Circulating Tumor Cells to Predict Microvascular Invasion and Dynamical Detection Indicate Prognosis of Hepatocellular Carcinoma

Background: The number of preoperative CTC was correlated with the presence of MVI. The change of postoperative CTC count can predict the prognosis of patients. But the change trend of postoperative CTC is controversial. Methods: A total of 137 patients were recruited for the study. Preoperative blood samples of all patients were collected to detect CTC. The time points for blood collection were before the operation, during operation, and at one week, one month, two months, three months, six months, and one year after surgery. The power of predicting the presence of MVI was analyzed by the receiver operating characteristic curve (ROC). According to the survival status, 137 patients were divided into three groups: no recurrence, early recurrence, and non-early recurrence. Results: A threshold CTC value of 5 showed the most signicant power to predict the existence of MVI. In a multivariate analysis, the parameters of preoperative CTCs, AFP, and tumor diameter were independent predictors of MVI (P < 0.05). A CTC value greater than or equal to 5 has better predictive value than AFP > 400μg/L or tumor diameter. The number of intraoperative CTCs in the three groups did not increase compared to before surgery (P > 0.05). The number of CTC in the non-recurrent group and the non-early recurrent group decreased signicantly one week after surgery compared with intraoperative values (P < 0.001), although there was no statistical signicance in the group with early recurrence (P = 0.95). The mean CTC in the early recurrence group was higher than that in the other two groups (P <0.001). Conclusion: The preoperative CTC counts in peripheral blood of patients with HCC is closely correlated to MVI. Intraoperative manipulation for the lesion by the surgeon does not increase the number of CTC in peripheral blood. Surgical removal of the tumor decreases the number of CTC. The persistence of CTC at a high level ( ≥ 5) after surgery suggests a risk of early recurrence.

Background Hepatocellular carcinoma is one of the most common malignancies worldwide, and its high mortality makes it the second leading cause of cancer death. [1] The dismal prognosis of HCC has improved signi cantly over the last decade due to increased knowledge of HCC behaviour, improvements in staging systems and multiple therapeutic options. [2] Nevertheless, the prognosis of HCC remains very poor due to the high incidence of recurrence and metastasis, and the 5-year recurrence rate after curative treatment remains high (70%), with 15% of HCC patients developing extrahepatic metastasis [3,4]. One important reason is that tumor cells are able to penetrate the microvasculature, disseminate through the bloodstream to other sites and nally form metastatic tumors. Studies have suggested that microvascular invasion (MVI) in HCC is one of the most signi cant risk factors for recurrence and metastasis in HCC following curative surgical resection [5]. MVI is de ned as clusters of cancer cells observed microscopically in vessels located in the tumor capsule and surrounding liver parenchyma [6].
When major lump was removed, residual micro-metastases could form postoperatively and result in early recurrence. Recent studies have demonstrated that tumor diameter, tumor number, degree of differentiation and serum level of prothrombin induced by vitamin K absence-II (PIVKA-II) were predictors of MVI [7,8]. A study reported that independent predictors of MVI were tumor diameter >2 cm, alphafetoprotein (AFP) >200 ng/mL and PIVKA-II >40 mAU/ml [9]. Previous research reported that the incidence of MVI ranged from 15% to 57% in HCC specimens and was associated with tumor size, AFP and typical image features [10]. The presence of MVI increases the risk of recurrence and dramatically shortens long-term survival [11,12]. Although preoperative imaging can evaluate macrovascular invasion and is helpful for selecting the appropriate therapy, MVI was almost impossible to predict by traditional preoperative imaging and was con rmed only by histopathological diagnosis after operation. Therefore, the application of MVI predictor was still very limited when it came to selecting therapeutic strategies and predicting prognosis [13].
Circulating tumor cells obtained by the liquid biopsy technique can be used to evaluate the presence of MVI by examining the peripheral blood preoperatively. A previous study showed that patients with a preoperative CTC 7.5ml of >2 were more likely to have vascular invasion [14]. The detection of CTC could predict the presence of MVI and the prognosis of hepatocellular carcinoma.
In addition, resection and liver transplantation are still the mainstream methods for the treatment of hepatocellular carcinoma. Some studies showed that tumor cells tended to disseminate from the vascular portal and were driven into the blood stream when moving and rotating the liver. [15][16][17] Previous studied have shown that cancer cells probably dislodge from the primary focus into the portal venous circulation when surgeons perform resection and rotate the liver [15,18]. In contrast, some studies demonstrated that a signi cant decrease in CTC was observed after the primary tumor was resected when values were compared to those taken during preoperative status. [19] The correlation of postoperative CTC count with surgical procedures remains controversial.
At present, there are few studies on preoperative CTC as a predictive indicator for the existence of MVI, and there are also few studies on the changes of postoperative CTC number. In this paper, the optimal cutoff value of CTC for predicting MVI was found through preoperative CTC, and the change in the number of CTC after surgery was explained how postoperative recurrence and death were affected. This information will contribute to understanding the impact of changes in the number of CTCS on prognosis.
Even in the future, CTC can be used as one of the clinical indicators to monitor recurrence and death.

