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 CELLSEARCH. 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 EpCAM-positive 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. Clin Cancer Res; 23(1); 88-96. ©2016 AACR.

Fig. 1. Study workflow.

112 patients with metastatic breast cancer were recruited to the study. Blood was collected and analysed for CTC count and total cfDNA levels in all 112 patients. In 5 patients with high CTC count individual CTCS were isolated by DEPArray and subjected to targeted NGS in comparison with matched cfDNA and primary tumour tissue where available.

Fig. 2. Longitudinal follow up of circulating tumor DNA in 3 patients with high CTC counts.

(A - C) Total cfDNA and circulating tumor DNA levels. (A) patient CTCM138; (B) patient CTCM105; (C) patient CTCM292. Endocrine or cytotoxic therapies are indicated by colored shading. The number of cfDNA copies obtained from the blood sample, mutant allele frequency (AF), number of mutant copies and CTC count is given below each graph. NA = Not available or CellSearch failed. (D) Digital PCR detection of low frequency mutations in circulating tumour DNA. ESR1 p.Y537C mutation, patient CTCM138 (top) and KRAS p.G12D mutation, patient CTCM105 (bottom). Green dots represent HEX-labelled wild-type (WT), blue dots represent FAM-labelled mutant DNA and brown dots represent double positive droplets containing WT and mutant DNA. Grey dots represent empty droplets.