A total of 11 female patients with breast cancer from the Asan Medical Center affiliated to the University of Uslan College of Medicine (Seoul, Korea) were included in the present study. The median age of the patients was 44 years old (range, 34–56 years old). Cancer stage, and histologic and nuclear grade were evaluated based on the tumor-node-metastasis classification (17) of the American Joint Committee on Cancer (Table I). All medical data used in the present study were anonymized and obtained following approval of the present study by the Institutional Review Board of Asan Medical Center (Seoul, Korea) (clearance no. 2013-1048).

Blood (10 ml) from each patient was collected for a month (December 2014) in Acid Citrate Dextrose tubes (BD Vacutainer^®; BD Biosciences, San Jose, CA, USA) and processed within 4 h. Each blood sample was divided in half, with the CTCs from one half undergoing immunofluorescent staining and the CTCs from the other undergoing cancer gene panel analysis. CTC isolation was performed using a SMART BIOPSY™ SYSTEM Isolation kit (cat no. CIKW10; Cytogen, Inc., Seoul, Korea). Blood samples were incubated with an antibody cocktail from the SMART BIOPSY SYSTEM Isolation kit (Cytogen, Inc.) against WBCs and red blood cells for 20 min, and mixed with pre-activation buffer followed by density gradient centrifugation at 400 × g for 30 min at room temperature. The cell suspension containing CTCs was collected and gradually diluted with dilution buffer (Cytogen, Inc.). Diluted cell suspensions were filtered through a HDM chip (Cytogen, Inc.) as previously described (18). Cells on the HDM chip were collected and transferred to a microtube. An EVE™ Automated Cell counter (Nano EnTek, Inc., Seoul, Korea) was used to measure cell size prior to and following HDM chip filtration. For immunofluorescent staining, isolated cells were fixed on slides in 4% paraformaldehyde for 5 min at room temperature. For cancer gene panel analysis, isolated cells were pelleted and kept at −80°C until required.

Cells on slides were permeabilized with 0.2% Triton X-100 in PBS for 10 min and quenched with 0.3% hydrogen peroxide for 30 min at room temperature. Cells were then blocked with 1% bovine serum albumin in PBS for 30 min and incubated with primary antibodies for 90 min at 37°C, followed by secondary antibody incubation at same condition. The primary antibodies used were mouse anti-EpCAM (dilution, 1:200; catalog no. #2929; CST Biological Reagents Company Ltd., Shanghai, China) and rabbit anti-CD45 (dilution, 1:50; catalog no. SC53047; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). EpCAM signaling was amplified using the TSA™ kit (catalog no. T20922; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. The secondary antibody used against CD45 was goat anti-rabbit Alexa Fluor^® 594 (dilution, 1:100; catalog no. A11012; Invitrogen; Thermo Fisher Scientific, Inc.). The slides were mounted using Fluoroshield™ Mounting Medium with DAPI (ImmunoBioScience Corp., Mukilteo, WA, USA). Stained cells were observed and images captured using a fluorescent microscope (Eclipse Ti; Nikon Corporation, Tokyo, Japan) with a 400X objective. To identify EpCAM-positive and CD45-negative CTCs, the PC9 (EpCAM-positive) and KG-1 (CD45-positive) cell lines were used as positive controls.

A total of 100 H358-green fluorescent protein (GFP) cells were spiked into 1 ml of healthy volunteers' blood, which underwent the same CTC isolation protocol described above. The isolated cell suspension was transferred to a new dish and the number of GFP-positive cells were counted under a fluorescent microscope (Eclipse Ti; Nikon Corporation) within 30 min. Experiments were performed in triplicate. The CTC detection rate was determined as follows: CTC detection rate (%)=(No. of CTCs detected in patients/total patient no.)x100. CTC purity was calculated as follows: CTC purity (%)=(Detection of CTC no./total cell no.)x100.

MCF-7 cells were obtained from Asan Medical Center affiliated to the University of Uslan College of Medicine (Seoul, Korea), and were routinely maintained in RPMI-1640 medium (Gibco^®; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal calf serum and 1% antibiotics at 37°C in humidified, concentrated CO₂ (5%) atmosphere.

CTC cell pellets from patients were amplified using the REPLI-g Single Cell kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's protocol. Briefly, cell pellets were mixed with denaturing buffer and incubated at 65°C for 10 min. Following the addition of stop solution, denatured DNA samples were mixed with REPLI-g sc DNA polymerase and reaction buffer, and incubated at 30°C for 8 h, followed by further incubation at 65°C for 3 min.

