mRNA Expression of the Cancer-testis Antigens SSX1 and SSX4 in Human Hepatocellular Carcinomas

Objective: To detect the mRNA expression of the cancer-testis antigens (CT) SSX1 and SSX4 gene in human hepatocellular carcinomas (HCCs) and to investigate the specificity of their expression in HCCs. Methods: The mRNA expression of SSX1 and SSX4 in HCC tissues and the corresponding nearby liver tissues in 35 cases was detected by using RT-PCR; Six positive RT-PCR products were randomly selected and sequenced. Results: In all 35 HCC tissues, SSX1 in 27 cases (81%) and SSX4 in 23 cases (73%) were detected, and their expression was negative in the liver tissues nearby HCC and the non-tumor liver tissues (12 cirrhotic tissues and 15 normal tissues). In all 6 cases selected randomly, the results of DNA sequencing were identical with the cDNA sequence of SSX1 and SSX4 genes. The SSX1, SSX4 mRNA expression was not significantly correlated with age, sex, the tumor size, the level of tumor differentiation, the serum AFP level and the infection rate of HBV and HCV respectively (P>0.05). Conclusion: The SSX1, SSX4 mRNA expression was greatly specific in HCCs, which would not only provide the ideal target molecular sites for HCC tumor vaccines, but also establish the potential value of the polyvalent tumor-antigen vaccines for HCC therapy and its theory bases.

Reduced expression of E-cadherin/catenin complex in hepatocellular carcinomas

AIM： TO examine the immunoreactivity of E-cadherin and four subtypes of catenin family in human hepatocellular carcinomas （HCCs） and to investigate the correlation between expression of E-cadherin/ catenin complex and clinicopathologic parameters of HCC patients. METHODS： An immunohistochemical study for E-cadherin and catenins was performed on 97 formalin-fixed, paraffin-embedded specimens of HCC. RESULTS： Reduced expression of E-cadherin, ^-, 13-, y-catenin and p120 was observed in 69%, 76%, 63%, 71% and 73%, respectively. Both expressions of E-cadherin and catenin components were significantly correlated with tumor grade （P = 0.000）. It showed significant difference between expression of catenin members and tumor stage （P = 0.003, P = 0.017, P = 0.007 and P = 0.000, respectively）. The reduced expression of E-cadherin in HCCs was significantly correlated with intrahepatic metastasis （IM） and capsular invasion （P = 0.008, P = 0.03, respectively）. A close correlation was also observed between the expression of catenins and the tumor size （P = 0.002, P = 0.034, P = 0.016 and P = 0.000, respectively）. In addition, the expression of each catenin was found correlated with IM （P = 0.012, P = 0.049, P =0.026 and P = 0.014, respectively）. No statistically significant difference was observed between the expression level of E-cadherin/catenin complex and lymph node permission, vascular invasion and satellite nodules. Interestingly, only expression of p120 showed correlation with AFP value （P = 0.035）. The expression of E-cadherin was consistent with α-, β-, γ-catenin and p120 expression （P = 0.000）. Finally, the abnormal expression of E-cadherin/catenin complex was significantly associated with patients＇ survival （P = 0.0253, P = 0.0052, P = 0.003, P = 0.0105 and P = 0.0016, respectively）. Nevertheless, no component of E-cadherin/catenin complex was the independent prognostic factor of HCC patients. CONCLUSION： Down-regulated expressions of E-cadherin, catenins and p120 occur frequently in HCCs and contribute to the progression and development of tumor. It may be more exact and valuable to detect the co-expression of E-cadherin/catenin complex than to explore one of them in predicting tumor invasion, metastasis and patient＇s survival.

Molecular profiling of hepatocellular carcinomas by cDNA microarray

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. Conventional diagnosis and treatment of this malignancy have been dismal and should be complemented by novel tools. The development and progress of HCC are believed to be caused by the accumulation of genetic changes resulting in altered expression of thousands of cancer-related genes, which can be measured by globe genetic analysis. Gene expression profiling of HCC has been employed to elucidate hepatocarcinogenesis and disclose molecular mechanisms underlying complex clinical features.Identifying phenotype-associated genes/profiles has impacts on current diagnosis and management strategy of HCC. In spite of some pitfalls of this technology and challenges in improving the research process, scrutinous validation of profiling data of HCC combined with other approaches will eventually benefit the patients.

