Sensitive cytometry based system for enumeration, capture and analysis of gene mutations of circulating tumor cells

Buffer, reagents and antibodies

BD Pharm Lyse lysing solution and Lyse/Fix Buffer were purchased from BD Biosciences (San Jose, CA, USA). The FcR blocking reagent and CD45 Microbeads (Dynabeads, DYNAL) were purchased from Miltenyi Biotec (Bergisch-Gladbach, Germany) and Invitrogen (Carlsbad, CA, USA), respectively. The FITC-conjugated anti-cytokeratin (CK) mAb CK3-6H5, phycoerythrin (PE)-conjugated anti-CD326 (EpCAM) mAb 9C4, and Alexa Fluor 700-conjugated anti-CD45 mAb F10-89-4 were purchased from Miltenyi Biotec, BioLegend (San Diego, CA, USA) and AbD Serotec (Oxford, UK), respectively.

Cell culture

Non-small cell lung cancer cell lines H1650, H1975, PC-9 and PC-14, and breast cancer cell line BT20 were used. PC-9 and PC-14 were kindly provided by Professor Hayata of Tokyo Medical College (Tokyo, Japan). H1650, H1975 and BT20 were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (GIBCO, Life Technologies, Grand Island, NY, USA), penicillin, streptomycin and amphotericin B (10 000 U/mL, 10 mg/mL and 25 μg/mL, respectively [Sigma-Aldrich]) under a humidified atmosphere containing 5% CO₂ at 37°C.

Circulating tumor cell enumeration and capture instrumentation

The novel multicolor flow-cytometry-based CTC detection and capturing system of this article utilized FISHMAN-R (On-Chip Biotechnologies) and On-Chip Sort (On-Chip Biotechnologies), which is a cell-sorter integrated with FISHMAN-R. These devices use a disposable microfluidics chip and include a collection reservoir within which the sorted cells are stored (Fig. 1a–c). This design enables reduction of sample loss to the absolute minimum; specifically, the dead volume of analysis using this chip is <0.01 μL. This system therefore provides suitable conditions for the enumeration of rare cells such as CTC from patients' blood. The details of the FISHMAN-R and On-Chip Sort were described previously.8-10 Data analysis was performed using FlowJo software v7.6.5 (Tree Star, Ashland, OR, USA). The principle of sorting is described on the manufacturer's website (http://www.on-chip.co.jp/en/index.html).

Circulating tumor cell enrichment, immunofluorescent staining process and enumeration

Circulating tumor cell enrichment from 4 mL of peripheral blood includes hemolysis and elimination of leukocytes by using CD45 antibody-conjugated microbeads. After the enrichment process, the CTC-enriched samples were subsequently fixed and stained using anti-CK-FITC, anti-EpCAM-PE and anti-CD45-Alexa Fluor 700 antibodies. Then, the samples were applied to FISHMAN-R or On-chip Sort. A flowchart of this process including the CTC enrichment percentage is shown in Figure 2.

Our system defined CTC as CK+ and/or EpCAM+ and CD45− cells, with a diameter comparable to cell size. The parameter forward scatter/side scatter threshold was preset to allow detection of particles greater than approximately 6–8 μm in size so as to eliminate particles other than CTC, such as unbound microbeads and cell fragments.

Circulating tumor cells sorting and gene analysis of captured cells

Circulating tumor cell sorting was performed using On-chip Sort. DNA was isolated from sorted CTC by using the QIAamp DNA Micro Kit (QIAGEN, Hilden, Germany). Isolated DNA was analyzed using the Therascreen EGFR RGQ PCR kit and the PI3K Mutation Test kit, which are based on ARMS and Scorpions PCR technologies (QIAGEN). The Therascreen EGFR RGQ PCR kit amplifies and analyzes mutations in the EGFR gene including 19 deletions in exon 19 and amino acid changes of T790M in exon 20 or L858R in exon 21. The PI3K Mutation Test Kit amplifies and analyzes the PIK3CA gene, including mutations in exons 9 and 20, which lead to E542K, E545K or H1047R amino acid changes.

Patient measurements and blood sample collection in the circulating tumor cell enumeration study

We analyzed 46 patients with various cancers and 5 healthy donors. To determine the threshold level of our method, we also analyzed 13 healthy donor samples. Healthy donors were defined as persons who had never been diagnosed with cancer before study enrollment. These healthy donor samples were collected and analyzed in three independent institutions. All patients and healthy donors in the present studies below provided informed consent and their participation in the studies was approved by the institutional review committee of the National Cancer Center. This work was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).

Ten milliliters of peripheral blood was collected in a CellSave tube for CTC enumeration by CellSearch. A total of 5 mL of blood was collected in a standard 5-mL blood collection tube containing EDTA, and 4 mL of this blood was used for CTC enumeration by FISHMAN-R. The CellSave tube was delivered to an independent medical laboratory (GeneticLab, Sapporo, Japan), where CTC were enumerated using a CellSearch CTC Kit. To confirm the superiority of FISHMAN-R over CellSearch in detecting EpCAM-negative cells, 18 cells of the EpCAM-negative lung cancer cell line PC-14 were spiked into the blood of a healthy volunteer and the sample was analyzed in the same manner. In this study, all samples were analyzed by investigators who were blinded to the sample origin.

Method validation of circulating tumor cell sorting and mutation analysis in spiked blood samples

We previously reported that spiked-in rare tumor cells in blood can be sorted using On-chip Sort independent of EpCAM expression.9 To confirm the efficiency of On-chip Sort in capturing rare cells and to estimate the cell number threshold for mutation detection, we performed spike-in experiments using lung and breast cancer cell lines before the clinical trial.

