Abstract P2-01-18: Orthogonal assessment of PIK3CA and ESR1 mutation detection in longitudinal cfDNA samples from endocrine-resistant HR+/HER2- advanced breast cancer patients using dPCR and NGS-based SafeSEQ technology
Authors: Antras Jesus Fuentes , García-Barberán Vanesa , Moreno Fernando , Sloane Hillary , López-Cade Igor , De Luna Alicia , Pascual Alejandro , Ramirez-Ruda Carmen , Lorca Victor , Flores Paloma , Perez-Segura Pedro , Ocaña Alberto , Preston Jennifer , Quinn Hannah , Jones Frederick , Garcia-Saenz Jose Angel 
Published: March 2022

Abstract
Background: Indentification of actionable or therapy resistance-associated mutations may guide sequential treatments in Hormone Receptor-positive and HER2-negative (HR+/HER2-) Advanced Breast Cancer (ABC) patients with resistance to prior endocrine therapies. Molecular profiling from cell-free DNA (cfDNA) is progressively permeating the clinical setting as an alternative or adjunct to tissue biopsies. Digital PCR (dPCR) assays permit the detection of genomic alterations with high sensitivity but are associated with cumbersome workflows and limited genomic coverage. NGS-based cfDNA assays are being increasingly adopted, as they can offer broader coverage while maintaining competitive sensitivity. Methods:Mutation testing was performed on plasma samples obtained from patients with HR+/HER2- ABC who had recurred or progressed after receiving an aromatase inhibitor. Samples were collected at the start of new treatment, during therapy, and at progression. dPCR tests were carried out using QuantStudio3D Digital PCR at the Molecular Oncology Laboratory at Clínico San Carlos Hospital. dPCR targets included PIK3CA E542K, E545K, and H1047R, and ESR1 Y537S and D538G. Samples[CdS1] were also tested with the SafeSEQ Breast Cancer Panel to assess clinically relevant genomic regions across PIK3CA, ESR1, AKT1, TP53, KRAS, and ERBB2. SafeSEQ testing was performed in Sysmex Inostics’ CLIA-certified, CAP-accredited laboratory. Results: Mutational data obtained from both dPCR and SafeSEQ testing were available for concordance analysis in 107 samples from 50 patients for PIK3CA, and 86 samples from 41 patients for ESR1. Combined results showed PIK3CA mutations in 47 samples (43.9%), with H1047R (14%), E545K (11.2%), and E542K (9.3%) being the most frequent mutations detected. ESR1 mutations were present in 35 samples (32.7%), where D538G (19.6%) and Y537S (9.3%) were most commonly observed. Among samples with no mutations detected by dPCR, the expanded coverage of SafeSEQ enabled the detection of clinically relevant mutations in PIK3CA and ESR1 in 10 (9.3%) and 3 (2.8%) samples, respectively. The concordance rates for dPCR and SafeSEQ were 93.1% and 88.9% for PIK3CA and ESR1, with positive percent agreements (PPA) of 93.8% and 84.6%, and negative percent agreements (NPA) of 92.8% and 90.9% for PIK3CA and ESR1, respectively. A strong correlation was observed between MAF levels (Spearman's ρ = 0.87 [95% CI 0.79-0.93], p&lt;0.001) as well as number of mutant molecules per mL of plasma (ρ = 0.79 [95% CI 0.66-0.88], p&lt;0.001). When samples with MAFs&lt;0.1% were evaluated, lower concordance rate was detected (38.5%; N=13; 46.2% detected by SafeSEQ and 92.3% by dPCR). Finally, no significant difference between assays was found when comparing concordance rates among samples at different timepoints. Regarding longitudinal ctDNA data, 18 and 16 patients showed mutations in PIK3CA and/or ESR1 in plasma at least in one timepoint. Monitoring results were concordant in 50%/56.3% of patients for PIK3CA/ESR1 (during therapy and/or at progression time, 5.6%/18.8% of patients showed decrease or clearance of ctDNA and 38.9%/25% showed increase of ctDNA).Conclusion:In this cohort of AI-progressing HR+/HER2- ABC patients, dPCR and SafeSEQ methods showed high overall agreement for the detection of mutations in PIK3CA and ESR1, key genes for clinical decision-making and enrollment in clinical trials. The expanded coverage of SafeSEQ makes it a powerful tool to inform patient management considering the growing pool of clinically relevant genomic alterations in this type of patients. Efforts are ongoing to expand the analysis to a larger cohort and incorporate long-term outcomes and multivariate adjustment. Supported by AstraZeneca.
Citation Format: Jesus Fuentes Antras, Vanesa García-Barberán, Fernando Moreno, Hillary Sloane, Igor López-Cade, Alicia De Luna, Alejandro Pascual, Carmen Ramirez-Ruda, Victor Lorca, Paloma Flores, Pedro Perez-Segura, Alberto Ocaña, Jennifer Preston, Hannah Quinn, Frederick Jones, Jose Angel Garcia-Saenz. Orthogonal assessment of PIK3CA and ESR1 mutation detection in longitudinal cfDNA samples from endocrine-resistant HR+/HER2- advanced breast cancer patients using dPCR and NGS-based SafeSEQ technology [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 P2-01-18.