Improvement In Circulating Tumor Cells Detection And Its Value As A Predictor Of Postsurgical Recurrence Of Hepatocellular Carcinoma

Background & ObjectiveWith the improvements in diagnosis and comprehensive treatment based on surgery, the survival rate of hepatocellular carcinoma (HCC) has improved significantly. But intrahepatic and extrahepatic recurrence is still frequent, even after curative surgery. Therefore it is imperative to explore a clinical relevance predictor of HCC recurrence to optimize treatment. Traditional predictors of recurrence are tumor stage and tumor differentiation grade. The staging and grading can broadly reflect the differences in survival rates in different patients, but these indicators do not accurately predict the risk of recurrence and metastasis. Because Hepatoma cells cells released into the blood may be the first step in hematogenous metastasis of HCC, Circulating tumor cells(CTCs) must be a good predictor of HCC. Sensitive molecular techniques developed in recent years are able to enumerate CTCs in the peripheral blood easily and accurately. CTCs can currently be applied in a variety of malignancies, including HCC, such as breast cancer, prostate cancer and colon cancer. The clinical significance of CTCs in breast cancer, colon cancer, prostate cancer and other cancers has gradually been affirmed and detection of CTCs in breast cancer has even been the introduction of the latest TNM staging of breast cancer.We have recently developed a sensitive and specific system to magnetically separate CTCs in HCC patients, mediated by the interaction of the ASGPR with its ligand. In the system, HCC cells were bound by biotinylated asialofetuin, anASGPR ligand, and subsequently labeled by anti-biotin antibody-coated magnetic beads, followed by magnetic separation. The separated HCC cells were then identified by immunofluorescence staining using the hepatocyte-specific antibody Hep Par 1. We have applied this system to patients with HCC and statistic analyses showed that both the positivity rate and the number of CTCs were significantly correlated with tumor size, portal vein tumor thrombus, and the disease extent as classified by the TNM classification and the Milan criteria. In the first part of this study, We have used ethylenediaminetetraacetic acid (EDTA) instead of heparin for anticoagulation because we have found that the heparin could cause cell suspension gel of cells , which affected cells through the separation column, reducing the separation efficiency.The recovery rate of the original detection method significantly improved through the modified method. The second part of this work aims to continue to explore the value of the separation and detection system to predict the risk of recurrence of postoperative HCC, as a guide to develop individualized treatment plan.Methods1.Calcium chelating agent EDTA was used for anticoagulation instead of heparin in some steps of the original method. The recovery, specificity and sensitivity of the HCC CTCs separation and detection system were determined by the Hep3B cell spiking experiments.2. Naive patients with HCC who had hepatic function of Child-Pugh class A or B and had undergone first curative hepatic resection were eligible for this study.3. Peripheral blood samples for CTCs detection were obtained from all patients preoperatively and 2 weeks after the operation.4. HCC cells were bound by biotinylated asialofetuin and subsequently labeled by anti-biotin antibody-coated magnetic beads, followed by magnetic separation. The separated HCC cells were then identified by immunofluorescence staining using the hepatocyte-specific antibody Hep Par 1.5. Regular review and follow-up.6.Kaplan-Meier curves were generated for recurrence-free interval and subsequently compared with the log-rank test. The prognostic relevance of clinicopathologic variables including postoperative CTCs to overall recurrence was evaluated by univariate analysis with the log-rank test and by multivariate analysis with Cox's proportional-hazards model.Results1.The cell spiking experiments showed that the recovery of the modified method was higher than that of the original methed (P<0.05).2. CTCs was positive (defined as CTCs≥1) in 54 patients (43%) preoperatively and in 9 patients (17%) who belong to the early stage (I-II,TNM stage) .3. The preoperative positivity rate of CTCs were significantly correlated with tumor size, vascular invasion, intrahepatic cancer spread, and the disease extent as classified by the TNM classification, but positivity rate of CTCs were not correlated with age, sex, etiology, Child-Pugh class or serum AFP level.4. CTCs was positive in 41 patients (32.5%) two week postoperatively. Among the 54 patients with presence of CTCs at baseline, only 20 showed at least one CTC in the postoperative sample. When we analyzed the changes in CTCs detection between pre- and postoperative samples, we found that 71 patients had consistently negative (n = 51) or consistently positive (n = 20) results in both pre- and postoperative analysis. Tirty-four patients were CTCs positive before surgery but CTCs negative at day 14, while in 21 patients no CTCs were detected before surgery but they tested positive at day14.5. With a median follow-up period of 14 months (range, 1-17 months), HCC recurred in 27 of 126 patients (21.4%). The median time to recurrence was 9 months (range, 3-16 months).6. There was no significant difference in recurrence-free interval and recurrence rate between patients with and without preoperative positivity of CTCs.7. The recurrence-free interval was significantly shorter in patients with postoperative positivity of AFP mRNA than in those without.8. Of 11 variables assessed, 6 were significantly related to recurrence in univariate analysis: tumor number, tumor size, vascular invasion, intrahepatic cancer spread, surgical margin and postoperative CTCs. Finally, 3variables were identified as independent predictors for HCC recurrence by multivariate analysis: postoperative CTCs, vascular invasion and tumor size.ConclusionsCTCs were detectable in patients with early hepatocellular carcinoma. The preoperative positivity rate of CTCs were significantly correlated with tumor size, vascular invasion, intrahepatic cancer spread, and the disease extent as classified by the TNM classification. Postoperative CTCs is an independent predictor for HCC recurrence.CTCs detection by the asialoglycoprotein receptor-based system may help predict patients at risk of postsurgical recurrence and perform them further treatment and close follow-up.