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.

Automated summary

This study demonstrates the feasibility and accuracy of using droplet digital polymerase chain reaction (ddPCR) for screening ESR1 mutations in circulating tumor DNA of metastatic breast cancer patients. The results indicate that ddPCR has excellent performance and opens up new opportunities for therapeutic interventions.