Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Jan 6;31(1):151-163.
doi: 10.1158/1078-0432.CCR-24-1658.

Genomic and Epigenomic Analysis of Plasma Cell-Free DNA Identifies Stemness Features Associated with Worse Survival in Lethal Prostate Cancer

Pradeep S Chauhan  1 , Irfan Alahi  1   2 , Savar Sinha  3 , Elisa M Ledet  4 , Ryan Mueller  3 , Jessica Linford  1 , Alexander L Shiang  5 , Jace Webster  6 , Lilli Greiner  1 , Breanna Yang  3 , Gabris Ni  3 , Ha X Dang  6   7 , Debanjan Saha  6 , Ramandeep K Babbra  8 , Wenjia Feng  3 , Peter K Harris  3 , Faridi Qaium  1 , Dzifa Y Duose  9 , Sanchez E Alexander  9 , Alexander D Sherry  9 , Ellen B Jaeger  4 , Patrick J Miller  4 , Sydney A Caputo  4 , Jacob J Orme  10   11 , Fabrice Lucien  11   12   13 , Sean S Park  1   11 , Chad Tang  9 , Russell K Pachynski  6   14 , Oliver Sartor  10   13   15 , Christopher A Maher  6   7   14   16 , Aadel A Chaudhuri  1   2   11   12
Affiliations

Genomic and Epigenomic Analysis of Plasma Cell-Free DNA Identifies Stemness Features Associated with Worse Survival in Lethal Prostate Cancer

Pradeep S Chauhan et al. Clin Cancer Res. .

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 < 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.

PubMed Disclaimer

Conflict of interest statement

Declaration of Interests

P.S.C., I.A., and A.A.C. have patent filings related to cancer biomarkers. F.Q. has stock options in Centene, Gilead and Horizon Therapeutics, and has consulted for LiquidCell Dx. J.J.O. has served as a consultant/advisor to Nanotics and Partner Therapeutics, and has research support from Genentech. C.T. has grant support from Cancer Prevention & Research Institute of Texas (CPRIT), Department of Defense (DoD), NCCN, and received royalties from Wolters Kluwer and consulting fees and honoraria from Siemen Healthineer, Lantheus, Telix, Molli Surgical, and Boston Scientific. R.P. has licensed technology to Tempus Labs and has served as advisory to AstraZeneca, Bayer, BMS, Blue Earth Diagnostics, Dendreon, Genentech/Roche, Genomic Health, EMD Serono, Janssen, Merck, Pfizer, Sanofi-Aventis, Tempus, Tolmar Therapeutics, and has research funding from BMS, Exelixis, Janssen, Genentech/Roche and Pharmacyclics. O.S. has received institutional research funding from Advanced Accelerator Applications, Amgen, AstraZeneca, Bayer, In Vitae, Janssen, Lantheus, Merck, Novartis, Sanofi, and Point Biopharma. O.S. has received support for attending meetings/travels from AstraZeneca, Bayer, Lantheus and Sanofi. O.S. also serves on a data safety monitoring board or advisory board for Pfizer, Merck, Janssen, AAA, Novartis, and AstraZeneca. O.S. has stock or other ownership interests in AbbVie, Cardinal Health, Clarity Pharmaceuticals, Convergent, Eli Lilly, Abbott, Ratio, United Health Group, and Telix. O.S. has served as a consultant or advisor to Advanced Accelerator Applications, Amgen, ART BioScience, Astellas Pharma, AstraZeneca, Bayer, Clarity Pharmaceuticals, EMD Serono, Fusion Pharmaceuticals, Isotopen Technologien, Janssen, MacroGenics, Novartis, Pfizer, Point Biopharma, Ratio, Sanofi, Telix Pharmaceuticals, and TeneoBio. O.S. has received payment for expert testimony from Sanofi. O.S has patents for Saposin C and receptors as targets for treatment of benign and malignant disorders (U.S. patent awarded January 23, 2007; patent no. 7,166,691); has provided expert testimony for Sanofi. C.A.M. has licensed technology and served as consultant to Tempus. A.A.C. has patent filings related to cancer biomarkers, and has licensed technology to Droplet Biosciences, Tempus Labs and to Biocognitive Labs. A.A.C. has served as a consultant/advisor to Roche, Tempus, Geneoscopy, NuProbe, Illumina, Daiichi Sankyo, AstraZeneca, AlphaSights, DeciBio, Guidepoint, Invitae and Myriad Genetics. A.A.C. has received honoraria from Roche, Foundation Medicine, and Dava Oncology. A.A.C. has stock options in Geneoscopy, research support from Roche, Illumina and Tempus Labs, and ownership interests in Droplet Biosciences, CytoTrace Biosciences and LiquidCell Dx.

