Survivin-positive circulating tumor cells as a marker for metastasis of hepatocellular carcinoma

BACKGROUNDCirculating tumor cells (CTCs) and survivin are indicators for tumor stage and metastasis, as well as epitheliomesenchymal transition, in various cancers, including hepatocellular cancer (HCC). AIMTo explore the potential of survivin-positive CTCs, specifically, as a marker for tumor progression in HCC patients.METHODSWe examined the survivin expression pattern in CTCs obtained from 179 HCC patients, and investigated the in vitro effects of survivin silencing and overexpression on the proliferation and invasion of HCC cells. CTC count and survivin expression in patient samples were examined using RNA in situ hybridization.RESULTSAll 179 patients were positive for CTC markers, and 94.41% of the CTCs were positive for survivin. The CTC and survivin-positive CTC counts were significantly higher in the HCC patients than in the normal controls, and were significantly associated with tumor stage and degree of differentiation. Further, survivin overexpression was found to induce HepG2 cell proliferation, reduce apoptosis, and improve invasive ability.CONCLUSIONSurvivin shows upregulated expression (indicative of anti-apoptotic effects) in HCC. Thus, survivin-positive CTCs are promising as a predictor of HCC prognosis and metastasis, and their accurate measurement may be useful for the management of this cancer.     

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 CD45antibody 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(P s<0.05).The expression rates of either ASGPR or CPS1were 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 CPS1identified more HCC cells than either antibody alone.However,these antibodies did not detect any tumor cells in blood samples spiked with the human breastcancer 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.     

Recent progress in molecular mechanisms of postoperative recurrence and metastasis of hepatocellular carcinoma

Hepatectomy is currently considered the most effective option for treating patients with early and intermediate hepatocellular carcinoma (HCC). Unfortunately, the postoperative prognosis of patients with HCC remains unsatisfactory, predominantly because of high postoperative metastasis and recurrence rates. Therefore, research on the molecular mechanisms of postoperative HCC metastasis and recurrence will help develop effective intervention measures to prevent or delay HCC metastasis and recurrence and to improve the long-term survival of HCC patients. Herein, we review the latest research progress on the molecular mechanisms underlying postoperative HCC metastasis and recurrence to lay a foundation for improving the understanding of HCC metastasis and recurrence and for developing more precise prevention and intervention strategies.     

Therapeutic strategies for post-transplant recurrence of hepatocellular carcinoma

Despite stringent selection criteria, hepatocellular carcinoma recurrence after liver transplantation (LT) still occurs in up to 20% of cases, mostly within the first 2–3 years. No adjuvant treatments to prevent such an occurrence have been developed so far. However, a balanced use of immunosuppression with minimal dose of calcineurin inhibitors and possible addition of mammalian target of rapamycin inhibitors is strongly advisable. Moreover, several pre- and post-transplant predictors of recurrence have been identified and may help determine the frequency and duration of post-transplant follow-up. When recurrence occurs, the outcomes are poor with a median survival of 12 mo according to most retrospective studies. The factor that most impacts survival after recurrence is timing (within 1–2 years from LT according to different authors). Several therapeutic options may be chosen in case of recurrence, according to timing and disease presentation. Surgical treatment seems to provide a survival benefit, especially in case of late recurrence, while the benefit of locoregional treatments has been suggested only in small retrospective studies. When systemic treatment is indicated, sorafenib has been proved safe and effective, while only few data are available for lenvatinib and regorafenib in second line. The use of immune checkpoint inhibitors is controversial in this setting, given the safety warnings for the risk of acute rejection.     

Monitoring disease progression and treatment efficacy with circulating tumor cells in esophageal squamous cell carcinoma: A case report

This study investigated whether changes in circulating tumor cell (CTC) numbers reflect tumor progression and treatment efficacy in esophageal squamous cell carcinoma (ESCC). A 47-year-old male patient with ESCC is presented in this case study. The patient was evaluated for a series of serum tumor markers and subjected to radiological examinations before and after surgery and during follow-up over the course of five years. In addition, the CTCs in 7.5 mL of peripheral blood were enriched by magnetic-activated cell sorting negative selection and identified by immunofluorescence staining. Serum tumor markers remained within normal ranges and were discordant with imaging scans during the follow-up. Initially, one CTC was detected in the peripheral blood sample, and 14 were observed seven days after the operation. After 12 wk, subcutaneous metastases and bone metastases occurred, and the number of CTCs increased to 84. After 48 wk, lung metastases were noted, and the CTC level was 21. At 104 wk, the number of CTCs was 14, and disease recurrence was detected by positron emission tomography-computed tomography. The CTC counts were in accord with the imaging studies at several time points. The additional information provided by CTC enumeration could thus facilitate monitoring of disease status and treatment efficacy and provide support for treatment decisions.     