EFFECT OF MATRINE ON EXPRESSION OF HCCR1 AND HCCR2 PROTEINS IN CULTURAL HUMAN HEPATOCELLULAR CARCINOMAS CELLS

Objective： To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas （HCC） cells at the level of gene and protein. Methods： Three methods, representational difference analysis （RDA） of cDNA, microarrays and fluorescence in situ hybridization （FISH） were used to detect levels of mRNA and protein expression of HCCR1 and HCCR-2 before and after treatment of matrine. Results： Matrine had inhibitory effect on the mRNA and protein expression of HCCR1 and HCCR2 in cultural HCC cells. Conclusion： Matrine has inhibitory effect on gene transcription, protein expression of HCCR 1 and HCCR2 in cultural HCC cells.

Hepatic Resection Combined with Radiofrequency Ablation versus Hepatic Resection Alone for Multifocal Hepatocellular Carcinomas:A Meta-analysis

This meta-analysis aimed to comprehensively assess the efficacy and safety of hepatic resection combined with radiofrequency ablation versus hepatic resection（HR） alone for the treatment of multifocal hepatocellular carcinomas（HCC）. A literature search was conducted from the database including MEDLINE, Embase, Cochrane Central Register of Controlled Trials（CENTRAL） and China Biology Medicine（CBM） disc. The primary outcomes included the 1-, 3-, 5-year overall survival（OS） and disease-free survival（DFS） rate. The secondary outcomes contained the intraoperative parameters and postoperative adverse events（AEs）. These parameters were all analyzed by Rev Man 5.3 software. After carefully screening relevant studies, four retrospective studies of high quality involving 466 patients（197 in the combined group and 269 in the HR group） were included in this study. The pooled results showed that the 1-, 3-, 5-year OS rate in the combined group were comparable with those in the HR group（OR=0.77, 0.96, 0.88; P=0.33, 0.88, 0.70, respectively）. Similarly, there was no significant difference in 1-, 3-, 5-year DFS rate between the combined group and the HR alone group（OR=0.57, 0.83, 0.72; P=0.17, 0.37, 0.32, respectively）. And the intraoperative parameters and postoperative AEs were also comparable between the above two cohorts. However, two included studies reported that tumor often recurred in the ablation site in the combined group. The present meta-analysis indicated that the HR combined with RFA could reach a long-term survival outcome similar to curative HR for multifocal HCC patients. And this therapy may be a promising alternative for these patients with marginal liver function or complicated tumor distribution. Furthermore, high quality randomized controlled trials（RCTs） are imperative to verify this conclusion.

Down-regulated expression of atypical PKC-binding domain deleted asip isoforms in human hepatocellular carcinomas

Asip is a mammalian homologue of polarity protein Par-3 of Caenorhabditis elegans and Bazooka of Drosophila melanogaster. Asip/Par-3/Bazooka are PDZ-motif containing proteins that localize asymmetrically to the cell periphery and play a pivotal role in cell polarity and asymmetric cell division. In the present study, we have cloned human asip cDNA and its splicing variants by 5’-RACE and RT-PCR using candidate human EST clones which have a high homology to rat asip cDNA. The full-length cDNA of human asip encodes a 1,353 aa protein exhibiting 88% similarity to the rat one. Human asip is a single copy gene consisting of at least 26 exons and localizing in human chromosome 10, band p11.2, with some extraordinarily long introns. All exon/intron boundary nucleotides conform to the "gt-ag" rule. Three main transcripts were detected by Northern blot analysis, and at least five variants, from alternative splicing and polyadenylation, have been identified by RT-PCR and liver cDNA library screening. Exon 17b deleted asip mRNAs expressed ubiquitously in normal human tissues, including liver, on RT-PCR analysis. However, they were absent from most human liver cancer cell lines examined. More interestingly, the expression of exon 1 7b deleted variants was down regulated in 52.6% (10/19) clinic specimens of human hepatocellular carcinomas (HCCs), compared with the surrounding nontumorous liver tissues from the same patients. The presence of various splicing transcripts, the variation of their distribution among different tissues and cells, and their differential expressions in human HCCs suggest that human Asip isoforms may function in different context.

