Integrative analyses of the cfDNA fragmentome and TCR repertoires capture chemo-immunotherapy response in diffuse pleural mesothelioma

Background: Immunotherapy-containing therapies have shown promise for patients with unresectable diffuse pleural mesothelioma (DPM), yet there are no reliable strategies to monitor therapy response. By expanding the compendium of cancer-associated alterations profiled, including genome-wide fragmentation patterns, cell-free DNA (cfDNA) whole genome sequencing (WGS) enables tracking of tumor dynamics. In tandem, while understudied, T cell clone dynamics may be informative in capturing early immunotherapy response. Methods: Using 314 tumor and peripheral blood biospecimes, we analyzed serial plasma cfDNA samples (n=135) from 55 patients with unresectable DPM, who received durvalumab with platinum-based chemotherapy (NCT02899195). Following cfDNA extraction and genomic library preparation, cfDNA at baseline (C1D1), Cycle 2 Day 1 (C2D1), and Cycle 5 Day 1 (C5D1) underwent low-coverage (1-2x) whole genome sequencing. Tumor- and mutation-naive estimates of tumor fraction were derived by applying the DELFI tumor score (DELFI-TS) model, which integrates genome-wide fragmentation patterns and chromosomal arm aneuploidy. In parallel, we performed TCR Vbeta CDR3 next-generation sequencing on tumor (n=43) and peripheral serial blood (n=136) samples. The TCR repertoire was characterized using clonality, TCR clonotype/cluster dynamics, and the Morisita-Horn similarity index to assess repertoire similarity across samples. Clinical outcomes were assessed by radiographic response, progression-free (PFS), and overall survival (OS). Results: Patients with distant metastasis (M1) had higher DELFI-TS levels (p=0.038). At baseline, DELFI-TS levels were numerically higher for radiographic non-responders (SD/PD) vs responders (CR/PR). Using the 92 th percentile of DELFI-TS in non-cancer cfDNA control samples to determine the limit of blank, patients with detectable baseline DELFI-TS (ctDNA⁺) had shorter PFS and OS (log-rank p<0.001). Patients that attained a radiographic response had more clonal peripheral TCR repertoires at both baseline and on-therapy timepoints compared to non-responders (p=0.037 at C1D1; p=0.007 at C2D1; p=0.042 at C5D1). Morisita-Horn similarity between C1D1 and on-therapy was lower in non-responders than responders (p=0.019 for C1D1 vs C2D1; p=0.036 for C1D1 vs C5D1). In contrast, a more diverse intra-tumoral TCR repertoire was noted for patients with an OS of 12 or more months (p=0.018). Conclusions: Our findings provide proof-of-concept that cfDNA fragmentomic analyses can quantify pre-treatment cfDNA tumor fraction that may capture clinical outcomes with chemo-immunotherapy response for patients with DPM. Longitudinal analyses of peripheral TCR repertoires can further differentiate responding from non-responding DPM, supporting the notion that joint analyses may more accurately capture immunotherapy response.