Development and Analytical Validation of a Two-Step Circulating Tumor DNA Workflow for Monitoring Camizestrant Response in ER+ Breast Cancer PDX Models

Abstract Circulating tumor DNA (ctDNA) enables minimally invasive tumor burden monitoring and is a mainstay of clinical response assessment, yet standardized workflows for patient-derived xenograft (PDX) models remain constrained by low murine plasma volumes. To bridge preclinical pharmacodynamic monitoring with clinical ctDNA readouts, we developed and validated a two-step workflow combining droplet digital PCR (ddPCR) quantification of human-specific LINE-1 retrotransposon elements with reflexed targeted next-generation sequencing (NGS). Applied to an ESR1 -mutant ER+ breast cancer PDX model (CTC174), LINE-1 levels correlated with tumor volume and growth rate. Treatment with the next-generation oral SERD camizestrant produced a > 97% reduction in plasma ctDNA versus vehicle. Samples exceeding the LINE-1 threshold were reflexed to targeted NGS, which detected ESR1 and PIK3CA mutations in control but not camizestrant-treated plasma. This workflow enables rational sample prioritization, concurrent variant surveillance, and exploratory profiling from minimal plasma, providing a preclinical analogue of clinical liquid biopsy monitoring.