A pilot study of cdc6 as a biomarker for circulating tumor cells in patients with lung cancer

Abstract Background Cell division cycle 6 (cdc6) is a key factor of DNA replication initiation license system and a proto‐oncogene. It has been detected in some tumor tissues to aid cancer diagnosis in many research projects. However, it remains unclear that if cdc6 could be detected in the peripheral blood, just like liquid biopsy, in solid tumor patients. The aim of this study is to investigate the possibility of cdc6 as a biomarker for circulating tumor cells in patients with lung cancer. Methods We first detected the expression of cdc6 in peripheral blood mononuclear cells (PBMCs) and tumor cells by in situ hybridization with cdc6 RNA probe. Then, we examined the expression of cdc6 in PBMCs from health individual, mononuclear cells from cord blood, or A549 cell line by RT‐qPCR. Furthermore, we used RT‐qPCR to test the cdc6 expression in PBMCs from tumor patients (test group) and non‐tumor individuals as a control group. Chi‐square test with Fisher's exact test was used to analyze the statistical significance of the difference. P < .05 is considered as statistically significant difference. Results When compared the cdc6 expression in cells from difference sources, we found that A549 tumor cell line had the strongest expression of cdc6, samples from cord blood showed the least expression level, indicating the detection strategy of RT‐qPCR is reasonable. Using this method, we studied whether cdc6 in Peripheral blood could be detected as a biomarker by examining cdc6 expression from PBMCs of patients with lung cancer. We found 20% of patients with lung cancer were cdc6 positive in PBMCs, whereas only 4.26% was found positive in the control group (P = .039, P < .05). Conclusion Cell division cycle 6 has a potential to be used as a circulating tumor cell biomarker for lung cancer. Further study in clinical application is still broad needed.


| INTRODUC TI ON
Tumor metastasis is a basic feature and an important sign for malignancy, and also a major cause for death. [1][2][3][4] Hence, early detection and prevention of tumor metastasis are critical for tumor treatment and prevention. It is an independent factor for evaluating cancer disease to detect the tumor cells from peripheral blood. [5][6][7][8] Researchers are developing methods for tumor cell enrichment from peripheral blood. A variety of methods, such as immunohistochemistry, flow cytometry, and RT-PCR, have been used in detecting samples from prostatic cancer, breast cancer, non-small-cell lung cancer, and colorectal cancer. 9 which could possibly be used as a wide spectrum tumor cell biomarker to monitor the cell proliferation status in peripheral blood of patients. cdc6 is a key factor of DNA replication license system, DNA replication initiation complex, which plays an important role to ensure DNA replicate only once during the one cell cycle. 12,13 Several studies have showed that cdc6 has the character of oncogene and also plays an important role in the evaluating tumor grade, screening tumor cell and predicting prognosis by probing it in the tissue or exfoliated cells. [14][15][16][17] Its expression and detection in peripheral blood of patients with tumor remain unknown. In this study, we studied feasibility of using cdc6 as biomarker for circulating tumor cell by examining the expression level of cdc6 in the PBMCs from health individuals, mononuclear cells from cord blood and a tumor cell line A549 with in situ hybridization, reverse transcript quantity polymerase chain reaction (RT-qPCR) technique. After the method established, we validate it with samples from non-operating lung cancer patients.

| Cell culture
Cells from A549 cells line (ATCC ® CCL-185™) were cultured in McCoy's 5A Medium modified with 10% FBS and 1% penicillin and streptomycin (Thermo Fisher Scientific) and maintained in a humidified incubator (5% CO 2 ) at 37°C. The cells were harvested for further experiments when their confluency reached 80%.

| RT-qPCR
Total RNA was extracted with TRIZOL reagent (Thermo Fisher

| Peripheral blood mononuclear cells (PBMCs) separation
About 2 mL whole blood anticoagulated with EDTA was added in 4 mL hemolytic agent (65 mol/L NH 4 Cl, 10 mmol/L KHCO 3 , 0.13 mmol/L EDTA), stood in ice for 15 minutes, and then centrifuged at 1700 g for 15 minutes at 4°C. After discarding supernatant then adding 2 mL hemolytic agent again, and repeating as above steps, the pellet was transferred into a new 1.5-mL EP tube and washed with PBS buffer twice at 1700 g for 8 minutes. The pellet was stored at −80°C for the following test.