Transcatheter arterial chemoembolization followed by immediate radiofrequency ablation for large solitary hepatocellular carcinomas

AIM: To assess the technical safety and efficacy of transcatheter arterial chemoembolization (TACE) combined with immediate radiofrequency ablation (RFA) for large hepatocellular carcinomas (HCC) (maximum diameter ≥ 5 cm). METHODS: Individual lesions in 18 patients with HCCs (mean maximum diameter: 7.5 cm; range: 5.1-15.5 cm) were treated by TACE combined with percutaneous RFA between January 2010 and June 2012. All of the patients had previously undergone one to four cycles of TACE treatment. Regular imaging and laboratory tests were performed to evaluate the rate of technical success, technique-related complications, local-regional tumor responses, recurrence-free survival time and survival rate after treatment.RESULTS: Technical success was achieved for all 18 visible HCCs. Complete response (CR) was observed in 17 cases, and partial response was observed in 1 case 1 mo after intervention. The CR rate was 94.4%. Local tumors were mainly characterized by coagulative necrosis. During follow-up (2-29 mo), the mean recurrencefree survival time was 16.8 ± 4.0 mo in 17 cases of CR. The estimated overall survival rate at 6, 12, and 18 mo was 100%. No major complications were observed. Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood of 17 patients transiently increased on the third day after treatment (ALT 200.4 ± 63.4 U/L vs 24.7 ± 9.3 U/L, P < 0.05; AST 228.1 ± 25.4 U/L vs 32.7 ± 6.8 U/L, P < 0.05). Severe pain occurred in three patients, which was controlled with morphine and fentanyl. CONCLUSION: TACE combined with immediate RFA is a safe and effective treatment for large solitary HCCs. Severe pain is a major side effect, but can be controlled by morphine.

Role of surgical resection for multiple hepatocellular carcinomas

AIM:To clarify the role of surgical resection for multiple hepatocellular carcinomas(HCCs)compared to transarterial chemoembolization(TACE)and liver transplantation(LT). METHODS:Among the HCC patients who were managed at Yonsei University Health System between January 2003 and December 2008,160 patients who met the following criteria were retrospectively enrolled:(1) two or three radiologically diagnosed HCCs;(2)no radiologic vascular invasion;(3)Child-Pugh class A;(4) main tumor smaller than 5 cm in diameter;and(5) platelet count greater than 50 000/mm3.Long-term outcomes were compared among the following three treatment modalities:surgical resection or combined radiofrequency ablation(RFA)(n=36),TACE(n=107), and LT(n=17).The survival curves were computed using the Kaplan-Meier method and compared with a log-rank test.To identify the patients who gained a survival benefit from surgical resection,we also investigated prognostic factors for survival following surgical resection.Multivariate analyses of the prognostic factors for survival were performed using the Cox proportional hazard model. RESULTS:The overall survival(OS)rate was significantly higher in the surgical resection group than in the TACE group(48.1%vs 28.9%at 5 years,P<0.005). LT had the best OS rate,which was better than that of the surgical resection group,although the difference was not statistically significant(80.2%vs 48.1%at 5 years,P=0.447).The disease-free survival rates were also significantly higher in the LT group than in the surgical resection group(88.2%vs 11.2%at 5 years, P<0.001).Liver cirrhosis was the only significant prognostic factor for poor OS after surgical resection. Clinical liver cirrhosis rates were 55.6%(20/36)in the resection group and 93.5%(100/107)in the TACE group.There were 19 major and 17 minor resections. En bloc resection was performed in 23 patients,multisite resection was performed in 5 patients,and combined resection with RFA was performed in 8 patients. In the TACE group,only 34 patients(31.8%)were recorded as having complete remission after primary TACE.Seventy-two patients(67.3%)were retreated with repeated TACE combined with other therapies. In patients who underwent surgical resection,the 16 patients who did not have cirrhosis had higher 5-year OS and disease-free survival rates than the 20 patients who had cirrhosis(80.8%vs 25.5%5-year OS rate, P=0.006;22.2%vs 0%5-year disease-free survival rate,P=0.048).Surgical resection in the 20 patients who had cirrhosis did not provide any survival benefit when compared with TACE(25.5%vs 24.7%5-year OS rate,P=0.225).Twenty-nine of the 36 patients who underwent surgical resection experienced recur-rence.Of the patients with cirrhosis,80%(16/20) were within the Milan criteria at the time of recurrence CONCLUSION:Among patients with two or three HCCs, no radiologic vascular invasion,and tumor diameters≤ 5 cm,surgical resection is recommended only in those without cirrhosis.