小肝细胞癌术后早期复发的危险因素分析

目的 探讨小肝细胞癌（sHCC）患者术后早期复发的危险因素。方法 收集2004至2006年上海东方肝胆外科医院手术切除肿瘤的sHCC患者75例。选取20项临床和病理学参数,以2年为早期复发的时限,应用COX风险比例模型进行单因素和多因素分析,筛选影响早期复发的独立危险因素。结果 本组75例sHCC患者经手术切除肿瘤后,1～5 a累积肿瘤复发率分别为10.7%、46.7%、76.0%、86.7%和92.0%;经COX风险比例模型分析,发现术前AFP＞400 μg/L（HR=2.477,95% CI=1.100～5.576,P=0.029）和肿瘤分布超过半肝（HR=5.801,95% CI=1.831～18.379,P=0.003）是sHCC患者术后早期复发的独立危险因素。结论 对于术前AFP＞400 μg/L和肿瘤分布超过半肝的sHCC患者,应加强术后随访,及早发现复发肿瘤并给予积极的治疗,以改善患者预后。     

肝癌细胞裂解物致敏的树突状细胞瘤苗预防肝癌术后转移复发

目的探讨人自体肝癌细胞裂解物致敏的树突状细胞(D C)瘤苗预防肝癌术后转移复发的价值。方法从肝癌患者外周血单核细胞中诱导D C,用重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF) 和重组人白细胞介素-4(rhIL-4)刺激活化,经自体肝癌细胞裂解物致敏D C制备瘤苗。将3 0例肝癌术后患者随机分为D C瘤苗治疗组1 5例,对照组1 5例。对两组病例治疗前后免疫功能、临床疗效、肿瘤转移复发率及生存率进行观察比较。结果DC瘤苗治疗后外周血CD 3 、CD4 /CD 8 及自然杀伤细胞比率较治疗前明显升高(P<0.0 5),且明显高于对照组治疗后的相应水平(P<0.05)。DC瘤苗治疗后血清IL-1 0水平明显下降(P<0.0 5)。随访18个月,DC瘤苗治疗组的转移复发率为13.3 3%,明显低于对照组53.33%(P <0.05);而治疗组的生存率为93.33%,明显高于对照组60.00%(P<0.05)。结论肝癌术后行自体肝癌细胞裂解物致敏的DC瘤苗治疗,可改善患者的免疫功能,降低肿瘤转移复发率,提高患者术后生存率,为肝癌的免疫治疗开辟一条新途径。     

Tumor vaccine against recurrence of hepatocellular carcinoma

AIM: To investigate the effects of autologous tumor vaccine on recurrence of hepatocellular carcinoma (HCC). METHODS: Sixty patients with HCC who had undergone curative resection, were randomly divided into HCC vaccine group and control group. Three vaccinations at 2-wk intervals were performed after curative hepatic resection. Delayed-type- hypersensitivity (DTH) test was performed before and after vaccination. Primary endpoints were the time of recurrence. RESULTS: Four patients in control group and 6 patients in HCC vaccine group were withdrawn from the study. The vaccine containing human autologous HCC fragments showed no essential adverse effect in a phase II clinical trial and 17 of 24 patients developed a DTH response against the fragments. Three of 17 DTH-positive response patients and 5 of 7 DTH- negative response patients had recurrences after curative resection. After the operation, 1-, 2- and 3-year recurrence rates of HCC vaccine group were 16.7%, 29.2% and 33.3%, respectively. But, 1-, 2- and 3-year recurrence rates of the control group were 30.8%, 53.8% and 61.5%, respectively. The time before the first recurrence in the vaccinated patients was significantly longer than that in the control patients (P<0.05). CONCLUSION: Autologous tumor vaccine is of promise in decreasing recurrence of human HCC.     

Local recurrence of hepatocellular carcinoma after radiofrequency ablation

A 51-year-old Chinese male with a 20-year history of hepatitis B was diagnosed with hepatocellular carcinoma in the right anterior portion of the liver, sized 3.5 cm × 3.2 cm, and was treated with radiofrequency ablation (RFA) on December 18, 2001. The patient did not receive antiviral therapy for hepatitis B virus after RFA. The treated lesion reduced gradually and reached its minimum size of 1.7 cm × 1.5 cm seven years later on November 18, 2008. However computed tomography findings revealed that a recurr...     