| Simulation sample
About 50 and 100 cells from A549 cell line were added into two tubes containing 1 mL whole blood from health people, respectively, which were anticoagulated with EDTA and named mix sample 1 and mix sample 2. PBMCs were separated according to the method previous mentioned.

| In situ hybridization with cdc6 RNA probe
In situ hybridization with cdc6 RNA probe was complied with the procedure of Enhanced Sensitive ISH Detection Kit I (BOSTER WUHAN). About 1.15 × 10 5 cells were evenly spread on the slide treated with 0.1% DEPC and 10% polylysine, and further fixed with

| Collection of clinical samples
A total of 63 patients were enrolled in this study, including 40 patients with lung cancer, 23 patients with benign pulmonary disease (Table 1). A total of 24 health volunteers, patients with benign pulmonary diseases, and patients with lung cancer were enrolled as control groups and experiment group, respectively. About 2 mL peripheral blood samples anticoagulated with EDTA were collected from the median cubital vein of these individuals.
To avoid tissue cell contamination, the first tube of blood was not used in the study. The inclusion criteria of subjects were shown on the diagram (Figure 1). Approval for the use of human subjects was obtained from the research ethics committee of Guang'anmen Hospital (Beijing, China).

| Statistical analysis
Statistical analysis was performed using SPSS17.0 for windows. Chisquare test with Fisher's exact test was used. P < .05 is considered to indicate a statistically significant difference.

| Preparation of cdc6 RNA probe
A DNA fragment of cdc6 gene was cloned into PEASY-T3 for RNA probe preparing. The cdc6 sequence was from the GenBank, and the PEASY-T3-cdc6 clone was verified by sanger sequencing ( Figure S1). The segment of cdc6 for RNA probe preparing was cloned at the downstream of T7 promoter on which cdc6 RNA probe was labeled with Dig by in vitro transcription with T7 RNA polymerase.

| Validation of cdc6 expression by in situ Hybridization
We first asked if our scientific strategy is feasible by examining the

| Expression level of cdc6 in the tumor cells and PBMCs by RT-qPCR
After confirming cdc6expression by hybridization, we next asked if cdc6could be detected by a high-throughput method, qPCR. We used RT-qPCR to examine the cdc6 expression in A549 cell line, and mononuclear cells from peripheral blood or cord blood of health donors. Results are summarized in Table 2

| Lung cancer patients have high cdc6 expression in PBMCs
Finally, we utilized the established high-throughput qPCR method to detect samples from patients with lung cancer. The results are summarized in Table 3. We found that 8 out of 40 (20%) patients

| D ISCUSS I ON
In this study, we examined cdc6 expression in PBMCs from health   26,27 We also observed two subjects whose cdc6 was positive. One was from health individual, the other was an inpatient with lung infection, whose CT for chest and abdomen showed that lymph nodes of mediastinum and abdomen were enlarged, and whose serum CYFRA-211 and SCC level were higher than reference value. Cdc6 tests were performed for three times with different PBMCs from this patient, and all of the results were positive. Based on these evidences, the patient should be consider a patient with cancer, but his family members refused to accept further examination because of age factor (87 years old). The other was a young female, and we later found that she was a patient with mild thalassemia.
No evidence supported that she was a patient with tumor, except several erythrocytoblasts in peripheral blood. Theoretically, the two cdc6-positive subjects should be excluded from the study, and thus, no cdc6-positive subjects had been observed in control group, which shows the high specificity of the study. Further study will be focusing on how to increase the sensitivity of the methodology, and whether it can be used as a new way for risk evaluation of tumor relapse or metastasis, and the monitoring of the therapeutic effect with enlarged cases.

ACK N OWLED G M ENTS
This work was supported by GAMH2003S65.