An Optimized Workflow to Evaluate Estrogen Receptor Gene Mutations in Small Amounts of Cell-Free DNA

The detection of mutated genes in cell-free DNA (cfDNA) in plasma has emerged as an important minimally invasive way to obtain detailed information regarding tumor biology. Reliable determination of circulating tumor–derived DNA, often present at a low quantity amidst an excess of normal DNA in plasma, would be of added value for screening and monitoring of cancer patients and for hypothesis-generating studies in valuable retrospective cohorts. Our aim was to establish a workflow to simultaneously assess four hotspot estrogen receptor mutations (mESR1) in cfDNA isolated from only 200 μL of plasma by means of uniplex or multiplex pre-amplification combined with digital PCR.