Trial design
This study was an observational clinical study and did not intervene in subgroups. 137 HCC patients were enrolled in the study. When we investigated the relationship between preoperative CTC number and the incidence of MVI, we grouped the patients according to the positive and negative groups of MVI. When we studied the relationship between postoperative CTC and recurrence, we divided patients into non-recurrence group, early recurrence group and non-early recurrence group. The same inclusion and exclusion criteria, surgical criteria, and CTC testing criteria were followed throughout the study. Technology. The inclusion criteria were 1) de nitive pathological diagnosis of HCC based on the World Health Organization criteria; 2) curative resection, de ned as complete macroscopic removal of the tumor with negative (R0) margins [20]; 3) no prior anticancer treatment 4) age between 18 and 80 years. The exclusion criteria were 1) distant metastasis; 2) Child-Pugh C liver disease. In addition, 18 patients with benign liver disease were enrolled as negative controls. The institutional review board approved the study protocol, and all patients provided written informed consent.

Surgical methods
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 UICC TNM classi cation. An anatomic resection was de ned as the complete removal of at least one Couinaud segment containing the focus. A partial resection was de ned as removal of the complete tumor plus a rim of non-neoplastic liver parenchyma. Proper hepatic vascular control techniques including selective in ow occlusion (SIO) maneuver and intermittent Pringle maneuvers (IPs) were used to reduce bleeding in 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. [21].
IPs encircling the hepatoduodenal ligament were performed with cycles of clamping and unclamping times of 15 minutes and 5 minutes, respectively. We enrolled 11 patients with portal vein thrombus (PVTT): seven were type I, three were type II, one was type III, and none were type IV patients.

CTC Detection
Five-milliliter blood samples were drawn to detect CTC by the isolation by size of epithelial tumor cells (ISET) method. The time points for blood collection were pre-operation (30 minutes before anesthesia), intra-operation (30 minutes after tumor removal) and at one week, one month, two months, three months, six months, and one year post-operation. The CellSearch System is the gold standard for detecting and counting CTCs [22]. The CellSearch System used to detect tumor cells relies on magnetic beads coated with anti-EpCAM monoclonal antibody to immunomagnetically capture away from the peripheral blood cells. However, only 35% of HCC patients express EpCAM [23]; therefore, many false-negative results will certainly occur. In 1999, Vona et al. [24] used the ISET to detected CTC in HCC patients, which was able to identify CTC by analysing the morphology and molecular characterization of circulating tumor cells; meanwhile, they demonstrated how tumor micro-emboli diffuse into the peripheral blood during surgery. This involves blood ltration and analysis by microscopy using standard histopathological/cytomorphological criteria [24,25]. The ISET technique-membrane ltration and separation of tumor cells-is based on differences in size and deformability between tumor cells and blood cells. The speci c tumor cell stain also identi es the captured CTC. In this study, the ISET technique combined with blood ltration was applied to isolate CTCs, and histopathological criteria were used to analyse the CTC by microscopy [26,27]. The ISET instrument ltered the blood to capture CTC by a polycarbonate membrane with an 8 µm pore. An experienced cancer cytologist identi ed the degree of malignancy using histopathological criteria. CTCs are de ned 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 shrivelled nuclear borders; d) nucleus-to-cytoplasmic ratio >0.8; e) giant nucleoli and f) non-homogeneous nuclear staining. Cells meeting at least 4 of these criteria were identi ed as CTCs.
In addition, if giant nucleoli or abnormal karyotypes appeared and at least 2 other criteria were met, the cells were also identi ed as CTCs.

