NGS analysis of the liquid biopsy in metastatic breast cancer patients: A pilot study

Pamela Pinzani 1, Francesca Salvianti, Mario Pazzagli
1University of Florence, Italy

Circulating tumor cells (CTCs) and cell-free DNA (cfDNA), released in the bloodstream from primary tumor and metastases, are considered a “liquid biopsy” of the tumor reflecting disease complexity and evolution at any stage.
In a previous study we demonstrated the feasibility of a proto- col for the mutational analysis at the single cell level in patients affected by metastatic breast cancer.
The aim of the present study is the optimization of a protocol for the detection of somatic mutations in cfDNA by Next Genera- tion Sequencing (NGS) and the comparison with data obtained from single circulating tumor cells isolated from the same blood draw. Notwithstanding that the two components of the liquid biopsy, CTCs and cfDNA, have been extensively studied separately, few studies compared the information obtained from these two differ- ent compartments for a personalized therapeutic approach.
Cell-free DNA was extracted from 2ml plasma by using the QIAamp circulating nucleic acid kit (QIAgen). DNA samples were sequenced by Ion s5 Sequencer by using the ion AmpliSeq Can- cer Hotspot Panel v2 (Thermofisher Scientific) targeting hotspot regions in 50 oncogenes and tumor suppressor genes which are frequently mutated in cancer patients.
Results obtained from cfDNA of 5 metastatic breast cancer patients have been compared with those of 3–5 CTCs isolated from the same blood draw and the primary tumor.
From the comparison we evidenced the presence of some con- cordant mutations between tissue and liquid biopsy, but also discrepancies.
In at least one patient no common somatic mutation was found between tissue and liquid biopsy. In one subject two hotspot mutations in TP53 were evidenced in CTCs and primary tumor but not in cfDNA while another mutation in the same gene was common to the tissue and cfDNA but could not be found in the CTCs.
This preliminary study suggests that CTCs and cfDNA represent two complementary aspects of the liquid biopsy. Our results sup- port the feasibility of the use of the liquid biopsy as a useful tool in personalized medicine for the management of metastatic breast cancer patients.

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