Mutation Analysis of Cell-Free DNA and Single Circulating Tumor Cells in Metastatic Breast Cancer Patients with High Circulating Tumor Cell Counts

Abstract

Purpose: The purpose of this study was to directly compare mutation profiles in multiple single circulating tumor cells (CTC) and cell-free DNA (cfDNA) isolated from the same blood samples taken from patients with metastatic breast cancer (MBC). We aimed to determine whether cfDNA would reflect the heterogeneity observed in 40 single CTCs.Experimental Design: CTCs were enumerated by CELL-SEARCH. CTC count was compared with the quantity of matched cfDNA and serum CA15-3 and alkaline phosphatase (ALP) in 112 patients with MBC. In 5 patients with !100 CTCs, multiple individual EpCAM-positive CTCs were isolated by DEPArray and compared with matched cfDNA and primary tumor tissue by targeted next-generation sequencing (NGS) of about 2,200 mutations in 50 cancer genes.Results: In the whole cohort, total cfDNA levels and cell counts (!5 CTCs) were both significantly associated with overall survival, unlike CA15-3 and ALP. NGS analysis of 40 individual EpCAMpositive CTCs from 5 patients with MBC revealed mutational heterogeneity in PIK3CA, TP53, ESR1, and KRAS genes between individual CTCs. In all 5 patients, cfDNA profiles provided an accurate reflection of mutations seen in individual CTCs. ESR1 and KRAS gene mutations were absent from primary tumor tissue and therefore likely either reflect a minor subclonal mutation or were acquired with disease progression.Conclusions: Our results demonstrate that cfDNA reflects persisting EpCAM-positive CTCs in patients with high CTC counts and therefore may enable monitoring of the metastatic burden for clinical decision-making.

Blood-based biomarkers in breast cancer: From proteins to circulating tumor cells to circulating tumor DNA

“…87 To the authors' knowledge, no study has yet evaluated a potential value for measurement of ER/PR in CTC. As in plasma, ESR1 mutations have also been detected in CTC, 88 but it is unclear how such mutations related to those in tumor tissue or ctDNA.…”

Analysis of circulating tumor DNA in breast cancer as a diagnostic and prognostic biomarker

“…Several studies have shown that plasma levels of ctDNA are significantly higher in those who had higher stages of breast cancer, nodal involvement or larger tumors [38,52], and a recent study showed an association with overal survival as well [80]. However, more evidence is needed to establish the prognostic value of ctDNA concentration in breast cancer patients.…”

Liquid biopsy - emergence of a new era in personalized cancer care

“…CTCs are released into the bloodstream during metastatic spread of the cancer through blood and are present as single cells or clusters [13]. They have been detected in various metastatic carcinomas including lung [14], breast [15,16], prostate [17], and colorectal cancer [18] but are extremely rare in healthy subjects and patients with nonmalignant diseases [19]. Even in patients with metastatic cancer, they occur on average at a frequency of 1 in 100 million cells and are mixed with approximately 10 million leukocytes and 5 billion erythrocytes per 1 ml blood [20].…”