Follow-up and Tumor Recurrence
The patients were surveilled every month with ultrasonography and AFP during the rst six months after surgery and every three months thereafter. Patients were scheduled to have a CT scan every six months and an MRI every year. Recurrence was diagnosed by computed tomography scans, magnetic resonance imaging, digital subtraction angiography, and elevated serum alpha-fetoprotein level. Follow-up was terminated on October 30, 2019. We de ned recurrence within one year after surgery as early recurrence.
[28] The time to recurrence was de ned as the interval between resection and the diagnosis of intrahepatic recurrence or extrahepatic metastasis end points [29]. Governmental death registration and telephone follow-ups were used to determine the patient's survival status. The mean follow-up time (mean ±SD) was 25.2 ± 6.6 months (median, 25.5 months; range, 11.6-34.3 months). Tumor recurrence was used as the end point of the study. Among the 137 patients, 60 were in the non-recurrence group, 35 were in the non-early recurrence group, and 42 were in the early recurrence group. Among all 77 recurrent patients, 65 had intrahepatic recurrence only, while 12 had both intrahepatic recurrence and extrahepatic metastasis.

Statistical Analysis
Data are presented as the mean ± 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. The change of CTC counts in subgroups were analysed using a paired-samples t-test. Multivariate analysis was performed using logistic regression model. Kaplan-Meier analysis was used to generate the survival curves and Log-rank test was used to compare patient survival between subgroups, P < 0.05 was considered statistically signi cant. Statistical analyses were performed with SPSS version 19.0.    Figure 1a. Eighteen patients with benign hepatic tumors had 0 CTC (P < 0.001). A comparison of CTC counts between MVI positive and MVI negative patients is shown in Figure 1b. The difference in the mean blood CTC 5mL levels between the MVI positive group and the MVI negative group was statistically signi cant (6.8 ± 5.1 versus 2.9 ± 2.5, P < 0.001). AFP, tumor diameter, and preoperative CTC count were included to predict the presence of MVI. ROC curves of AFP, tumor diameter, preoperative CTC count, and multiparameter combination were drawn (Figure 1c) (Figure 1d).

Comparison the change of CTC count caused by surgery in the Subgroups
All patients were divided into three groups: no recurrence, early recurrence and non-early recurrence. The CTC counts at each time point for the three groups are shown in Figure 2a. Figure 2b showed that intraoperative CTC did not increase across the three groups compared to pre-surgical values (2.7 versus 3.1, P = 0.233; 4.9 versus 5.5, P = 0.169; 7.3 versus 7.5, P = 0.714). The number of CTC in the nonrecurrent group and the non-early recurrent group decreased signi cantly one week after surgery compared with that in the intraoperative group (1.1 versus 3.1, P < 0.001; 1.2 versus 5.5, P < 0.001), although there was no statistical signi cance in the group with early recurrence (7.6 versus 7.5, P = 0.95). Mean CTC was de ned as the average number of CTC for all time points. Figure 2c demonstrates that the mean CTC count of the three groups was statistically different (1.4 versus 4.0 versus 7.1, P <0.001).
Using a mean CTC of 5 as the cut-off value, all patients were divided into two groups. The survival curve showed that the survival time of group CTC ≥ 5 was signi cantly shorter than that of group CTC count < 5 (14.7 month versus not reached, P < 0.001) (Figure 2d). The proportion of early recurrence for the CTC > 5 group was also higher than that of group CTC count < 5 (84.8% versus 3.3%, P < 0.001). When the mean value of CTC is greater than 5 and continues to be greater than 5 after surgery, it strongly indicates risk of early postoperative recurrence. Extrahepatic metastasis occurred in 12 of the 137 patients. We de ned the difference between postoperative CTC 1 week and intraoperative CTC as △CTC. We found that the number of CTC in the non-extrahepatic metastasis group decreased 1 week after surgery, but the number of CTC in the extrahepatic metastasis group increased abnormally 1 week after surgery(-2.4 ± 2.5 versus 2.3 ± 2.8, P < 0.001).

