Observation of circulating tumour cells in patients with non-small cell lung cancer by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction in peroperative period

Abstract

Purpose: To assess whether surgical manoeuvre or resection of lung cancer could lead to haematogenous dissemination of malignant cells. In the mean time, the relationship between the sequence of vessel ligation and the haematogenous dissemination of cancer cells during operation was determined.

Methods: Exploiting cytokeratin 19 (CK19)/carcinoembryonic antigen (CEA) mRNA as markers, 69 peripheral blood samples were collected from 23 consecutive patients with non-small cell lung cancer (NSCLC) who underwent surgical resection with curative intention in preoperative, intraoperative and postoperative period, respectively. Before the operation, all patients were randomly assigned to one of the two surgical procedure groups according to the order of vessel ligation, PV-first group and PA-first group. Additionally, the ten patients with benign lung disease served as control subjects undergoing surgical resection. The quantity and timing of the shedding of lung cancer cells into the circulation of patients were also monitored by fluorescent quantitative-reverse transcriptase-polymerase chain reaction before, during and after surgery.

Results: (1) The CK19 diagnostic test: the value of CK19 mRNA in operation was significantly higher than that of preoperation (5.246+/-0.196 vs. 4.472+/-0.164, P=0.000) and postoperation (5.246+/-0.196 vs. 4.694+/-0.177, P=0.013). The values between adenocarcinoma and squamous carcinoma were strikingly different (4.9110+/-1.0315 vs. 4.1891+/-0.4126, t=2.364, P=0.028). The values between PV-first group and PA-first group during perioperative period appear to be different (4.503 vs. 5.085, P=0.086). Before operation, of the 23 cases studied, 14 cases were positive (60.9%). Surprisingly, circulating epithelial cells were detected in two patients resected for benign lung disease. (2) The CEA diagnostic test: the level of CEA mRNA ascended continuously within this period. The postoperative values were significantly higher than those of preoperation (4.874 vs. 4.483, P=0.000) and those of operative day (4.874 vs. 4.537, P=0.000). The values between PV-first group and PA-first group appear to reach statistical significance (4.397 vs. 4.817, P=0.075). At the same time, there was a correlation between preoperative T-stage and perioperative CEA mRNA (4.267 vs. 4.760, P=0.025). Among the 23 cases, 10 cases were positive (43.5%). Both patients with benign lung disease served as control subjects undergoing surgical resection and the volunteers were negative.

Conclusions: A considerable proportion of patients who appear to have resectable NSCLC might be regarded as having systemic disease, which is often undetectable by current tumour staging method. In terms of a marker used for the NSCLC patients who undergo operation, CEA is more suitable than CK19. The CK19-expressing epithelial cells are released intraoperatively into the circulation, meanwhile CEA-expressing tumour cells are disseminated mostly postoperatively. Surgical manipulation could promote the release of tumour cells into the bloodstream, but the ligation of pulmonary vein before the ligation of the pulmonary artery may partly prevent such release during surgery.

Fig. 1

The ROC curve of CK19 diagnostic test. The curve analysis is based on a plot of sensitivity as a function of 1-specificity. The area under the curve was 0.965, indicating high accuracy. In this study, the upper value of the CI of the median of the volunteer group was considered as the cut-off value, which is 4.120. Under this standard, the sensitivity of the diagnostic test was 90%, and the specificity was 84%

Fig. 2

Standard curve for LC-5 cell line dilutions. The graph shows the Ct value versus the log of the number of CK19 mRNA, measured in triplicate. The standard curve shows four orders of linear dynamic range

Fig. 3

Typical amplification plot. The graph of the increment of fluorescence reporter signal (ΔRn) versus cycle number during PCR shows three stages: baseline, exponential phase and plateau. The Ct value is calculated by determining the point at which the fluorescence exceeds an arbitrary threshold limit. For each reaction tube, the fluorescence signal of the reporter dye (FAM) is divided by the fluorescence signal of the passive reference dye (ROX), to obtain a ratio defined as the normalized reporter signal (Rn). ΔRn represents the normalized reporter signal (Rn) minus the baseline signal

Fig. 4

The comparison of CK19mRNA level in blood at different time points during the perioperative period. The level of CK19 mRNA in PB at different time points. The points show the value of the day before operation, the operating day and the seventh day after operation, respectively. The values of results were expressed as log₁₀ CK19mRNA copies per millilitre serum. The sign D _−1, D ₀ and D ₇ represent the day before surgery, the operating day and the seventh day after surgery, respectively

Fig. 5

The comparison of preoperative CK19 mRNA values between lung adenocarcinoma and squamous cell carcinoma

Fig. 6

The effect of sequence of vessel ligation on perioperative CK19 mRNA level. The sign D ₋₁, D ₀ and D ₇ represent the day before surgery, the operating day and the seventh day after surgery, respectively. PV–PA represent patients whose PV was ligated first followed by PA ligation, meanwhile PA–PV represent patients whose PA was ligated first followed by PV ligation

Fig. 7

The ROC curve of the CEA diagnostic test. The curve analysis is based on a plot of sensitivity as a function of 1-specificity. In this study, the upper value of the CI of the median of the volunteer group was considered as the cut-off value, which is 4.6151. Under this standard, the sensitivity of the diagnostic test was 91%, and meanwhile the specificity is 87%

Fig. 8

Comparison of the trend of CK19 mRNA and CEA mRNA in PB within the perioperative period. The sign D ₋₁, D ₀ and D ₇ represent the day before surgery, the operating day and the seventh day after surgery, respectively

Fig. 9

The comparison of preoperative CEA mRNA values between ADC and SCC

Fig. 10

The effect of sequence of vessel ligation on perioperative CEA mRNA in blood. The sign D ₋₁, D ₀ and D ₇ represent the day before surgery, the operating day and the seventh day after surgery, respectively. V–A represent patients whose pulmonary vein was ligated first followed by pulmonary artery ligation, meanwhile A–V represent patients whose pulmonary artery was ligated first followed by pulmonary vein ligation