Circulating Tumor-Cell-Associated White Blood Cell Clusters in Peripheral Blood Indicate Poor Prognosis in Patients With Hepatocellular Carcinoma

Abstract

Aim: Circulating tumor cells (CTC) are a precursor to metastasis in several types of cancer and are occasionally found in the bloodstream in association with immune cells, such as white blood cells (WBCs). CTC-associated WBC (CTC-WBC) clusters can promote CTC appreciation and metastasis, suggesting that patients with CTC-WBC clusters found in the peripheral blood may have a worse prognosis. However, it is unclear whether CTC-WBC clusters are present in the peripheral blood of patients with hepatocellular carcinoma (HCC) and suggest a poor prognosis for HCC. Methods: We collected peripheral blood from 214 patients with HCC from January 2014 to December 2016. CanPatrol™ CTC analysis technology was used to isolate and count CTCs and CTC-WBC clusters in the patients' peripheral blood. Chi-squared analysis was used to calculate the correlation between the CTC-WBC clusters and clinicopathological characteristics. Kaplan-Meier survival analysis and Cox regression analysis were used to assess patient prognosis. Results: We used CanPatrol™ CTC analysis technology to count different types of CTCs and CTC-WBC clusters. The results showed that CTC-WBC clusters and tumor size (P = 0.001), tumor number (P = 0.005), portal vein tumor thrombus (P = 0.026), BCLC stage (P < 0.001), AFP level (P = 0.002), and total number of CTCs (P < 0.001) were statistically related. Cox regression analysis revealed that CTC-WBC clusters are an independent prognostic indicator of DFS (HR = 1.951, 95%CI:1.348-2.824, P < 0.001) and OS (HR = 3.026, 95%CI:1.906-4.802, P < 0.001) in HCC patients. Using Kaplan-Meier analysis, we found that positive CTC-WBC cluster patients had significantly shorter DFS and OS than patients with negative CTC-WBC (P < 0.001 and P < 0.001, respectively). Conclusions: CTC-WBC clusters in the peripheral blood are an independent predictor of DFS and OS, and their presence indicates poor prognosis in patients with HCC.

Keywords: CTC-WBC cluster; Kaplan-Meier plot; circulating tumor cells; hepatocellular carcinoma; prognosis.

Figure 1

Flowchart of the diagnosis of patients enrolled in the study. *Death from non-HCC causes included 2 cases of cerebral hemorrhage, 2 cases of myocardial infarction, and 1 case of traffic accident.

Figure 2

Examples of CTCs, WBCs, and CTC-WBC clusters under automated fluorescent microscope imaging. Epithelial CTCs stained with EpCAM or CK8/18/19 (red). Mesenchymal CTCs stained with Vimentin or Twist (green). WBCs stained with CD45 (white). Nuclei stained with DAPI (blue). DAPI 40,6-diamidino-2-phenylindole.

Figure 3

Survival analysis of hepatocellular carcinoma (HCC) patients. CTC-WBC cluster-positive status is associated with a poorer outcome in patients with HCC. Kaplan-Meier analysis revealed significant differences in disease-free survival (P < 0.001) (A) and overall survival (P < 0.001) (B) between preoperative CTC-WBC cluster-positive and -negative patients in an HCC cohort. Survival analysis of HCC patients using the Kaplan-Meier method. P-values were determined using the log-rank test. DFS, disease-free survival; OS, overall survival. Extended Data Figure 1 ROC curve showing the effectiveness of the use of total CTCs and CTC-WBC clusters in the diagnosis of HCC.