Circulating tumor cells participate in the formation of microvascular invasion and impact on clinical outcomes in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide. Although the treatment strategies have been improved in recent years, the long-term prognosis of HCC is far from satisfactory mainly due to high postoperative recurrence and metastasis rate. Vascular tumor thrombus, including microvascular invasion (MVI) and portal vein tumor thrombus (PVTT), affects the outcome of hepatectomy and liver transplantation. If vascular invasion could be found preoperatively, especially the risk of MVI, more reasonable surgical selection will be chosen to reduce the risk of postoperative recurrence and metastasis. However, there is a lack of reliable prediction methods, and the formation mechanism of MVI/PVTT is still unclear. At present, there is no study to explore the possibility of tumor thrombus formation from a single circulating tumor cell (CTC) of HCC, nor any related study to describe the possible leading role and molecular mechanism of HCC CTCs as an important component of MVI/PVTT. In this study, we review the current understanding of MVI and possible mechanisms, discuss the function of CTCs in the formation of MVI and interaction with immune cells in the circulation. In conclusion, we discuss implications for potential therapeutic targets and the prospect of clinical treatment of HCC.

Thymosin ß4, a potential marker of malignancy and prognosis in hepatocellular carcinoma.

BACKGROUND: The lack of effective early diagnostic markers is an obstacle in clinical diagnosis and treatment of hepatocellular carcinoma (HCC). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an increasing popular approach for identification of clinically relevant parameters including biomarkers. PATIENTS AND METHODS: 540 subjects, including 274 HCC, 119 liver cirrhosis, 89 hepatitis, and 58 healthy volunteers were enrolled. MALDI-TOF MS was used to select potential novel biomarkers from serum of HCC patients. Its clinical application was evaluated by experiments and clinical data analysis. RESULTS: We identified Thymosin ß4 (Tß4) in serum by MALDI-TOF MS. The expression of Tß4 was detected up-regulating in HCC cells and tissues which enhanced motility of HCC cells. More important, the level of serum Tß4 was significantly elevated in HCC patients. The AUROC showed the optimum diagnostic cut-off was 1063.6 ng/mL, ROC and 95% CI of Tß4 (0.908; 0.880-0.935) were larger than that of serum AFP (0.712; 0.662-0.762; p < 0.001). The sensitivity (91.3% vs 83.1%) and specificity (81.2% vs 20.3%) of serum Tß4 were higher than alpha-fetoprotein (AFP). In AFP-negative HCC, the sensitivity could reach to 80.5%. ROC analysis showed serum Tß4 had a better performance compared with AFP in distinguishing early-stage and small HCC. Tß4 is correlated with TNM stage (p = 0.016) and vascular invasion (p = 0.005). Survival analysis indicated the survival time of Tß4 positive patients was shorter (p < 0.001). Cox analysis suggested Tß4 could be an independent factor for HCC prognosis. CONCLUSION: Tß4 may serve as a novel biomarker for HCC diagnosis and prognosis.

[Effects of saline-water furrow irrigation on the stability of soil water-stable aggregates in cotton field]. / 咸水沟灌对棉田土壤水稳性团聚体稳定性的影响.

It is of great importance to explore the effects of saline-water furrow irrigation on soil water-stable aggregates for safe and efficient utilization of saline water resources. We conducted a long-term cotton experiment with six levels of saline-water furrow irrigation (1, 2, 4, 6, 8, 10 g·L-1) since 2006 and analyzed the variations of soil salinity and water-stable aggregates in the 10th and 15th years under saline irrigation. The results showed that soil salinity in the 0-30 cm layer at the ditch increased with increasing salinity level of irrigation water. There were significant differences between the 6, 8, 10 g·L-1 and 1 g·L-1 treatments. Soil salinity in each treatment increased gradually with increasing soil depth. Saline-water furrow irrigation tended to reduce the stability of soil water-stable aggregates. When the salinity level of the irrigation water was ≥6 g·L-1, the mass fraction of macroaggregates (>0.25 mm), the mean weight diameter and geometric mean diameter of water-stable aggregates significantly decreased. In contrast, the fractal dimension and mean weight specific surface area increased significantly. The stability of soil water-stable aggregates decreased with soil depth in all treatments. Under the condition of saline-water furrow irrigation for several years, there was no accumulation of soil salinity and instability of water-stable aggregates in the 0-30 cm soil layer at the ditch with each passing year. With the irrigation scheduling of this study, saline-water furrow irrigation with salinity ≤4 g·L-1 did not affect soil salinity and water-stable aggregate stability of cotton field in this area.

Anti-tumor activity and mechanism of oligoclonal hepatocellular carcinoma tumor-infiltrating lymphocytes in vivo and in vitro.

Objective: To explore a method for culturing hepatocellular carcinoma and tumor-infiltrating lymphocytes (HCC-TIL) and investigate the mechanism of TIL in killing tumors. Methods: The distribution of regulatory T cells (Treg) in HCC was detected by immunohistochemistry. Conventional TIL and oligoclonal TIL were isolated by the traditional method of enzyme digestion combined with mechanical treatment for whole HCC and micro HCC tissue block culturing method. MTT was used to compare the killing activity of TIL. Flow cytometry was used to analyze the proportion of CD8+ T cells and Treg cells in TIL. Tumor-bearing mice were established, and TIL adoptive immunotherapy was performed. Results: Treg cells were mainly distributed in the stroma of HCC. In vitro experiments showed oligoclonal TIL had higher cytotoxicity to tumor cells which negatively correlated with the proportion of Treg cells. In vivo experiments showed oligoclonal TIL had a higher anti-tumor effect. IFN-γ in peripheral blood and the positive rate of intratumoral lymphocytic infiltration in oligoclonal TIL group were both higher. TGF-ß and IL-10 in peripheral blood and the positive rate of intratumoral FoxP3 and IL-17 were both lower than those in conventional TIL group. Conclusion: The oligoclonal TIL culture method could obtain TIL with higher purity, and cytotoxicity to tumor cells was associated with Treg cells. The oligoclonal TIL had cytotoxicity to autologous HCC cells and significant inhibitory effect on the growth of transplanted tumors. The mechanism might be associated with the inhibition of Treg cells proliferation, increase of IFN-γ secretion, and decrease of TGF-ß, IL-10, and IL-17 secretion.

