Cell-free DNA fragmentomes capture response to immuno-radiotherapy in metastatic non-small cell lung cancer

Background: Timely assessment of response to immune checkpoint inhibition (ICI) is critical but often limited by the heterogeneity of radiographic responses. Mutation-based analyses of cell-free DNA (cfDNA) circumvent these challenges, but are, in turn, prone to artifacts arising from clonal hematopoiesis. Plasma cfDNA fragmentome analyses using low-pass whole genome sequencing (WGS) may enable a scalable approach to evaluate systemic tumor burden in a tumor- and mutation-naïve manner. Methods: cfDNA fragmentome analyses were performed following low-pass WGS of 244 plasma samples from 62 patients treated with pembrolizumab +/- radiotherapy (NCT02492568). A locked instance of the DELFI-TF, a random forest regression model based on genome-wide fragmentation patterns and aneuploidy, was applied to determine cfDNA fragmentome-based estimates of tumor fraction. The 95 th percentile of tumor fraction in a non-cancer reference set established the limit of blank (LOB). Fragmentome-based landmark molecular response was defined as ctDNA below LOB at 6 weeks. Baseline tumor samples (n=24) patients were analyzed by RNA sequencing to characterize transcriptomic profiles stratified by cfDNA fragmentome profiles. Clinical outcomes were evaluated by RECIST 1.1 (at 6 and 12 weeks), progression-free survival (PFS), and overall survival (OS). Results: At baseline, DELFI-TF values were correlated with radiographic tumor burden (R=0.32, P=0.017). Notably, tumors from patients with high DELFI-TF showed an enrichment in gene sets related to cell cycle, DNA replication and repair (adjusted P<0.05), suggesting that fragmentome TF accurately captured cellular turnover. At 6 weeks, 73% (8 out of 11) of patients with radiographic response attained fragmentome molecular response, while the subset of patients with radiographically stable or progressive disease was more heterogeneous in their fragmentome molecular response (24 out of 50, 48%). Fragmentome molecular response was more concordant with best overall response (BOR) at 12 weeks (Fisher's exact P=0.018). Analysis of baseline tumors from patients achieving fragmentome molecular response revealed an inflamed tumor microenvironment (adjusted P-value <0.001). Among patients with stable or progressive disease at the first radiographic evaluation, fragmentome response predicted longer PFS (logrank P=0.0096) and OS (logrank P=0.012). Similarly, fragmentome molecular response predicted PFS (logrank P=7.4e-5) and OS (logrank P=0.00028) across the entire cohort. Conclusions: Plasma cfDNA fragmentome-derived tumor fraction reflects cellular turnover and lung cancer biology within the context of immunotherapy, while also enabling reliable, cost-effective, and scalable molecular response evaluations.