Figures

Fig 1.
Fig 1.. Study overview.
Blood samples were collected from 129 mCRPC patients from three independent institutions, including samples collected prior to the initiation of AR-targeted therapy and during treatment. EnhanceAR-Seq applied to plasma cell-free DNA was used for the detection of genomic alterations and risk stratification of mCRPC patients. Genome-wide EM-seq for 43 patients was done using plasma collected prior to the initiation of first-line AR-targeted therapy. Nucleosome profiling of binding sites for 377 transcription factors in plasma cfDNA was done using Griffin to identify pathways enriched in AR-altered lethal mCRPC patients. scRNA-seq data from 14 separate mCRPC patients was analyzed to identify stemness markers using CytoTRACE, which were used to define a stemness signature that was validated in both plasma EM-seq and tumor tissue bulk RNA-seq data. AR, androgen receptor gene; EM-seq, enzymatic methylation sequencing; EnhanceAR-Seq, Enhancer and neighboring loci of Androgen Receptor Sequencing; mCRPC, metastatic castration-resistant prostate cancer; RNA-seq, RNA sequencing; scRNA-seq, single-cell RNA sequencing. Figure created with BioRender.com.
Fig. 2.
Fig. 2.. Genomic characterization of mCRPC plasma cell-free DNA.
(A) Genomic alterations detected in plasma cfDNA including the androgen receptor (AR) and enhancer region upstream of AR in pre-ARSI and on-ARSI plasma collected from mCRPC patients. Progression-free and overall survival Kaplan-Meier analysis according to AR/enhancer alteration status in plasma collected (B-C) before starting first-line ARSIs and (D-E) during first-line ARSIs. p values were calculated by the log-rank test and hazard ratios (HRs) by the Mantel-Haenszel method. ARSI, androgen-receptor signaling inhibitor; cfDNA, cell-free DNA; mCRPC, metastatic castration-resistant prostate cancer.
Fig. 3.
Fig. 3.. Plasma cell-free DNA methylome of AR/enhancer altered lethal mCRPC.
(A) Distribution of hypomethylated DMRs in genic regions of pre-treatment plasma cfDNA of AR/enhancer altered lethal mCRPC patients (n = 18) compared to AR/enhancer wild-type patients (n = 25). (B) Promoter-level methylation rate of DMRs in cfDNA from AR/enhancer altered lethal (n = 18) versus AR/enhancer wild-type (n = 25) mCRPC patients. (C) Top 50 hypomethylated DMRs in genic regions of pre-treatment plasma cfDNA from AR/enhancer altered lethal versus wild-type mCRPC patients. Methylation levels are shown as a heatmap with scale bar shown on the right. Student’s t test was used to calculate the p value in the violin plot in B. ARSI, androgen-receptor signaling inhibitor; cfDNA, cell-free DNA; DMR, differentially methylated region; mCRPC, metastatic castration-resistant prostate cancer.
Fig. 4.
Fig. 4.. Nucleosome profiling of plasma cell-free DNA in AR/enhancer altered lethal mCRPC patients.
Nucleosome profiling of 10,000 TFBSs associated with 377 transcription factors from GTRD (Methods) to infer transcription factor activity from plasma cfDNA. Central coverage profiles for transcription factors (A,B) HOXB13 and (C,D) FOXO1 in AR/enhancer altered lethal versus AR/enhancer wild-type mCRPC patients. Data in B,D are represented as box and whisker plots, with p values calculated by Student’s t test. (E) Top 20 transcription factors with binding sites found to be most accessible in AR/enhancer altered lethal mCRPC patients versus AR/enhancer wild-type mCRPC, and (F) gene set enrichment analysis of these 20 transcription factors with –log10(q) and hit count in the query list shown. * represents p < 0.05 calculated by Student’s t test. AR, androgen receptor gene; cfDNA, cell-free DNA; GTRD, Gene Transcription Regulation Database; mCRPC, metastatic castration-resistant prostate cancer; TFBS, transcription factor binding site.
Fig. 5.
Fig. 5.. Prognostic stemness signatures associated with lethal mCRPC.
(A) Schema describing the stemness analysis workflow. (B) UMAP decomposition of scRNA-seq data from 14 mCRPC patients with the adenocarcinoma cluster comprised of 835 cells highlighted. (C) Top 10 differentially expressed genes in most stem-like cells and least stem-like cells after single-cell analysis of mCRPC adenocarcinoma cells by CytoTRACE (Methods). Comparison of promoter-level methylation of the (D) most and (E) least stem-like genes with mCRPC type after metagene analysis (Methods). Data are shown as box and whisker plots, with p values calculated by Student’s t test. (F-G) Stemness measured from plasma EM-seq (Methods) correlated with worse survival outcomes by Kaplan-Meier analysis. (H) Validation of the stemness signature in an external cohort of 80 mCRPC patients with tumor tissue profiled by bulk RNA-seq. For Kaplan-Meier analyses, p values were calculated by the log-rank test and hazard ratios by the Mantel-Haenszel method. EM-seq, enzymatic methylation sequencing; HR, hazard ratio; mCRPC, metastatic castration-resistant prostate cancer; RNA-seq, RNA sequencing; scRNA-seq, single-cell RNA sequencing; TFBS, transcription factor binding site; UMAP, Uniform Manifold Approximation and Projection.
See this image and copyright information in PMC

