Abstract P1-07-01: Real time detection of ESR1 mutation in blood by droplet digital PCR in the PADA-1 trial: Feasibility and cross-validation with NGS

Abstract
Background: Mutations of ESR1 (ESR1mut), the gene encoding for Estrogen Receptor (ER) alpha, have been functionally characterized as driving resistance to aromatase inhibitors (AI) given to in ER+ HER2- metastatic breast cancer (mBC). In the randomized multicenter phase 3 PADA-1 trial (NCT03079011), ER+ HER2- mBC patients treated with first line AI+Palbociclib were screened every two months for ESR1mut in blood (bESR1mut), by subjecting cell-free circulating DNA (cfcDNA) to a droplet digital PCR (ddPCR) assay. Upon the onset of a rising (i.e., increasing or appearing) bESR1mut, patients with no concomitant disease progression were randomized between keeping the same regimen (AI-Palbociclib) or switching to Fulvestrant-Palbociclib. We report herein the sample flow of this first-in-its-class trial, from April 2017 to March 2021, and the cross-validation of ddPCR results with NGS. Methods: At each time point, 20 mL of blood were drawn into BCT Streck® tubes. cfcDNA was extracted from 4 mL of plasma. bESR1mut testing was centralized in 2 platforms using the same ddPCR assay, which combines a drop-off ddPCR, targeting the clustered hotspot L536, Y537 and D538 mutations found in exon 8, with an unconventional ddPCR interrogating specifically the E380Q mutation, located in exon 5 (Jeannot et al, Oncogene 2020). Results were expressed as the number of ESR1mut copies detected per mL of plasma together with the Mutant Allele Frequency (MAF). As a control, we submitted 200 ESR1mut+ (by ddPCR) left-over samples to an amplicon-based Next Generation Sequencing (NGS) assay covering all ESR1 exons - with a nominal sensitivity of >0.5% MAF. Results: From 03/2017 to 03/2021, 1,017 MBC patients have been included in 83 centers and 12,552 blood samples have been collected. The median time of delivery to central platforms was 1 day (range: [0-11]). A median volume of 9.5 mL [2-20] of plasma was obtained after 2 centrifugations. bESR1mut results were notified to investigators with a median turnaround time of 13 days [1-813] (32 days in 2017, 9 days in 2019, 8 days in 2021). Among the 12,525 available ddPCR results: N=77 (<1%) were considered as non-contributive (failed); N=11,533 (92%) samples were bESR1mut-; 267 (2%) samples allowed to find a rising bESR1mut (first occurrence of bESR1mut in a given patient under AI-Pal, triggering randomization in the absence of concomitant progression), while 648 (6%) other bESR1mut+ samples were not labeled as rising (e.g. repeated samplings in randomized patients, etc.). Samples with rising bESR1mut had a median MAF of 0.83% [0.11-35] and a median number of 14.5 ESR1mut copies/mL [4-1225]. The 200 bESR1mut+ samples by ddPCR submitted to NGS had a median MAF of 1.39% [0.1-46.6] and a median number of 24 ESR1mut copies/mL [4-6,493]. 168 samples (84%) were confirmed by NGS as displaying an ESR1mut in exon 5 and/or 8, of which 50 samples harbored polyclonal mutations. MAFs retrieved by NGS and ddPCR showed an excellent intraclass correlation coefficient (ICC=0.93; 95%CI[0.85;0.97]). Lack of ESR1mut detection by NGS in 32 samples could be primarily attributed to low MAF (MAF<0.5% by ddPCR, N=14/32; MAF<1%, N=29/32) and/or low coverage (<1,000x) (N=11/32). Conclusion: PADA-1 demonstrated that large scale, real time ESR1mut screening is feasible. The ddPCR assay yielded to valid results in a short turnaround time and at a limited cost. The primary objective of PADA-1, which tested the actionability of bESR1mut, will be reported in another presentation. Funding: Pfizer, INCA PRT-K (Grant nb PRT-K19-110)
Citation Format: Céline Callens, François-Clément Bidard, Anais Curto-Taribo, Olfa Trabelsi-Grati, Samia Melaabi, Suzette Delaloge, Anne-Claire Hardy-Bessard, Thomas Bachelot, Florian Clatot, Thibault De La Motte Rouge, Jean-Luc Canon, Laurent Arnould, Fabrice André, Sandrine Marques, Marc-Henri Stern, Jean-Yves Pierga, Anne-Vincent Salomon, Emmanuelle Jeannot, Frederique Berger, Ivan Bieche, Anne Pradines. Real time detection of ESR1 mutation in blood by droplet digital PCR in the PADA-1 trial: Feasibility and cross-validation with NGS [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-07-01.