Abstract 4631: Deciphering immunotherapy response variability and immune-related toxicities through longitudinal multimodal ctDNA analysis in advanced melanoma

Abstract Background: Immune checkpoint inhibitors (ICIs) have revolutionized treatment of advanced cutaneous melanoma by leveraging the host immune system to target and eliminate tumour cells. However, predictive biomarkers for clinical response to ICIs remain elusive, limiting the ability to stratify patients effectively. The heterogeneity in treatment outcomes, coupled with the limited sensitivity and timeliness of radiographic assessments, particularly in patients with stable disease, underscores the urgent need for molecularly guided, minimally invasive approaches for real-time monitoring of therapeutic efficacy. Biomarkers, such as circulating tumour DNA (ctDNA) and T-cell clonal dynamics, offer promise not only in assessing tumour burden and dynamics but also potentially in providing early indicators of immune-related adverse events (irAEs). Methods: We analysed 70 longitudinal plasma samples from 13 patients with resectable or unresectable, Stage III melanoma from the MELResist study. These patients were treated with either pembrolizumab or a combination of ipilimumab and nivolumab. PD1/PDL1 expression in FFPE tumour samples was quantified using digital pathology, and deep whole-genome sequencing (around 30x coverage) was performed on tumour tissue to profile genomic alterations. By integrating ctDNA analysis (>10x WGS coverage) with matched tumour samples using the cfDNAPro, ichorCNA and INVAR2 pipelines, we conducted serial WGS-based monitoring to evaluate molecular response dynamics as measured by copy number alterations, fragmentomics and patient-specific tumour mutations respectively. Additionally, irAEs were assessed through longitudinal tracking of the peripheral T-cell repertoire (TCR) using TCR-sequencing of buffy coat samples, alongside with plasma protein expression profiling via the Olink Explore panel. Results: In evaluating ctDNA molecular response, we found a copy number fraction ranging from 0 to 42% by ichorCNA analysis, with higher fractions at later timepoints correlating with poorer overall survival. Fragmentomic analysis revealed changes in 4-mer end motif frequencies and the ratio of short-to-long fragments (100-150 bp vs. 151-220 bp). Specifically, a decreased CCCA motif frequency and an increased short-to-long fragment ratio at later timepoints were associated with poorer overall survival. Conclusion: ctDNA dynamics during immunotherapy for advanced melanoma reflect clinical outcomes. Changes in ctDNA can be identified using tumour informed methods with high sensitivity but at additional cost or tumour agnostic methods with lower logistical burden. Together with our assessment of T-cell clonal dynamics and proteomic changes, these approaches will offer valuable insights for monitoring therapeutic benefits and immune-related adverse events (irAEs) during immunotherapy. Citation Format: Zhao Cheng, Sarah McDonald, Hannah Thomas, Ze Zhou, Paulius Mennea, Haichao wang, Dmitry Shcherbo, Christina Zhang, Grainne McAndrew, Fazlur Talukdar, Wendy N. Cooper, Hui Zhao, Nitzan Rosenfeld, Philippa G. Corrie, Amit Roshan. Deciphering immunotherapy response variability and immune-related toxicities through longitudinal multimodal ctDNA analysis in advanced melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4631.