Common telomerase reverse transcriptase promoter mutations in hepatocellular carcinomas from different geographical locations

AIM: To determine the mutation status of human telomerase reverse transcriptase gene(TERT) promoter region in hepatocellular carcinoma(HCC) from different geographical regions.METHODS: We analyzed the genomic DNA sequences of 59 HCC samples comprising 15 cell lines and 44 primary tumors,collected from patients living in Asia,Europe and Africa.We amplified a 474 bp DNA fragment of the promoter region of TERT gene including the 1295228 and 1295250 sequence of chromosome 5 by using PCR.Amplicons were then sequenced by Sanger technique and the sequence data were analyzed with by using DNADynamo software in comparison with wild type TERT gene sequence as a reference.RESULTS: The TERT mutations were found highly frequent in HCC.Eight of the fifteen tested cell lines displayed C228 T mutation,and one had C250 T mutation with a mutation frequency up to 60%.All of the mutations were heterozygous and mutually exclusive.Ten out of forty-four tumors displayed C228 T mutation,and additional five tumors had C250 T mutation providing evidence for mutation frequency of 34% in primary tumors.Considering the geographic origins of HCC tumors tested,TERT promoter mutation frequencies were higher in African(53%),when compared to non-African(24%) tumors(P = 0.056).There was also a weak inverse correlation between TERT promoter mutations and murine double minute 2 single nucleotide polymorphism 309 TG polymorphism(P = 0.058).Mutation frequency was nearly two times higher in established HCC celllines(60%) compared to the primary tumors(34%).CONCLUSION: TERT promoter is one of most frequent mutational targets in liver cancer,and hepatocellular carcinogenesis is highly associated with the loss of telomere-dependent cellular senescence control.

Laparoscopic resection vs laparoscopic radiofrequency ablation for the treatment of small hepatocellular carcinomas: A single-center analysis

AIMTo compare survival and recurrence after laparoscopic liver resection (LLR) and laparoscopic radiofrequency ablation (LRFA) for the treatment of small hepatocellular carcinoma (HCC).METHODSBetween June 1, 2005 and November 30, 2010, 46 patients (62.26 &#x000b1; 8.55 years old; female/male: 12/34) treated for small HCC were enrolled following strict criteria. Patients with better liver function and larger tumors were referred for LLR (n = 24), while those with poorer liver function and multiple tumors were referred for LRFA (n = 22), and they were then followed for similar durations (44.74 &#x000b1; 21.3 mo for LLR vs 40.27 &#x000b1; 30.8 mo for LRFA).RESULTSThe LLR and LRFA groups were homogeneous with regard to age, sex, etiology of liver cirrhosis, and AFP levels. The overall survival (OS) and disease-free survival (DFS) probability was 0.354 and 0.260, respectively. A significantly higher OS was observed in the LLR group (LLR: 0.442; LRFA: 0.261; P = 0.048), whereas no statistical difference was found for DFS (LLR: 0.206; LRFA: 0.286; P = 0.205). In the LRFA group was treated a greater number of nodules (LLR: 1.41 &#x000b1; 0.77; LRFA: 2.72 &#x000b1; 1.54; P &#x0003c; 0.001). Cox regression analysis found the number of intraoperative HCC nodules as the unique variable statistically significant for OS (hazard ratio: 2.225; P &#x0003c; 0.001). The rank-hazard plot showed a steeper increase of relative hazard for intraoperative nodules &#x0003e; 2.CONCLUSIONOur preliminary results confirm the superiority of hepatic resection on thermoablation in the treatment of small HCC in selected patients, when both approaches are made laparoscopically. LLR showed better results compared to LRFA in terms of OS. These data need to be confirmed by further studies on a larger number of patients.

