Digital PCR-based plasma DNA methylationassayfor noninvasive molecular subtyping of small cell lung cancer: A prospective feasibility study

Background: Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with limited treatment options and poor survival outcomes. Although recent studies highlight the clinical relevance of molecular subtypes for therapeutic stratification, tissue-based subtyping is often infeasible because most patients receive chemotherapy rather than surgical resection. This study aimed to develop and clinically evaluate a digital PCR assay for subtype-specific methylation profiling using plasma samples. Methods: Twenty patients with pathologically confirmed SCLC were prospectively enrolled at Seoul National University Boramae Medical Center from April 2023 to September 2025. Plasma samples were collected prior to systemic therapy, and molecular subtypes were classified as NEUROD1-type (N-type; n = 1), ASCL1-type (A-type; n = 16), POU2F3-type (P-type; n = 3), or SCLC-I type (I-type; n = 0). Plasma-derived molecular subtypes determined by the digital PCR-based methylation assay were compared with these clinical classifications. Results: Subtype-associated methylation markers were identified through integrative analysis of publicly available methylation array and expression datasets from the National Cancer Institute Small Cell Lung Cancer Screening Project. These candidate loci were integrated into a 4-plex digital PCR assay, which demonstrated clear subtype-enriched methylation patterns across representative SCLC cell lines. In 20 prospectively collected plasma samples, all targets were successfully quantified with sufficient clinical performance. Plasma-derived molecular subtypes showed overall concordance of 85.0% (17/20) with clinical classifications, with detection sensitivities of 100.0% (1/1) for N-type, 87.5% (14/16) for A-type, and 66.7% (2/3) for P-type cases. These findings support the feasibility of plasma-based methylation profiling for noninvasive SCLC molecular subtyping. Conclusion: This study demonstrates the feasibility of using a digital PCR-based methylation assay to classify SCLC molecular subtypes from plasma. Larger validation cohorts are warranted to refine cutoffs and confirm clinical utility.