Clinical applications of next-generation sequencing-based ctDNA analyses in breast cancer: defining treatment targets and dynamic changes during disease progression

Abstract

The advancements in the detection and characterization of circulating tumor DNA (ctDNA) have revolutionized precision medicine and are likely to transform standard clinical practice. The non-invasive nature of this approach allows for molecular profiling of the entire tumor entity, while also enabling real-time monitoring of the effectiveness of cancer therapies as well as the identification of resistance mechanisms to guide targeted therapy. Although the field of ctDNA studies offers a wide range of applications, including in early disease, in this review we mainly focus on the role of ctDNA in the dynamic molecular characterization of unresectable locally advanced and metastatic BC (mBC). Here, we provide clinical practice guidance for the rapidly evolving field of molecular profiling of mBC, outlining the current landscape of liquid biopsy applications and how to choose the right ctDNA assay. Additionally, we underline the importance of exploring the clinical relevance of novel molecular alterations that potentially represent therapeutic targets in mBC, along with mutations where targeted therapy is already approved. Finally, we present a potential roadmap for integrating ctDNA analysis into clinical practice.

Keywords: breast cancer; circulating tumor DNA; liquid biopsy; precision medicine; targeted therapies; therapeutic targets.

Fig. 1

ctDNA dynamics along the clinical course of breast cancer. The blue line represents ctDNA variations over disease progression and the orange line represents the emergence of new mutations during treatment. ddPCR, digital droplet PCR; MRD, molecular residual disease; NGS, next‐generation sequencing; qPCR, quantitative PCR; WES, whole‐exome sequencing; WGS, whole‐genome sequencing. Adapted from [142]. Figure created with BioRender.com.

Fig. 2

Tumor‐informed vs. tumor‐naive approaches. This figure gives an overview of strategies for liquid biopsy based in clinical practice. In orange the applications of tumor‐informed assays are highlighted, in green the use of tumor‐naïve assays is shown. Adapted from [143, 144]. Figure created with BioRender.com.

Fig. 3

List of currently clinically relevant ESR1 mutations in patients with endocrine resistant ER‐positive breast cancer (as analyzed in the current studies leading to approval or upcoming approval of SERDs in. development). ER, estrogen receptor; SERD, selective estrogen receptor degrader. Figure created with BioRender.com.

Fig. 4

Six examples for patients with mBC with ESR1 molecular testing results. Six patient examples are illustrated to demonstrate the spectrum of results that may be observed from routine molecular profiling of plasma DNA in the clinical setting. The same 77‐gene CGP panel was employed for all cases. This assay has been validated to reliably detect variants down to a VAF of 0.5%, indicating that any variant with a VAF below this percentage is below the technical LOD. In the left‐most panel, the alterations detected are listed alongside their VAFs, which are provided as a percentage in parentheses. An asterisk next to the VAF designates that this variant is below the assay LOD. In addition, the TF in plasma was estimated with a standard algorithm (ichorCNA), which has an LOD of 3%, and is provided in the box above the list of detected variants. In the middle column, a brief orientation and interpretation of the results is provided, which should help understand the technical and biological considerations that play a role in the clinical decision. The third column provides a brief justification of the treatment implications. As an official guideline or decision tree for the implementation of ESR1 mutation testing results is currently lacking, the justification here serves as more of a high‐level interpretation of the results for clinicians. Importantly, this highlights the utility of the molecular tumor board in performing an interdisciplinary, comprehensive evaluation of the results within the individual patient context to derive the most evidence‐based decision. CGP, comprehensive genomic profiling; LOD, limit of detection; mBC, metastatic breast cancer; TF, tumor fraction; VAF, variant allele frequency. Figure created with BioRender.com.

Fig. 5

Key steps for integrating liquid biopsy assays into routine clinical workflows (adapted from [50]).