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Detection of Rare Mutations in CtDNA Using Next Generation Sequencing
Published on: August 24, 2017
Standardized Analytical Verification of ctDNA ESR1 Mutation Testing in Metastatic HR+/HER2- Breast Cancer: A European
Federico Rojo1, Valentina Guarneri2,3, Nicola Fusco4,5
1Pathology Department, IIS-Fundación Jiménez Díaz University Hospital-UAM-CIBERONC, Madrid, Spain. frojo@fdj.es.
Introduction:
Activating ESR1 mutations are a major mechanism of resistance to aromatase inhibitors in hormone receptor-positive, HER2-negative metastatic breast cancer (mBC). International guidelines, including those from ASCO, NCCN, and ESMO, recommend liquid biopsy as the preferred approach for ESR1 mutation testing at progression on endocrine therapy, with digital PCR (dPCR) and next-generation sequencing (NGS) as the preferred analytical platforms. Although elacestrant was approved by the U.S. Food and Drug Administration together with Guardant360® Dx as its companion diagnostic, European regulatory frameworks allow the use of validated in-house assays for ESR1 testing, which are increasingly being implemented across clinical laboratories. To support the clinical implementation of ESR1 testing and improve analytical standardization in routine practice, we performed a European multicentre analytical verification study using dPCR- and NGS-based liquid biopsy workflows.
Methods:
Six referral institutions participated in this study. All laboratories verified dPCR workflows and four also verified NGS-based assays using standardized reference materials containing clinically relevant ESR1 mutations. Limit of detection (LoD) and limit of blank (LoB) were determined in each laboratory according to locally validated workflows following CLSI-based verification procedures. Analytical sensitivity and specificity were assessed across platforms, focusing on the two most frequently tested ESR1 hotspot mutations, p.Y537S and p.D538G. Total DNA input ranged from 10 to 30 ng per reaction for dPCR assays, while NGS input followed platform-specific requirements.
Results:
LoD values ranged from 0.01% to 0.07% variant allele frequency (VAF) for dPCR assays and from 0.01% to 0.1% for NGS-based workflows, depending on platform characteristics and DNA input. Limit of blank values were low across laboratories, with minimal background signal detected in plasma samples from healthy donors. Comparable analytical performance was observed across different platforms when assays were performed under validated laboratory conditions.
Discussion:
These results demonstrate that both dPCR and NGS-based liquid biopsy workflows can be successfully implemented for ESR1 mutation testing in routine clinical practice using locally validated assays. This multicentre verification study provides practical guidance on assay verification, DNA input requirements, and key analytical parameters required to ensure reliable ESR1 mutation detection across different European laboratories. Robust analytical verification of ESR1 testing may improve diagnostic reliability and support personalized treatment strategies for patients with hormone receptor-positive, HER2-negative metastatic breast cancer.