Discussion
At present, surgeons consider hepatectomy and liver transplantation the optimal therapies to improve prognosis in HCC. Unfortunately, the high recurrence rate (50%-70% at 5 years) is still discouraging [30]. The most signi cant reason for the dismal prognosis is vascular invasion. Previous research has demonstrated that vascular invasion is a vital risk factor for the prognosis of HCC patients after curative hepatectomy [31]. Disappointingly, routine diagnostic methods are unable to identify the HCC patient subpopulation at high risk of having microvascular invasion preoperatively. Anatomic resection independently improves long-term survival in patients with T1-T2 HCC, probably due to the elimination of venous tumor thrombi within the resected domain [32]. An anatomic resection is recommended when the size of the HCC ranges from 2 cm to 5 cm [33]. However, almost all patients with HCC have liver cirrhosis, and excessive removal of non-neoplastic liver parenchyma can lead to liver dysfunction and the morbidities of ascites, jaundice and hypoalbuminemia. Thus, preoperatively predicting the existence of vascular invasion is crucial to the selection of surgical methods. Liquid biopsy technique can be used to evaluate the presence of MVI by examining peripheral blood preoperatively. Previous studies have shown that patients with a preoperative CTC 7.5mL of ≥2 were more likely to have vascular invasion [14]. In the present study, we found that patients with preoperative CTC 5mL counts of ≥5 were more likely to have vascular invasion. Compared to AFP and tumor diameter, the preoperative CTC count was the most signi cant predictor of MVI and its diagnostic power is superior to the multi-parameter combination. CTC could predict the presence of MVI and help guide the options for surgical methods, thus improving the prognosis of hepatocellular carcinoma.
It is controversial that surgical manipulation can drive the CTC into blood, resulting in the dissemination of tumor cells. Previous studies showed that the CTC value tended to decrease postoperatively until it was maintained at normal levels. [19,34] Others studies have demonstrated that the increase of postoperative CTC indicated a poor prognosis. [35,36] We found that the intraoperative CTC level of patients in the three groups did not increase compared to the preoperative CTC level. The results showed that intraoperative manipulation by the surgeon does not drive CTC into the blood stream resulting in the dissemination of tumor cells. In the non-recurrence and non-early recurrence subgroups, we found that the level of CTC at one week after surgery decreased sharply compared to intraoperative levels and remained at a low level until the recurrence. The result suggested that removal of the tumor was one of the reasons for the decrease in CTC one week after surgery and that recurrence will lead to a subsequent increase. However, in the early recurrence subgroup, the CTC levels did not decrease one week after surgery and remained at a consistently high level. In our data, of the 12 patients with extrahepatic metastasis, nine occurred within one year after surgery. In addition, in the extrahepatic metastasis group, CTC increased abnormally 1 week after surgery. However, in the early recurrence subgroup, the CTC levels did not decrease one week after surgery and remained at a consistently high level. In our data, of the 12 patients with extrahepatic metastasis, nine occurred within one year after surgery. By analyzing the dynamic change in the number of CTC, we found that the average CTC level of patients with early recurrence was higher than that of other subgroups; patients with an average CTC level greater than 5 had a signi cantly higher risk of early recurrence and a signi cantly shorter survival time than other subgroups.
The limitations of this study are small samples size, short follow-up time, and a single study center. A multicenter, big samples, randomized clinical trial should be designed to illustrate the prognostic signi cance of CTC in HCC.

Conclusion
The preoperative CTC counts in peripheral blood of patients with HCC is closely correlated to MVI.

Availability of data and materials
All data generated or analyzed during this study are available from the corresponding author.

Competing interests
The authors declare that they have no competing interests.

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