循环肿瘤细胞及游离DNA甲基化在肝细胞癌患者中的研究进展

随着对肿瘤认识的不断深入,人们发现在原发肿瘤形成和生长的早期阶段,肿瘤细胞即可以脱离原发肿瘤组织释放到外周血形成循环肿瘤细胞,同样在肿瘤形成的早期阶段就会出现肿瘤细胞的坏死和凋亡,这些凋亡或坏死的肿瘤细胞也可以释放其DNA入外周血形成血浆或血清游离的DNA,因此对肿瘤患者循环肿瘤细胞及游离DNA的分析被认为是实时的"液相活检",肿瘤患者中的循环肿瘤细胞具有非常强的异质性,我们可以根据其物理和生物学性质采用不同的技术对其进行富集和检测;可以借助现代分子生物学手段对循环游离DNA进行提取,并对其遗传学和表观遗传学的异常改变进行分析,这可为肿瘤的早期诊断、疗效评估、复发监测及预后判断提供重要的信息.本文结合本课题组的研究重点,就循环肿瘤细胞及游离DNA及其甲基化在肝细胞癌患者的研究进行综述.     

Prognostic value of postoperative complication for early recurrence after curative resection of hepatocellular carcinoma

Background: Postoperative complications may adversely affect oncological outcomes. The aim of this study was to evaluate the impact of postoperative complications on early-phase recurrence after curative resection for hepatocellular carcinoma(HCC).Methods: We included 145 HCC patients who underwent initial and curative resection between January2004 and December 2013. Postoperative complications of grade III or higher based on Clavien–Dindo classification were defined as clinically relevant postoperative complications. Recurrence within two years after hepatectomy was defined as early-phase recurrence.Results: Thirty-eight patients(26%) developed postoperative complications. The only predictive factor for postoperative complication was longer operative duration(P = 0.037). The disease-specific survival rate of patients with complication was lower than that of patients without complications(P = 0.015). Earlyphase recurrence was observed in 20/38(53%) patients who suffered postoperative complications and36/107(34%) patients with no complications, which was statistically significant(P = 0.039). Multivariate analysis identified four factors contributing to early-phase recurrence: high serum AFP level(P = 0.042),multiple tumors(P 0.001), poor differentiation(P = 0.036) and presence of postoperative complication(P = 0.039).Conclusions: Postoperative complication is an independent prognostic factor for early-phase recurrence after curative resection of HCC. Close observation of patients with postoperative complications may be a necessary treatment strategy for HCC.     

Inhibitory effect of dimeric β peptide on the recurrence and metastasis of hepatocellular carcinoma in vitro and in mice

AIM: To block the adhesion of tumor cells to the extracellular matrix, and prevent tumor metastasis and recurfence, the dimer of the β peptide (DLYYLMDLSYSMKGGDLYYLMDLSYSMK, β2) was designed and synthesized and its anti-adhesion and anti-invasion effects on hepatocellular carcinoma cells were assessed. Additionally,its influence on the metastasis and recurrence of mouse hepatocellular carcinoma was measured.METHODS: The anti-adhesion effect of β2 on the highly metastatic hepatocellular carcinoma cell line HCCLM6 cells and fibronectin (FN) was assayed by the HTT assay. The inhibition of invasion of HCCLM6 cells by β2 was observed using a Transwell (modified Boyden chamber)and matrigel. Using the hepatocellular carcinoma metastasis model and LCI-D20 nude mice, the influence of β2 on the metastasis and recurrence of hepatocellular carcinoma after early resection was investigated.RESULTS: HCCLM6 cells co-incubated with 100 μmol/L,50 μmol/L, 20 μmol/L or 10 μmol/L β2 for 3 h showed an obvious decrease in adhesion to FN. The adhesion inhibition ratios were 11.8%, 21.7%, 29.6% and 48.7%,respectively. Additionally, HCCLM6 cells cultured with 100 μmol/L β2 had a dramatic decrease in cell invasion.β2 was also observed to inhibit the incisal edge recurfence and the distant metastasis of nude mice hepatocellular carcinoma after early resection (P ＜ 0.05).CONCLUSION: The β2 peptide can specifically block the adhesion and invasion of HCCLM6 cells, and can inhibit HCC recurrence and metastasis of LCI-D20 model posthepatectomy in vivo. Thus, β2 should be further studied as a new anti-tumor drug.     

