Mutational analysis of single circulating tumor cells by next generation sequencing in metastatic breast cancer

Abstract

Circulating Tumor Cells (CTCs) represent a "liquid biopsy" of the tumor potentially allowing real-time monitoring of cancer biology and therapies in individual patients.The purpose of the study was to explore the applicability of a protocol for the molecular characterization of single CTCs by Next Generation Sequencing (NGS) in order to investigate cell heterogeneity and provide a tool for a personalized medicine approach.CTCs were enriched and enumerated by CellSearch in blood from four metastatic breast cancer patients and singularly isolated by DEPArray. Upon whole genome amplification 3-5 single CTCs per patient were analyzed by NGS for 50 cancer-related genes.We found 51 sequence variants in 25 genes. We observed inter- and intra-patient heterogeneity in the mutational status of CTCs.The highest number of somatic deleterious mutations was found in the gene TP53, whose mutation is associated with adverse prognosis in breast cancer.The discordance between the mutational status of the primary tumor and CTCs observed in 3 patients suggests that, in advanced stages of cancer, CTC characteristics are more closely linked to the dynamic modifications of the disease status.In one patient the mutational profiles of CTCs before and during treatment shared only few sequence variants.This study supports the applicability of a non-invasive approach based on the liquid biopsy in metastatic breast cancer patients which, in perspective, should allow investigating the clonal evolution of the tumor for the development of new therapeutic strategies in precision medicine.

Keywords: breast cancer; circulating tumor cells; next generation sequencing; single cell sequencing; somatic mutations.

Figure 1. Workflow for single CTC detection and molecular analysis

Each arrow represents one step of the procedure. Details on the methods are reported aside.

Figure 2. Image of a single CTC isolated from one patient as it appears in the image gallery of the DEPArray^™ instrument

The cell shows a positive fluorescent signal for DAPI (the DNA intercalating dye) and for the expression of cytokeratins (CK), while is negative for CD45.

Figure 3. Patients’ clinical outcome from the time of blood sampling

Figure 4. Number of sequence variants identified in the case study

(A) Number of novel/described variants per gene. (B) Number of deleterious/benign variants per gene.