This study was an observational clinical study. The patients were divided into MVI-positive and MVI-negative groups to evaluate the diagnostic power of parameters for the presence of MVI. We divided the patients according to the recurrence interval with regard to the relationship between postoperative CTC count and recurrence. The same inclusion and exclusion criteria, surgical criteria, and CTC testing criteria were followed throughout the study. No criteria were changed during the study..
Patients were recruited from the Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The inclusion criteria were as follows: 1) definitive pathological diagnosis of HCC based on the World Health Organization criteria; 2) curative resection, defined as the complete macroscopic removal of the tumor with negative margins (R0); 3) no prior anticancer treatment; and 4) age between 18 and 80 years. The exclusion criteria were as follows: 1) distant metastasis; and 2) Child-Pugh C liver disease. In addition, 18 patients with benign liver disease were enrolled as negative controls. All procedures performed in this study abided by the Declaration of Helsinki. The institutional review board approved the study protocol, and all patients provided written informed consent.
All surgeries were accomplished by a team who was able to professionally implement hepatectomy. The surgical principles, including anatomic resection and partial resection, were followed according to the corresponding TNM classification. Proper hepatic vascular control techniques, including the selective inflow occlusion (SIO) maneuver and intermittent Pringle maneuvers (IPs), were used to reduce bleeding during liver resection. The SIO maneuver is described by the following procedure: dissecting the portal vein, proper hepatic artery, right and left hepatic arteries, and bile ducts followed by continuously blocking the hepatic artery in the tumor-bearing lobe with a bulldog clamp. IPs encircling the hepatoduodenal ligament were performed with cycles of clamping and unclamping times of 15 minutes and 5 minutes, respectively. We enrolled twenty-eight patients with portal vein tumor thrombus (PVTT): 19 were type I, 6 were type II, 3 were type III, and none were type IV. The following definitions of PVTT were used: type I, tumor thrombus involving segmental branches of the portal vein or above; type II, tumor thrombus involving the right/left portal vein; and type III, tumor thrombus involving the main portal vein trunk (Cheng’s new classification system). The different therapeutic schedules of PVTT were schemed according to the corresponding type. Segmental hepatectomy was performed for type I, hemihepatectomy was performed for type II, and hepatectomy plus thrombectomy was performed for type III.
The CellSearchTM system is the gold standard for capturing CTCs based on epithelial cell adhesion molecules (EpCAMs), which represent aggressive stem cell-like CTCs. [23, 24]. However, a recent study showed that in the blood of a total of 14 HCC patients, the percentage of EpCAM-positive CTCs was only 8.03%. The principle of the isolation by size of epithelial tumor cells (ISET) method is to isolate tumor cells by analyzing their morphology and deformability and then identify tumor cells by special staining methods. In addition to the CellSearchTM system, the ISET method is a relatively mature technology used to study CTCs. As early as 10 years ago, the ISET method was applied in breast and lung cancer  clinical research. For HCC, the advantage of the ISET method is that it can capture EpCAM-negative tumor cells. However, it also has obvious disadvantages. In the whole study, the leucocytes and thrombus could have possibly blocked the filter pores. In our study, CTC detection was carried out in a total of 1032 blood samples (82 blood samples could not be drawn because of the patients, and the blood collection was missed). According to our statistics, due to blocked filter pores and polluted backgrounds, 8.2% (85/1032) of the blood samples failed at the isolation stage. As a result, tumor cells could not be identified. Therefore, a total of 167 CTC counts were missing from the data.
Blood samples (5 mL) were drawn to detect CTCs by the ISET method. The time points for blood collection were before surgery (30 minutes before anesthesia), during surgery (30 minutes after tumor removal) and at 1 week, 1 month, 2 months, 3 months, 6 months, and 1 year after surgery. This method involves blood filtration and analysis by microscopy using standard histopathological/cytomorphological criteria[28, 29]. The ISET instrument filtered the blood to capture CTCs with a polycarbonate membrane with an 8 µm pore, and at least two experienced cancer cytologists finally independently analyzed and synthesized the captured CTCs. CTCs were defined with respect to the following six characteristics: a) abnormal karyotypes, such as lobulated nuclei; b) cell diameter larger than 15 µm; c) irregular, dented or shriveled nuclear borders; d) nucleus-to-cytoplasmic ratio > 0.8; e) giant nucleoli; and f) nonhomogeneous nuclear staining. Cells meeting at least four of these criteria were identified as CTCs. In addition, if giant nucleoli or abnormal karyotypes appeared and at least two other criteria were met, the cells were also identified as CTCs. Fig. 1 shows typical microscopic images of CTCs.
Follow-up and Tumor Recurrence
The patients were followed up every month with ultrasonography and AFP during the first six months after surgery and every three months thereafter. The patients were scheduled to have a CT scan every six months and an MRI scan every year. Recurrence was diagnosed by computed tomography scans, magnetic resonance imaging, digital subtraction angiography, and elevated serum alpha-fetoprotein levels. Follow-up was terminated on October 30, 2019. We defined recurrence within one year after surgery as an early recurrence. The time to recurrence was defined as the interval between resection and the diagnosis of intrahepatic recurrence or extrahepatic metastasis. Governmental death registration and telephone follow-ups were used to determine the patient's survival status. The mean follow-up time was 25.2 ± 6.6 months (median, 25.5 months; range, 11.6-34.3 months). Among the 137 patients, 60 were in the nonrecurrence group, 35 were in the non-early recurrence group, and 42 were in the early recurrence group. Among all 77 patients in the recurrence groups, 65 had intrahepatic recurrence only, while 12 had both intrahepatic recurrence and extrahepatic metastasis.
The data are presented as the mean ± standard deviation (SD). Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive value of the preoperative peripheral blood CTC count for the presence of MVI. Student’s t-test was used for comparisons between groups where appropriate. Multivariate analysis was performed using the logistic regression model. Kaplan-Meier analysis was used to generate survival curves, and the log-rank test was used to compare patient survival between subgroups; P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 19.0 for Windows (IBM).