Expression of heat shock proteins (HSP27, HSP60, HSP70, HSP90, GRP78, GRP94) in hepatitis B virus-related hepatocellular carcinomas and dysplastic nodules

AIM: Expression of heat shock proteins (HSPs) is frequently up-regulated in hepatocellular carcinoma (HCC), which evolves from dysplastic nodule (DN) and early HCC to advanced HCC. However, little is known about the differential expression of HSPs in multistep hepatocarcinogenesis. It was the purpose of this study to monitor the expression of HSPs in multistep hepatocarcinogenesis and to evaluate their prognostic significance in hepatitis B virus (HBV)related HCC.METHODS: Thirty-eight HCC and 19 DN samples were obtained from 52 hepatitis B surface antigen-positive Korean patients. Immunohistochemical and dot immunoblot analyses of HSP27, HSP60, HSP70, HSP90, glucoseregulated protein (GRP)78, and GRP94 were performed and their expression at different stages of HCC development was statistically analyzed.RESULTS: Expression of HSP27, HSP70, HSP90, GRP78, and GRP94 increased along with the stepwise progression of hepatocarcinogenesis. Strong correlation was found only in GRP78 (Spearman's r= 0.802). There was a positive correlation between the expressions of GRP78, GRP94, HSP90, or HSP70 and prognostic factors of HCC. Specifically, the expression of GRP78, GRP94, or HSP90 was associated significantly with vascular invasion and intrahepatic metastasis.CONCLUSION: The expressions of HSPs are commonly up-regulated in HBV-related HCCs and GRP78 might play an important role in the stepwise progression of HBVrelated hepatocarcinogenesis. GRP78, GRP94, and HSP90 may be important prognostic markers of HBV-related HCC, strongly suggesting vascular invasion and intrahepatic metastasis.

Concurrent and subsequent radiofrequency ablation combined with hepatectomy for hepatocellular carcinomas

Partial hepatectomy has long been the standard treatment modality for patients with hepatocellular carcinoma(HCC),although the majority of patients with HCCs are not candidates for curative resection.Radiofrequency ablation(RFA) has been widely used as the preferred locoregional therapy.RFA and hepatectomy can be complementary to each other for the treatment of multifocal HCCs.Combining hepatectomy with RFA permits the removal of larger tumors while simultaneously ablating any smaller residual tumors.By using this combination treatment,more patients might become candidates for curative resection.For treating recurrent tumors involving the liver after hepatectomy,RFA has been performed recently instead of transcatheter arterial chemoembolization or ethanol ablation.Many retrospective studies on the combination of RFA and hepatectomy demonstrate favorable results of effectiveness and safety.However,further investigation of prospective design will be needed to confirm these encouraging results.

Sub-classification of intermediate-stage(Barcelona Clinic Liver Cancer stage-B) hepatocellular carcinomas

Hepatocellular carcinoma(HCC), the fifth most common cancer in the world, shows increasing incidence worldwide. Curative treatments such as hepatectomy,liver transplantation, and radiofrequency ablation are applied in only 30%-60% of cases. Most remaining patients receive transarterial chemoembolization(TACE).Patients with intermediate-stage HCCs are regarded as good candidates for TACE. However, the intermediate stage includes non-homogeneous patients. Some movements are underway to stratify patients using prognostic factors to identify patient groups exhibiting greater benefit from TACE than other patient groups.This review describes two substaging systems that subclassify intermediate-stage HCCs and discusses the importance of dividing intermediate-stage patients.

Analysis of N-ras gene mutation and p53 gene expression in human hepatocellular carcinomas

IM To study the relationship between Nras gene mutation and p53 gene expression in the carcinogenesis and the development of human hepatocellular carcinomas (HCC).METHODS The Nras gene mutation and the p53 gene expression were analyzed in 29 cases of HCC by polymerase chain reactionsingle strand conformation polymorphism (PCRSSCP) and immunohistochemistry.RESULTS Thirteen cases of HCCs were p53 positive (448%), which showed a rather high percentage of p53 gene mutation in Guangxi. The aberrations at Nras codon 2-37 were found in 7931% of HCCs and 8077% of adjacent nontumorous liver tissues. More than 2 point mutations of Nras gene were observed in 22 cases (7586%). Twelve cases (4137%) of HCCs showed both Nras gene mutation and p53 gene expression.CONCLUSIONS Nras gene and p53 gene may be involved in the carcinogenesis and the development of HCC. That 38% of HCCs with Nras gene mutation did not express p53 protein indicates that some other genes or factors may participate in the carcinogenesis and the development of HCC.