Various numbers of PC-9 cells and BT-20 cells were picked up using a micropipette under an inverted microscope and were then added to a blood sample (4 mL). After these spiked samples were processed using our CTC enrichment method, the spiked cells were captured using On-chip Sort. The number of captured cells was visually counted under a fluorescence microscope, and we then calculated the sorting efficiency. To estimate the cell number threshold for detection of EGFR deletion mutations or the PIK3CA variant H1047R mutation in sorted cells, various numbers of PC-9 cells, H1650 cells and BT20 cells were spiked into 4 mL of blood, and the spiked cells were captured using On-chip Sort. Mutations in the captured cells were then analyzed using the gene analysis method described in the subsection above titled “Circulating tumor cells sorting and gene analysis of captured cells.”

Patient measurements and blood sample collection in the circulating tumor cells sorting and mutation analysis study

We analyzed 4 lung cancer and 4 breast cancer patients. The EGFR mutation status had been confirmed in surgical or biopsied specimens of the lung cancer patients before enrolment in this study. Breast cancer patients were enrolled without testing PIK3CA mutation status. Peripheral blood (4 mL) for this study was drawn from the study participants. CTC sorting using On-chip Sort and gene mutation analyses of captured cells were performed as described in the subsection above titled “Circulating tumor cells sorting and gene analysis of captured cells.”

Statistical analysis

EZR (Saitama Medical Center, Jichi Medical University), which is a graphical user interface for R (The R Foundation for Statistical Computing, version 2.13.0), was used for statistical analyses.11

Circulating tumor cell enumeration study

We compared the sensitivity of our system with that of CellSearch for the detection of CTC in the blood of patients with various types of cancers. Eighteen patients with non-small cell lung cancer, 4 with small cell lung cancer, 13 with breast cancer, 5 with cancer of unknown primary, 3 with cervical cancer, 1 with peritoneum cancer, 1 with endometrial cancer and 1 with head and neck cancer were enrolled. Patient clinical data and the number of CTC detected in each patient examined using FISHMAN-R and CellSearch are shown in Table 1. The result of 13 healthy donor samples determined the threshold level of our system as 3 CTC/4 mL (Fig. 3f[i]). The number of CTC detected per patient using FISHMAN-R ranged from 0 to 829 (median, 5)/4 mL, and the CTC number of 65.2% (30/46) of the samples was above the threshold level. In contrast, using CellSearch, the CTC number of only 28.3% (13/46) of the samples was greater than the threshold level determined in a previous study (≥2/7.5 mL).1 When the blood sample of a healthy donor that was spiked with 18 EpCAM-negative PC-14 cells was analyzed, while FISHMAN-R detected 10 of the spiked cells, CellSearch did not even detect a single spiked cell. In all of the other blood samples from the healthy donors, the number of CTC detected by FISHMAN-R and CellSearch was not greater than the threshold of each system (Fig. 3f[ii]). The results showed that our system was significantly more sensitive than that of the CellSearch system for the detection of CTC in breast cancer and non-small cell lung cancer patients (P = 0.0342 and 0.00558, Wilcoxon test; Fig. 3a,c). Although not reaching a level of statistical significance, the CTC detection sensitivity in the other cancer types, including small cell lung cancer and cancer with unknown primary, showed a trend toward being greater than that of the CellSearch system (Fig. 3b,d,e).

Sorting efficiency and mutation analysis of sorted cells in spiked blood samples

PC-9 cells (5, 20 or 100 cells) and BT-20 cells (10, 25, 50 or 100 cells) were spiked into 4 mL of peripheral blood, and the spiked cells were then recovered from the blood using On-chip Sort. Recovered cells were visually distinguished from leukocytes (Fig. 4a) and were then counted under a fluorescent microscope. Regression analysis of the number of captured cells versus the number of spiked cells produced a correlation coefficient (R²) of 0.9029 (Fig. 4b). The mean capture rates were 101.35 ± 15.3% for PC-9 and 77.0 ± 8.1% for BT-20 (Fig. 4c). To estimate the minimal number of CTC that are required for detection of mutations using our method, we performed further spike-in experiments. PC-9 cells (5, 10 or 100 cells) and H1650 cells (5, 10 or 30 cells), which have the EGFR exon 19 deletion mutation, and BT20 cells (10, 20 or 50 cells) that express the PIK3CA variant H1047R mutation were spiked into 4 mL of peripheral blood. Following processing of each sample in a manner similar to that described for CTC enrichment (Fig. 2), the spiked cells were sorted from the peripheral blood using On-chip Sort, which successfully sorted spiked cells from all samples. EGFR deletion mutation was detected in samples spiked with 5, 10 or 100 PC-9 cells and 5, 10 or 30 H1650 cells using the method described in the Materials and Methods. The PIK3CA variant H1047R mutation was detected in samples spiked with 20 or 50 BT20 cells. The same results were obtained twice in independent experiments.

Circulating tumor cell sorting and mutation analysis study

The patient clinical data and the number of CTC detected in each patient in the CTC mutation analysis study are shown in Table 2. A typical isolated CTC with a few leukocytes is shown in Figure 5a. In some cases, EpCAM-negative/CK-positive CTC could be detected (Fig. 5b). EGFR deletion mutation was detected in 2 of 4 lung cancer patients and the PIK3CA variant H1047R mutation was detected in 1 of 4 breast cancer patients using PCR of the captured CTC (Table 2). Archival primary tumor tissue of the patient in whom the PIK3CA mutation was detected in captured CTC was analyzed for PIK3CA mutation; however, the same mutation was not detected.