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Background: Early-stage, node-negative, hormone receptor positive (HR+) HER2 negative (HER2-) breast cancer (BC) patients comprise a subset with good prognosis. Based on gene expression panels, these patients may skip chemotherapy and complete adjuvant endocrine therapy as an individual genomic basis assessment. However, up to 15% of patients categorized as non-high risk by Oncotype DX (Recurrence Score, RS≤25) may experience disease relapse. The field of cancer epigenomics is emerging as a dynamic tool to complement prognostic expression analyses and to provide further insight into the biological underpinnings of early HR+/HER2- BC. Methods: A genome-wide DNA methylation profiling in tumor tissue samples, from node-negative HR+/HER2- BC patients with available Oncotype DX RS data, was analyzed using the Illumina MethylationEPIC 850K BeadChip. We evaluated all differential methylation patterns independently of their position to establish a bioinformatic analytical pipeline. The online tools KM Plotter and ROC Plotter were used to assess the impact of survival outcomes (5-year relapse-free survival, RFS) and response to therapy of the candidate genes. Individual patient data from TCGA was used to analyze the correlation between methylation and mRNA levels in HR+/HER2- BC patients. Statistical significance was defined by p Citation Format: Jesus Fuentes-Antrás, Vanesa García-Barberán, Nicolas Costa-Fraga, Fernando Moreno, Aida Bao-Caamano, Aitor Rodríguez-Casanova, Alfonso López de Sá, Alicia De Luna, Igor Lopez-Cade, Carmen Ramirez-Ruda, Alejando Pascual, Pedro Perez-Segura, Balázs Győrffy, Alberto Ocaña, Angel Diaz-Lagares, Jose Angel Garcia-Saenz. Genome-wide DNA methylation analysis identifies novel biomarkers associated with risk of relapse beyond oncotype DX recurrence-score risk assessment within HR+/HER2- early-stage breast cancer patients [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 P3-05-06.

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 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.