Iodized oil uptake assessment with cone-beam CT in chemoembolization of small hepatocellular carcinomas

AIM:To evaluate the utility of assessing iodized oil uptake with cone-beam computed tomography(CT)in transarterial chemoembolization(TACE)for small he-patocellular carcinoma(HCC).METHODS:Cone-beam CT provided by a biplane flat-panel detector angiography suite was performed on eighteen patients(sixteen men and two women;41-76 years;mean age,58.9 years)directly after TACE for small HCC(26 nodules under 30 mm;mean diam-eter,11.9 mm;range,5-28 mm).The pre-procedural locations of the tumors were evaluated using tripha-sic multi-detector row helical computed tomography(MDCT).The tumor locations on MDCT and the iodized oil uptake by the tumors were analyzed on cone-beam CT and on spot image directly after the procedures.RESULTS:All lesions on preprocedural MDCT were de-tected using iodized oil uptake in the lesions on cone-beam CT(sensitivity 100%,26/26).Spot image depictediodized oil uptake in 22 of the lesions(sensitivity 85%).The degree of iodized oil uptake was overestimated(9%,2/22)or underestimated(14%,3/22)on spot image in f ive nodules compared with that of cone-beam CT.CONCLUSION:Cone-beam CT is a useful and conve-nient tool for assessing the iodized oil uptake of small hepatic tumors(< 3 cm)directly after TACE.

Expression of PTEN,PPM1A and P-Smad2 in hepatocellular carcinomas and adjacent liver tissues

AIM： To investigate the expressions of PTEN, PPMIA and P-Smad2 in hepatocellular carcinoma （HCC） and their significance.METHODS： The expressions of PTEN, PPMIA and P-Smad2 in 31 HCC tissues, 25 adjacent liver tissues and 13 non-tumor liver tissues were detected by using Envision immunohistochemical technique. RESULTS： The positive expression （64.52%） and staining intensity （4.19 ± 3.31） of PTEN in the cytoplasm of HCC were significantly lower and weaker than those in the adjacent or non-tumor liver tissues （97.37%, 7.88 ± 0.93; 100%, 7.77 ± 0.93, respectively） （P 〈 0.05）, and its staining intensity in the cytoplasm of HCC, which belongs to Edmondson pathologic grades Ⅱ-Ⅲ and above, was also lower than that of grade I and I-Ⅱ. Furthermore, its location in the nucleus or cytoplasm of liver cells was negatively correlated with the progression of liver disease （r = -0.339, P = 0.002）; most of PPMIA might be only expressed in the nucleus of adjacent liver tissues, non-HCC tissues or Edmondson grade I and I - Ⅱ HCC, but it was mainly expressed in the cytoplasm of HCC with Edmondson grade ≥ Ⅱ, weakly or negatively expressed in the nucleus （P 〈 0.05）, and its location was negatively correlated with the progression of liver disease （r = -0.45, P = 0.0000）. P-Smad2, which was mostly located in the nucleus and cytoplasm of grade I and I -Ⅱ HCC, surrounding or non-tumor liver tissues, was only in the nucleus of HCC with Edmondson grade Ⅱ and above （P 〈 0.001）, and its location was positively correlated with the disease progression （r = 0.224, P = 0.016）. Spearman correlation analysis revealed that P-Smad2 was significantly negatively correlated with PTEN and PPMIA （r = -0.748, P = 0.000; r = -0.366, P = 0.001, respectively）; and PTEN and PPMIA were positively correlated with HCC carcinogenesis （r = 0.428, P = 0.000）.CONCLUSION： The aberrant location of expression and staining intensity of PTEN, PPMIA and P-Smad2 in HCC and their relationship might have an impact on the pathogenesis of HCC.