Intrahepatic splenosis mimics hepatocellular carcinoma in a patient with chronic hepatitis B: A case report and literature review.

BACKGROUND: Splenosis is a benign and relatively uncommon condition caused by trauma or splenectomy or other procedures involving splenic tissue. It is usually asymptomatic, and often diagnosed accidentally, especially misdiagnosed as malignant tumor. METHODS: A 54-year-old man with prior history of chronic hepatitis B virus infection and underwent splenectomy for traumatic splenic rupture following a traffic accident 23 years previously was admitted to our hospital and found a hepatic mass in the right upper quadrant during an imaging examination. The diagnosis of his was not clear and finally he agreed to receive a surgical treatment. RESULTS: During the operation, we found a mass in the right posterior lobe of the liver and a hard nodule on the right side of the diaphragm, both were completely resected, and postoperative histopathologic examination revealed that all excised tissues were proved to have histological structure typical for the spleen. CONCLUSIONS: The occurrence of intrahepatic splenosis is rare with only few cases previously reported in the literature. It is a benign disease and sometimes difficult to distinguish from diseases of the liver. The need for positive surgical resection of splenosis is still controversial.

Randomized Controlled Trial of Hepatic Portal Reocclusion as a New Option for Detecting Bile Leakage during Hepatic Resection.

BACKGROUND/AIMS: To explore the possibility and feasibility of hepatic portal reocclusion for detecting bile leakage during hepatectomy. METHODS: Data were prospectively collected from 200 patients who underwent hepatectomy alone for removal of various benign or malignant tumors between March 2014 and November 2014. The surgical procedure used a conventional method for all patients, and one additional step (hepatic portal reocclusion) was included in group B. The postoperative outcomes of the patients in group A (subjected to the traditional procedure) and group B (subjected to hepatic portal reocclusion) were compared during the same period, and the incidence rates of postoperative bile leakage and other complications in the 2 groups were also analyzed. RESULTS: The incidence of postoperative bile leakage in group B was significantly lower than that in group A (1.0 vs. 9.2%, p = 0.009), although no significant differences in postoperative indicators of liver dysfunction and other complications were observed between the 2 groups (p > 0.05). CONCLUSIONS: Hepatic portal reocclusion effectively reduced the incidence of bile leakage compared to the traditional procedure, without significantly affecting liver function. Therefore, this method might be an alternative to other tests for bile leakage.

Improved method increases sensitivity for circulating hepatocellular carcinoma cells.

AIM: To improve an asialoglycoprotein receptor (ASGPR)-based enrichment method for detection of circulating tumor cells (CTCs) of hepatocellular carcinoma (HCC). METHODS: Peripheral blood samples were collected from healthy subjects, patients with HCC or various other cancers, and patients with hepatic lesions or hepatitis. CTCs were enriched from whole blood by extracting CD45-expressing leukocytes with monoclonal antibody coated-beads following density gradient centrifugation. The remaining cells were cytocentrifuged on polylysine-coated slides. Isolated cells were treated by triple immunofluorescence staining with CD45 antibody and a combination of antibodies against ASGPR and carbamoyl phosphate synthetase 1 (CPS1), used as liver-specific markers, and costained with DAPI. The cell slide was imaged and stained tumor cells that met preset criteria were counted. Recovery, sensitivity and specificity of the detection methods were determined and compared by spiking experiments with various types of cultured human tumor cell lines. Expression of ASGPR and CPS1 in cultured tumor cells and tumor tissue specimens was analyzed by flow cytometry and triple immunofluorescence staining, respectively. RESULTS: CD45 depletion of leukocytes resulted in a significantly greater recovery of multiple amounts of spiked HCC cells than the ASGPR(+) selection (Ps < 0.05). The expression rates of either ASGPR or CPS1 were different in various liver cancer cell lines, ranging between 18% and 99% for ASGPR and between 9% and 98% for CPS1. In both human HCC tissues and liver cancer cell lines, there were a few HCC cells that did not stain positive for ASGPR or CPS1. The mixture of monoclonal antibodies against ASGPR and CPS1 identified more HCC cells than either antibody alone. However, these antibodies did not detect any tumor cells in blood samples spiked with the human breast cancer cell line MCF-7 and the human renal cancer cell line A498. ASGPR(+) or/and CPS1(+) CTCs were detected in 29/32 (91%) patients with HCC, but not in patients with any other kind of cancer or any of the other test subjects. Furthermore, the improved method detected a higher CTC count in all patients examined than did the previous method (P = 0.001), and consistently achieved 12%-21% higher sensitivity of CTC detection in all seven HCC patients with more than 40 CTCs. CONCLUSION: Negative depletion enrichment combined with identification using a mixture of antibodies against ASGPR and CPS1 improves sensitivity and specificity for detecting circulating HCC cells.