancer-altered lethal mCRPC. Finally, we identified stemness genes by applying CytoTRACE to external single-cell RNA-seq data from mCRPC, and applied these to cfDNA via promoter-level methylation analysis. We then assessed these stem-associated genes in tumor bulk RNA-seq data from an independent mCRPC cohort. Results: AR/enhancer alterations were detected in 44% of pre-treatment cfDNA samples and correlated with significantly worse progression-free survival (PFS) (HR = 2.12, P = 0.01) and overall survival (OS) (HR = 2.48; P = 0.02). AR/enhancer alterations were detected in 19% of on-treatment cfDNA samples and were also associated with profoundly worse PFS (HR = 15.38, P = 0.0003) and OS (HR = 15.53, P = 0.002). Further, genome-wide cfDNA fragmentomic analysis revealed that binding sites for developmentally relevant transcription factors such as FOXA1, NKX-3 and HOXB13 were significantly more accessible in AR/enhancer-altered lethal mCRPC patients. Gene set enrichment analysis of the 20 most accessible transcription factors revealed several pathways associated with stem cell development. Interestingly, the 10 most stem-associated genes obtained using CytoTRACE applied to an external cohort of single-cell RNA-seq data from mCRPC (He et al. 2021) were promoter-hypomethylated in the plasma cfDNA of AR/enhancer-altered lethal mCRPC patients (P = 0.005). Further, metagene analysis of these stem-associated genes revealed worse PFS by Kaplan-Meier analysis (P = 0.04), which was confirmed by multivariate cox proportional hazards modeling. Our stemness metagene further stratified overall survival (P = 0.03) when applied to an external cohort of 80 mCRPC patients from Abida et al. 2019. Conclusions: Alterations in AR including the upstream enhancer are remarkably prognostic in mCRPC with pre-treatment predictive potential. The mechanism can be inferred via cfDNA epigenomic analysis and appears to involve increased tumor cell stemness in AR-altered lethal mCRPC. Citation Format: Pradeep Singh Chauhan, Irfan Alahi, Savar Sinha, Ryan Mueller, Alexander L. Shiang, Jace Webster, Ha X. Dang, Debanjan Saha, Lilli Greiner, Breanna Yang, Elisa M. Ledet, Ramandeep K. Babbra, Wenjia Feng, Peter K. Harris, Faridi Qaium, Ellen B. Jaeger, Patrick J. Miller, Sydney A. Caputo, Oliver A. Sartor, Russell K. Pachynski, Christopher A. Maher, Aadel A. Chaudhuri. Epigenomic analysis of plasma cell-free DNA identifies stemness features associated with worse prognosis in AR-altered lethal metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 983.