Preoperative evaluation and prediction of clinical scores for hepatocellular carcinoma microvascular invasion: a single-center retrospective analysis

IntroductionMicrovascular invasion (MVI) of is generally considered to be an important prognostic factor for hepatocellular carcinoma (HCC) after operation, An accurate prediction of MVI before operation is helpful for clinical decision-making before operation.Material and methodsA retrospective analysis of 227 cases of hepatocellular carcinoma patients after hepatectomy has been confirmed the pathological result whether there was MVI, and has been determined the independent risk factors of MVI. Based on these independent risk factors, we constructed a clinical scoring risk model for predicting MVI.ResultsAmong the 227 patients with HCC, 74 (34.6%) were MVI positive. Using receiver operating characteristic (ROC) curve and logistic regression model, we found that alpha-fetoprotein(AFP)≥158 ng/mL(odds ratio[OR] = 4.152,95% confidence interval [95%CI]:1.602∼10.760,p = 0.003), Des-γ-carboxy prothrombin (DCP)≥178mAU/mL(OR = 9.730,95%CI:3.392∼27.910,p < 0.001), circulating tumor cells (CTCs)≥3/3.2 ml(OR = 7.747,95%CI:3.019∼19.881,P < 0.001), maximum tumor diameter≥59 mm(OR = 3.467,95%CI:1.368∼8.669,p = 0.008) and tumor margin unsmoothness(OR = 0.235,95%CI:0.096∼0.573,p = 0.001) were independent risk factors for MVI, they predicted that the area under the curve of MVI was 0.752, 0.777, 0.857, 0.743 and 0.333, respectively. Based on these five independent risk factors, we constructed a clinical scoring risk model for predicting MVI. The model predicts that the area under the curve of MVI is 0.922, and its prevalence rate from 0 to 5 are 3.1%(1/32), 5.3%(4/76), 12.2%(5/41), 66.7%(20/30), 87.9%(29/33), 100%(15/15), respectively (P < 0.001).ConclusionBased on AFP, DCP, CTC, maximum tumor diameter and tumor margin unsmoothness, we constructed a model to predict the risk of MVI clinical score, so as to make a more accurate individualized treatment plan before operation, which has important clinical significance and application prospect to improve the curative effect of HCC.     

Role of microRNA-regulated cancer stem cells in recurrent hepatocellular carcinoma

Among the most common cancers, hepatocellular carcinoma (HCC) has a high rate of tumor recurrence, tumor dormancy, and drug resistance after initial successful chemotherapy or radiotherapy. A small subset of cancer cells, cancer stem cells (CSCs), exhibit stem cell characteristics and are present in various cancers, including HCC. The dysregulation of microRNAs (miRNAs) often accompanies the occurrence and development of HCC. miRNAs can influence tumorigenesis, progression, recurrence, and drug resistance by regulating CSCs properties, which supports their clinical utility in managing and treating HCC. This review summarizes the regulatory effects of miRNAs on CSCs in HCC with a special focus on their impact on HCC recurrence.     

介入治疗对原发性肝癌术后早期肝内复发转移的疗效

目的分析介入疗法应用于原发性肝癌术后早期肝内复发转移治疗中的疗效及安全性。方法选取凉山州第二人民医院2010年4月-2011年4月收治的78例原发性肝癌术后早期肝内复发转移患者为研究对象,随机分为观察组和对照组,每组39例。观察组采用5-氟尿嘧啶联合奥沙利铂进行介入治疗,对照组采用5-氟尿嘧啶联合奥沙利铂进行化疗治疗。对两组患者3~5个疗程的临床疗效、不良反应、治疗依从性及随访3个月、6个月、9个月及12个月的生存率进行统计学分析。结果观察组3~5个疗程的临床疗效分别为56.4%、71.8%、79.5%,均高于对照组(P0.05);在各个疗程的不良反应、治疗依从性比较中,观察组均优于对照组(P0.05);观察组在随访3个月、6个月、9个月及12个月的生存率较对照组高(P0.05)。结论介入疗法应用于原发性肝癌术后早期肝内复发转移治疗能够提高疗效,降低并发症,延长生存时间,从而提高治疗的依从性,具有重要的临床价值。     

Isolated metastasis to the gallbladder from hepatocellular carcinoma

Aim: Metastasis to gallbladder (GB) from hepatocellular carcinoma (HCC) is rare, and it is difficult to determine indications for surgery. We report eight cases of synchronous isolated GB metastasis, and analyze their features retrospectively. Methods: Among 439 HCC patients who underwent hepatectomy from 1998 to 2008 at our institution, 393 (89.5%) underwent concurrent cholecystectomy. Results: Among them, eight (1.8%) had GB metastasis without other distant metastases. None of these cases showed evidence of direct invasion. All cases had advanced portal vein thrombus (PVTT) and their main tumor located near the GB bed. Five cases had apparent tumor mass in the GB wall, and the other three cases had only tumor thrombus in the GB veins. Six cases were treated postoperatively with local infusion therapy with interferon, and three of them showed long‐term survival. Conclusion: Our eight cases of GB metastasis from HCC were closely related to PVTT. Surgical resection and multimodal treatment would be necessary for long‐term survival in cases with isolated GB metastasis.     