Genomic mutations were analyzed using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Thermo Fisher Scientific, Inc.), a next-generation sequencing assay that identifies multiple somatic mutations [2,800 Catalogue of Somatic Mutations in Cancer (COSMIC) mutations across 50 genes]. Genomic DNA was amplified using the aforementioned WGA protocol and the amplicons were purified using the Agencourt AM-Pure XP kit (Beckman Coulter, Inc., Brea, CA, USA), followed by end repair and ligation using Ion Xpress™ Barcode Adapters kit (catalog no., 4471250; Thermo Fisher Scientific, Inc.). The median fragment size and concentration of the final amplicon library were detected using a BioAnalyzer 2100 with Agilent High Sensitivity DNA kit (Agilent Technologies, Inc., Santa Clara, CA, USA).

The amplicon library was diluted to 10 pM with TE buffer and 5 µl of the library was used for automatic PCR; the Ion OneTouch™ system (catalog no. 4474779; Invitrogen; Thermo Fisher Scientific, Inc.) performed emulsion PCR reactions using Ion PGM™ Template OT2 200 kit following the manufacturer's protocol. The following cycling conditions were used: 80°C for 3 min; 18 cycles of (99°C for 20 sec, 58°C for 30 sec, 72°C for 1 min, 99°C for 20 sec, 56°C for 30 sec and 70°C for 1 min); and 10 cycles of (99°C for 20 sec, and 58°C for elongated durations from 3–20 min) with heat cover at 85°C. Subsequently, the emulsion PCR product was enriched using Dynabeads^® MyOne™ Streptavidin C1 beads (Invitrogen; Thermo Fisher Scientific, Inc.). The final enriched ion spheres were mixed with a sequencing primer and polymerase, and loaded onto five 316 chips (Ion PGM™ Sequencing 200 kit v2) in total. Base calling was generated using Torrent Suite software (version 3.0; Thermo Fisher Scientific, Inc.) with tmap-f3 indexing. BAM and FASTQ alignment files were generated based on the base calling results and were used for variant calling, including single nucleotide polymorphisms and insertions/deletions.

The software used for statistical analysis was IBM SPSS version 24 (IBM Corp., Armonk, NY, USA) and Student's t-test was performed. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the cell recovery rate of the HDM chip used, a spike-in test using H358-GFP cells was performed. This identified a cell recovery rate of 83.5%. (Fig. 1A). Furthermore, the average CTC size in the patients' blood prior to and following HDM chip filtration was compared. The average CTC size prior to filtration was 7.84±0.87 µm, which increased to 13.99±3.67 µm following filtration (Fig. 1B). This is in agreement with a previous study that reported that the average size of CTCs from patients with breast cancer was 13.1 µm (19), in addition to confirming that a HDM chip with a 6.5 µm pore size effectively enriched for CTCs in the present study.

EpCAM-positive CTCs were detected in the blood samples of patients with breast cancer (Fig. 2). PC9 (EpCAM-positive) and KG-1 (CD45-positive) cells were used as positive controls during this immunostaining, in addition to WBCs. The average CTC count from the 11 patients was 3.9 in 5 ml blood and the detection rate was 90.9% (10 out of 11) (Table II). The purity of CTCs isolated with the protocol used in the current study was 14.2% (range, 0–100; Table II). The average amount of total DNA obtained following WGA was 28.6 µg (range, 4.9–37.8 µg; Table II). Reference coverage (the average number of reads that cover each reference position) analysis demonstrated complete amplification of CTC DNA compared with the MCF7 breast cancer cell line (Fig. 3).

Cancer gene panel analysis was performed using the Ion Ampliseq Cancer Hotspot Panel v2. In total, ≥10 ng of DNA can be analyzed for 2,800 COSMIC mutations across 50 genes using this panel. Mutations in MCF7 breast cancer cells were analyzed as a control, with mutations detected in the following genes: PIK3CA, APC, PTEN, IDH2 and TP53 (data not shown). In patient samples, mutations were detected in the following genes: IDH2, TP53, NRAS, IDH1, PDGFRA, HRAS, STK11, EGFR, PTEN, MLH1, PIK3CA, CDKN2A, KIT and SMARCB1 (Table III). The median allele frequency across all patients and genes was 4 (range, 1.7–100%). Mutations with allele frequencies of between 2 and 5% were considered CTC-specific mutations and mutations with frequencies >50% were considered germline mutations. Although patient 6 tested negative for EpCAM-positive CTCs, COSMIC mutations were detected in the sample. However, patient 9 tested negative for all COSMIC mutations despite having the highest number of EpCAM-positive cells: ERBB4, FGFR3, APC, CSF1R, FGFR1, RET, FLT3 and STK11 (not included in COSMIC and data not shown).