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Updated: Jun 4, 2026
Cell-Free DNA Integrity Analysis in Urine Samples
Published on: January 5, 2017
Genomic and Epigenomic Analysis of Plasma Cell-Free DNA Identifies Stemness Features Associated with Worse Survival
Pradeep S Chauhan1, Irfan Alahi1,2, Savar Sinha3
1Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota.
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.
Insights
Biomarkers in cell-free DNA (cfDNA) can predict lethal metastatic castration-resistant prostate cancer (mCRPC). This study reveals that AR/enhancer alterations and stemness signatures in cfDNA are linked to poor survival in mCRPC patients.
Area of Science:
- Oncology
- Genomics
- Molecular Biology
Background:
- Metastatic castration-resistant prostate cancer (mCRPC) resistant to androgen receptor signaling inhibitors (ARSI) presents a significant clinical challenge.
- Effective biomarkers for predicting lethal mCRPC and understanding its underlying mechanisms are urgently needed.
Purpose of the Study:
- To investigate cell-free DNA (cfDNA) alterations as prognostic biomarkers in lethal mCRPC.
- To elucidate the molecular mechanisms associated with ARSI-resistant mCRPC lethality.
Main Methods:
- Targeted cfDNA sequencing was performed on 126 mCRPC patients.
- Genome-wide cfDNA methylation sequencing was conducted on 43 plasma samples.
- RNA sequencing data from tumor samples were analyzed to develop and validate a stemness signature.
Main Results:
- AR/enhancer alterations detected by cfDNA sequencing prior to first-line ARSI treatment correlated with significantly worse progression-free and overall survival.
- Plasma methylome analysis showed increased promoter-level hypomethylation in lethal mCRPC patients.
- A stemness signature inferred from cfDNA was validated in a separate cohort, indicating a link between stemness and mCRPC lethality.
Conclusions:
- Cell-free AR/enhancer alterations serve as a validated prognostic biomarker in lethal mCRPC.
- The lethality of mCRPC is associated with a reprogramming of developmental states towards increased stemness.
