Exploring the Utility of Cell-Free DNA Hydroxymethylation Profiling in Small-Cell Lung Cancer

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma characterized by poor survival. Despite a high tumor mutation burden, biomarker discovery in SCLC remains challenging due to rapid tumor plasticity and limited tissue availability, highlighting the promise of liquid biopsy-based approaches. Epigenetic dysregulation of DNA 5-hydroxymethylcytosine (5hmC) has emerged as a cancer hallmark. However, its role in SCLC remains largely unexplored. Here, we characterized the cell-free DNA (cfDNA) 5hmC landscape in SCLC and evaluated its potential applications. We profiled the cell-free hydroxymethylomes of 107 pre-treatment SCLC patients and 53 matched controls using the 5hmC selective chemical labeling (5hmC-Seal) assay. SCLC displayed higher global 5hmC levels and distinct enrichment at neurodevelopmental and synaptic pathways, consistent with the neuroendocrine identity of SCLC. Concordance between plasma and matched circulating tumor cell patient-derived xenograft (CDX) demonstrated that cfDNA 5hmC reflects tumor epigenetic states and correlates with transcriptomic-derived molecular subtypes. Elevated SCLC-specific 5hmC levels and extensive stage (ES) disease were associated with inferior survival, with ES disease showing enrichment of pathways linked to cellular plasticity and neurodevelopment. Together, these findings indicate that cfDNA 5hmC profiling has potential as a biologically informative and clinically relevant biomarker in SCLC, with possible applications in tumor subtyping and risk stratification.