肝癌根治术后肝内复发合并肝外转移积极手术治疗10例

目的:探讨对严格选择的肝癌根治术后肝内复发并肝外转移患者采取积极外科治疗策略的临床价值.方法:回顾性分析10例肝癌根治术后肝内复发伴肝外孤立转移灶病例的外科治疗情况.并与行保守治疗的肝癌根治术后肝内复发伴肝外转移的48例患者的生存情况进行比较.结果:Kaplan-Meier生存分析显示积极外科治疗组复发后中位生存时间为45.0mo±5.1mo,保守治疗组复发后中位生存时间仅为12.0mo±2.1mo.Log-rank检验对两组生存时间比较,发现积极手术治疗组生存时间显著长于保守治疗组(P<0.01)结论:对于严格选择的病例采取肝外孤立转移灶切除联合肝内复发灶切除或局部治疗的积极外科治疗策略能够显著提高患者生存时间.     

Inhibitory effect of dimeric β peptide on the recurrence and metastasis of hepatocellular carcinoma in vitro and in mice

AIM：To block the adhesion of tumor cells to the extracellular matrix, and prevent tumor metastasis and recurrence, the dimer of the β peptide （DLYYLMDLSYSMKG- GDLYYLMDLSYSMK, β2） was designed and synthesized and its anti-adhesion and anti-invasion effects on hepa- tocellular carcinoma cells were assessed. Additionally, its influence on the metastasis and recurrence of mouse hepatocellular carcinoma was measured.
METHODS：The anti-adhesion effect of β2 on the highly metastatic hepatocellular carcinoma cell line HCCLM6 cells and fibronectin （FN） was assayed by the MTT as- say. The inhibition of invasion of HCCLM6 cells by β2 was observed using a Transwell （modified Boyden chamber） and matrigel. Using the hepatocellular carcinoma metas- tasis model and LCI-D20 nude mice, the influence of β2 on the metastasis and recurrence of hepatocellular carci- noma after early resection was investigated.
RESULTS：HCCLM6 cells co-incubated with 100 mmol/L, 50 mmol/L, 20 mmol/L or 10 mmol/L β2 for 3 h showed an obvious decrease in adhesion to FN. The adhesion inhibition ratios were 11.8%, 21.7%, 29.6% and 48.7%, respectively. Additionally, HCCLM6 cells cultured with 100 mmol/L β2 had a dramatic decrease in cell invasion. β2 was also observed to inhibit the incisal edge recur- rence and the distant metastasis of nude mice hepato- cellular carcinoma after early resection （P 〈 0.05）.
CONCLUSION：The β2 peptide can specifically block the adhesion and invasion of HCCLM6 cells, and can inhibit HCC recurrence and metastasis of LCI-D20 model pos-thepatectomy in vivo. Thus, β2 should be further studied as a new anti-tumor drug.     

Surgical resection of a solitary para-aortic lymph node metastasis from hepatocellular carcinoma

Lymph node (LN) metastases from hepatocellular carcinoma (HCC) are considered uncommon. We describe the surgical resection of a solitary para-aortic LN metastasis from HCC. A 65-year-old Japanese man with B-type liver cirrhosis was admitted for the evaluation of a liver tumor. He had already undergone radiofrequency ablation, transcatheter arterial chemoembolization, and percutaneous ethanol injection therapy for HCC. Despite treatment, viable regions remained in segments 4 and 8. We performed a right paramedian sectionectomy with partial resection of the left paramedian section of the liver. Six months later, serum concentrations of alpha-fetoprotein (189 ng/mL) and PIVKA-2 (507 mAU/mL) increased. Enhanced computed tomography of the abdomen revealed a tumor (20 mm in diameter) on the right side of the abdominal aorta. Fluorine-18 fluorodeoxyglucose positron emission tomography revealed an increased standard uptake value. There was no evidence of recurrence in other regions. Esophagogastroduodenoscopy and colonoscopy revealed no malignant tumor in the gastrointestinal tract. Para-aortic LN metastasis from HCC was thus diagnosed. We performed lymphadenectomy. Histopathological examination revealed that the tumor was largely necrotic, with poorly differentiated HCC on its surface, which confirmed the suspected diagnosis. After 6 mo tumor marker levels were normal, with no evidence of recurrence. Our experience suggests that a solitary para-aortic LN metastasis from HCC can be treated surgically.