Real-Time Detection of ESR1 Mutation in Blood by Droplet Digital PCR in the PADA-1 Trial: Feasibility and Cross-Validation with NGS.

The clinical actionability of circulating tumor DNA requires sensitive detection methods with a short turnaround time. In the PADA-1 phase 3 trial (NCT03079011), metastatic breast cancer patients treated with an aromatase inhibitor and palbociclib were screened every 2 months for activating ESR1 mutations in blood ( bESR1 mut ). We report the feasibility of the droplet digital polymerase chain reaction (ddPCR) and cross-validation with next-generation sequencing (NGS). bESR1 mut testing was centralized in two platforms using the same ddPCR assay. Results were reported as copies/mL of plasma and mutant allele frequency (MAF). We analyzed 200 positive ddPCR samples with an NGS assay (0.5-1% sensitivity). Overall, 12,552 blood samples were collected from 1017 patients from 83 centers. Among the 12,525 available samples with ddPCR results, 11,533 (92%) were bESR1 mut -negative. A total of 267 patients newly displayed bESR1 mut (26% patients/2% samples) with a median copy number of 14/mL (range: 4-1225) and a median MAF of 0.83% (0.11-35), 648 samples (20% patients/5% samples) displayed persistent bESR1 mut , and 77 (<1%) samples encountered a technical failure. The median turnaround time from blood drawing to result notification was 13 days (Q1:9; Q3:21 days). Among 200 ddPCR-positive samples tested, NGS detected bESR1 mut in 168 (84%); 25 of the 32 cases missed by NGS had low MAF and/or low coverage. In these 200 samples, bESR1 mut MAF by both techniques had an excellent intraclass correlation coefficient (ICC = 0.93; 95% CI [0.85; 0.97]). These results from a large-scale trial support the feasibility and accuracy of real-time bESR1 mut tracking by ddPCR, opening new opportunities for therapeutic interventions.