Comprehensive plasma-based epigenomic profiling from INTR@PID Lung 037 study samples to identify pathways associated with response and resistance to pembrolizumab in PD-L1–high NSCLC.

Abstract

e20547 Background: Pembrolizumab produces variable benefit in PD-L1-high metastatic NSCLC, and mechanisms of resistance remain insufficiently defined. Plasma-based epigenomic profiling could potentially identify pathway-level biology not captured by PD-L1 IHC or genomic mutations alone. An exploratory post hoc analysis was performed using comprehensive epigenomic profiling from 1 mL of plasma to characterize pathways associated with pembrolizumab response and resistance from INTR@PID Lung 037 study samples. Methods: 192 longitudinally collected plasma samples (baseline, on-treatment, and end-of-treatment [EOT]) were analyzed using Precede’s comprehensive epigenomic liquid biopsy platform. 99% of samples passed analyte-specific QC metrics and 55% samples had detectable ctDNA, consistent with previous reports. Differential promoter and enhancer activation and pathway enrichments were evaluated across PFS-stratified groups. Pathway activity dependence on ctDNA levels was used to investigate whether signals originated from tumor or stroma. Pathway activities were profiled across gain of function (GOF) and loss of function (LOF) alterations. Epigenomic activation of ADC targets was also assessed. Results: Baseline plasma from non-responders (lowest PFS tertile) showed enrichment of MYC and proliferative pathways, and an RNA-based intrinsic IFN-resistance signature, whereas responders showed reduced KRAS and EMT signalling. Despite patients being selected for PD-L1 expression ≥50%, higher epigenomic PD-L1 promoter activation trended with greater tumor shrinkage. Differential pathway activities for 86 mutations were identified, including 68 linked to immune-related programs (IFN, TNFα, JAK/STAT). NF1 and ARID1A LOF mutations exhibited immune-enriched profiles, whereas KEAP1 LOF and MDM2 GOF showed immune-depleted profiles, consistent with poor ICB response. Paired baseline-EOT samples showed acquired resistance characterized by squamous-like differentiation and keratinization pathways, consistent with lineage plasticity and immune exclusion. ADC target profiling demonstrated a subset of patients exhibiting co-activation of MUC1 and EGFR , suggesting potential suitability and combination potential for bispecific or targeted ADC strategies. Conclusions: Plasma epigenomic profiling resolved genes and pathways associated with pembrolizumab response, in PD-L1-high NSCLC, including intrinsic IFN-driven resistance and acquired squamous-like resistance. This approach also captured functional immune and tumor-associated biology not evident from PD-L1 IHC or mutation status. These findings provide a clear rationale for the use of plasma epigenomics for non-invasive monitoring, patient stratification, and future evaluation of combination and targeted strategies.