# Integrative dual ctDNA 5mC/5hmC methylomics and clonal reconstruction infer tumor transcription and resistance phenotypes in metastatic prostate cancer

## TL;DR

This study shows that analyzing DNA modifications in blood can reveal tumor characteristics and resistance patterns in prostate cancer patients.

## Contribution

The study introduces a method to infer gene-level transcription and resistance phenotypes from ctDNA 5mC/5hmC methylomics.

## Key findings

- Plasma transcriptomes distinguished adenocarcinoma from neuroendocrine prostate cancer phenotypes.
- A WNT5A-associated signature was linked to poor response to treatment.
- Two resistance trajectories were identified based on tumor heterogeneity and AR dependency.

## Abstract

Liquid biopsies can detect actionable mutations and infer broad tumor states from genome-wide cfDNA measurements, but quantitative transcriptome-like phenotyping at single gene resolution still largely requires tissue. Here, we asked whether 6-base whole-genome sequencing that jointly quantifies 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) could infer gene expression directly from plasma. We applied this framework to plasma from patients enrolled in a phase 2 clinical trial of the PARP inhibitor olaparib plus the PD-L1 inhibitor durvalumab for metastatic castration-resistant prostate cancer. Inferred plasma transcriptomes distinguished adenocarcinoma vs. neuroendocrine phenotypes and identified a noncanonical WNT5A-associated signature linked to poor clinical response. Integrating longitudinal cfDNA methylomic profiles with phylogenetic reconstruction further revealed two resistance trajectories: one featuring high tumor heterogeneity with persistent AR signaling, and another marked by an AR-independent, stem-like program with metabolic reprogramming. These findings demonstrate that ctDNA can inform phenotype-driven tumor biology at gene-level resolution, integrating epigenetic modifications, inferred transcriptional programs, and clonal dynamics as a function of treatment response.