Genomic profiling of isolated circulating tumor cells from metastatic breast cancer patients

Abstract

Molecular characterization of circulating tumor cells (CTC) from blood is technically challenging because cells are rare and difficult to isolate. We developed a novel approach to isolate CTCs from blood via immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE-FACS). Isolated CTCs were subjected to genome-wide copy number analysis via array comparative genomic hybridization (aCGH). In clinical studies, CTCs were isolated from 181 patients with metastatic breast cancer, 102 of which were successfully profiled, including matched archival primary tumor from five patients. CTCs revealed a wide range of copy number alterations including those previously reported in breast cancer. Comparison with two published aCGH datasets of primary breast tumors revealed similar frequencies of recurrent genomic copy number aberrations. In addition, serial testing of CTCs confirmed reproducibility and indicated genomic change over time. Comparison of CTCs with matched archival primary tumors confirmed shared lineage as well as some divergence. We showed that it is feasible to isolate CTCs away from hematopoietic cells with high purity through IE-FACS and profile them via aCGH analysis. Our approach may be used to explore genomic events involved in cancer progression and to monitor therapeutic efficacy of targeted therapies in clinical trials in a relatively noninvasive manner.

Fig. 1

Copy number analysis of BT474 cells isolated via isolated via immunomagnetic enrichment and FACS (IE/FACS). (A) Representative ACGH profiles generated from 1 to 50 BT474 cells and (B) unamplified BT474 DNA positive control. The log2 ratio value for each BAC clone is plotted on the y-axis. The x-axis represents the genomic position of each BAC clone on the array, with chromosome numbers indicated. Vertical solid lines indicate chromosome boundaries, and vertical red dashed line represents the centromeric region dividing each chromosome into the p- or short arm (to the left of centromere) and the q- or long arm (to the right of the centromere). Color represents copy number status: red=loss, green=gain, blue=amplification, and black=no change.

Fig. 2

Copy number analysis of CTCs. Genomic profiles from (A) 112 CTCs from patient #302 (B) 20 CTCs from patient #22 and (C) 7 CTCs from patient #108.

Fig. 3

Recurrent copy number aberrations in CTCs. Frequency plot of copy number alterations in (A) CTCs from 102 metastatic breast cancer patients, (B) in 62 primary breast tumors (Fridlyand et al 2006 (20)) and (C) 137 primary breast tumors (Chin et al 2006 (21)). Gains and losses are shown in red and blue, respectively.

Fig. 4

Serial copy number analysis of CTCs. Genomic profiles of CTCs from patient #303. (A) 74 CTCs at day 1. (B) 50 CTCs at day 128. (C) 50 CTCs at day 169 (Profiles from triplicate of 50 CTCs sorted independently from same blood draw are shown in Supplementary Figure 5).

Fig. 5

Copy number analysis of CTCs and matched primary tumor. (A) Profiles from 250 CTCs and the primary tumor of patient #4009. (B) Profiles from 20 CTCs isolated from two independent blood draws (days 1 and 42) and the primary tumor of patient #4013. (C) Genomic profiles from sorting replicates of 20 CTCs at day 1 isolated from the same blood sample, then amplified and arrayed independently as well as 20 CTCs at day 87 and the primary tumor of patient #462. The primary tumor showed a low level gain in HER2 (arrow).