Update of

Comment in

References

    1. Beer TM, Armstrong AJ, Rathkopf DE, Loriot Y, Sternberg CN, Higano CS, et al. Enzalutamide in metastatic prostate cancer before chemotherapy. N Engl J Med 2014;371(5):424–33 doi 10.1056/NEJMoa1405095. - DOI - PMC - PubMed
    1. Fizazi K, Scher HI, Molina A, Logothetis CJ, Chi KN, Jones RJ, et al. Abiraterone acetate for treatment of metastatic castration-resistant prostate cancer: final overall survival analysis of the COU-AA-301 randomised, double-blind, placebo-controlled phase 3 study. Lancet Oncol 2012;13(10):983–92 doi 10.1016/S1470-2045(12)70379-0. - DOI - PubMed
    1. Ryan CJ, Smith MR, Fizazi K, Saad F, Mulders PF, Sternberg CN, et al. Abiraterone acetate plus prednisone versus placebo plus prednisone in chemotherapy-naive men with metastatic castration-resistant prostate cancer (COU-AA-302): final overall survival analysis of a randomised, double-blind, placebo-controlled phase 3 study. Lancet Oncol 2015;16(2):152–60 doi 10.1016/S1470-2045(14)71205-7. - DOI - PubMed
    1. Scher HI, Fizazi K, Saad F, Taplin ME, Sternberg CN, Miller K, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med 2012;367(13):1187–97 doi 10.1056/NEJMoa1207506. - DOI - PubMed
    1. Antonarakis ES, Lu C, Wang H, Luber B, Nakazawa M, Roeser JC, et al. AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med 2014;371(11):1028–38 doi 10.1056/NEJMoa1315815. - DOI - PMC - PubMed

MeSH terms