Background: Mesenchymal circulating tumor cells (M-CTCs) may be related to tumor progression, and Ki67 expression is known to be involved in tumor proliferation. The aim of the present study was to explore the relationship between M-CTCs and Ki67 in hepatocellular carcinoma (HCC) and their ability to predict prognosis. Methods: Peripheral blood samples were obtained from 105 HCC patients before radical surgery. CTCs were isolated using CanPatrol enrichment and classified via in situ hybridization. Ki67 expression in HCC tissue was assessed through immunohistochemistry. Potential relationships of M-CTC, Ki67 with clinicopathological factors and prognosis were evaluated. Overall survival (OS) was analyzed using the Kaplan–Meier method and Cox regression. The prognostic efficacy of M-CTC, Ki67 and both together (M-CTC + Ki67) was assessed in terms of time-dependent receiver operating characteristic (ROC) curves and Harrell's concordance index. Results: Of the 105 patients, 50 were positive for M-CTCs (count ≥ 1 per 5 mL) and 39 showed high Ki67 expression (≥ 50% tumor cells were Ki67-positive). The presence of M-CTC was significantly associated with alpha-fetoprotein (AFP) ≥ 400 ng/mL (P = 0.007), tumor size ≥ 5 cm (P = 0.023), multiple tumors (P < 0.001), poorly differentiated tumors (P = 0.003), incomplete tumor capsule (P < 0.001), Barcelona Clinic liver cancer (BCLC) stage B or C (P < 0.001), microvascular invasion (MVI) (P = 0.05) and portal vein tumor thrombosis (PVTT) (P = 0.006). High Ki67 expression correlated with AFP ≥ 400 ng/mL (P = 0.015), tumor size ≥ 5 cm (P = 0.012), incomplete tumor capsule (P < 0.001), MVI (P = 0.001), PVTT (P = 0.003), advanced BCLC stage (P = 0.01), and vessel carcinoma embolus (VCE) (P = 0.001). M-CTC positively correlated with Ki67. Patients positive for M-CTCs had a significantly shorter OS than patients negative for them. Similarly, high Ki67 expression was associated with a significantly lower OS. The high-risk group (positive for M-CTCs and high Ki67 expression) had worse OS than the other groups (P < 0.0001). Uni- and multivariate analyses showed that OS was independently predicted by M-CTC [hazard ratio (HR) 1.115; P < 0.001], Ki67 (HR 1.666; P = 0.046) and the combination of both (HR 2.885; P = 0.008). Based on ROC curves and the concordance index, the combination of M-CTC and Ki67 was superior to either parameter alone for predicting the OS of HCC patients. Conclusions: The presence of M-CTC correlates with high Ki67 expression in HCC patients, and both factors are associated with poor prognosis. Furthermore, the combination of M-CTC and Ki67 is a useful prognostic indicator for predicting OS in patients with HCC after hepatectomy, performing better than either parameter on its own.

Original Article Gut microbiome dysbiosis in patients with hepatitis B virus-related hepatocellular carcinoma after extended hepatectomy liver failure Yu-Chong Peng, Jing-Xuan Xu, Chuan-Fa Zeng, Xin-Hua Zhao, Le-Qun Li, Lu-Nan Qi

Background & Aims: Hepatic resection is the first-line treatment for hepatocellular carcinoma. Whether to perform anatomical (AR) or non-anatomical resection (NAR) remains controversial. This retrospective study compares the outcomes according to the number and type of circulating tumor cells (CTC). Methods: The cohort included 136 patients with hepatocellular carcinoma treated with R0 resection between 2015 and 2017. CanPatrol CTC-enrichment technique was use to enrich and classify CTCs according to epithelial-to-mesenchymal transition phenotype. Results: 91.91% of total patients were CTC-positive, with 91.23% in the AR group and 92.41% in the NAR group. Tumor-free survival (TFS) did not differ significantly between two groups. However, TFS was significantly higher in patients with low CTCs count and mesenchymal- and epithelial/mesenchymal-negative phenotypes. As for the incidence and types of recurrence, high pre-resection CTC count and mesenchymal- and epithelial/mesenchymal-positivity were significantly associated with extrahepatic and multi-intrahepatic recurrence. Higher morbidities for hepatic failure and ascites were observed in patients treated by AR. Conclusion: AR may be more beneficial than NAR only in patients with low CTC count and mesenchymal- and epithelial/mesenchymal-negative phenotypes. For patients with a high CTC count, the balance between operative risk and prognostic benefit is more important than the resection method performed.

Background and Aims: Extrahepatic recurrence (EHR) is one of the major reasons for the poor prognosis of hepatocellular carcinoma (HCC). The present study aimed to develop and assess the performance of predictive models by using a combination of presurgical circulating tumor cell (CTCs) data and clinicopathological features to screen patients at high risk of EHR to achieve precise decision‐making.Patients and Methods: A total of 227 patients with recurrent HCC and preoperative CTC data from January 2014 to August 2019 were enrolled. All patients were randomly assigned to one of two cohorts: development or validation. Two preoperative and postoperative nomogram models for EHR prediction were developed and multi‐dimensionally validated.Results: Patients with EHR had generally lower recurrence‐free survival (p < 0.001), and overall survival (p < 0.001), and significantly higher CTC counts (epithelial CTCs, epithelial/mesenchymal hybrid CTCs, and mesenchymal CTCs count, all p < 0.05) than those without EHR. Univariate and multivariate analyses revealed that EHR was associated with four risk factors in the development cohort: total CTC count (p = 0.014), tumor size (p = 0.028), node number (p = 0.045), and microvascular invasion (p = 0.035). These factors were incorporated into two nomogram models (preoperative and postoperative), which reliably predicted EHR through multidimensional verification (e.g., calibration plot, receiver operating characteristic analysis, decision curve analysis, and clinical impact curve analysis) in the development and validation cohorts, respectively. With threshold of scores of 100.3 and 176.8 before and after surgery respectively, both nomograms were able to stratify patients into two distinct prognostic subgroups (all p < 0.05).Conclusion: The present study proposed two nomogram models integrating presurgical CTC counts and clinicopathological risks and showed relatively good predictive performance of EHR, which may be beneficial to the clinical practice of HCC recurrence. Further multicenter studies are needed to assess its general applicability.