Inactivation of the tumor suppressor Krüppel-like factor 6 (KLF6) by mutation or decreased expression in hepatocellular carcinomas

Background and aim： The Krueppel-like transcription factor KLF6 is a novel tumor-suppressor gene. It was inactivated in human prostate cancer and other tumors tissue, as the result of frequent mutation and loss of heterozygosity （LOH）. However, there is no data reporting the levels of KLF6 both mRNA and protein in hepatocellular carcinomas （HCCs）. We therefore detected mutations and expression of KLF6 in HCC tissues and further observed the effect of it on cell growth in HCC cell lines. Methods： We analyzed the exon-2 ofKLF6 gene by direct DNA sequencing, and detected the expression of KLF6 by RT-PCR and Western blot in 23 HCC tissues and corresponding nontumorous tissues. Loss of growth suppressive effect of the HCC-derived KLF6 mutant was characterized by in vitro growth curves plotted, flow cytometry and Western blotting. Results： KLF6 mutations were found in 2 of 23 HCC tissues and one of mutations was missense. Expression ofKLF6 mRNA or protein was down-regulated in 8 （34.7%） or 9 （39.1%） of 23 HCC tissues. Wild-type KLF6 （wtKLF6） inhibited cellular proliferation and prolonged G1 -S transition by inducing the expression of p21WAF 1 following stable transfection into cultured HepG2 cells, but tumor-derived KLF6 mutant （mKLF6） had no effects. Conclusion： Our findings suggest that KLF6 may be involved in pathogenesis of HCC.

Radiotherapy for multiple brain metastases from hepatocellular carcinomas

A 78-year-old man with liver cirrhosis was found to have multiple hepatocellular carcinomas （HCCs） and underwent 3 sessions of transcatheter arterial chernoernbolization. Fourteen months after diagnosis, the patient presented with left herniparesis. Contrast- enhanced magnetic resonance imaging showed multiple metastases with ring-shaped enhancement in the cerebrum and cerebellum. There were no metastases to other organs. The metastatic lesions almost completely disappeared after whole-brain radiotherapy with a total dose of 50 Gy. Neurologic symptoms decreased, and the patient＇s quality of life improved. The patient underwent 2 more sessions of transcatheter arterial chemoembolization. Twelve months after the diagnosis of brain metastasis, the patient remains alive. The present case indicates that radiotherapy can improve quality of life and prolong survival in some patients with brain metastases from HCCs.

The “No-touch” technique improves the survival of patients with advanced hepatocellular carcinomas treated by liver transplantation: A single-center prospective randomized controlled trial

Background:Liver transplantation(LT)is the best treatment for patients with hepatocellular carcinoma(HCC).However,the surgical technique needs to be improved.The present study aimed to evaluate the“no-touch”technique in LT.Methods:From January 2018 to December 2019,we performed a prospective randomized controlled trial on HCC patients who underwent LT.The patients were randomized into two groups:a no-touch technique LT group(NT group,n=38)and a conventional LT technique group(CT group,n=46).Operative outcomes and survival in the two groups were analyzed.Results:The perioperative parameters were comparable between the two groups(P>0.05).There was no significant difference between the two groups in disease-free survival(DFS)(P=0.732)or overall survival(OS)(P=0.891).Of 36 patients who were beyond the Hangzhou criteria for LT,the DFS of the patients in the NT group was significantly longer than that in the CT group(median 402 vs.126 days,P=0.025).In 31 patients who had portal vein tumor thrombosis(PVTT),DFS and OS in the NT group were significantly better than those in the CT group(median DFS 420 vs.167 days,P=0.022;2-year OS rate 93.8%vs.66.7%,P=0.043).In 14 patients who had diffuse-type HCCs,DFS and OS were significantly better in the NT group than those in the CT group(median DFS 141 vs.56 days,P=0.008;2-year OS rate 75.0%vs.33.3%,P=0.034).Multivariate analysis showed that for patients with PVTT and diffusetype HCCs,the no-touch technique was an independent favorable factor for OS(PVTT:HR=0.018,95%CI:0.001-0.408,P=0.012;diffuse-type HCCs:HR=0.034,95%CI:0.002-0.634,P=0.024).Conclusions:The no-touch technique improved the survival of patients with advanced HCC compared with the conventional technique.The no-touch technique may provide a new and effective LT technique for advanced HCCs.

Lenvatinib for large hepatocellular carcinomas with portal trunk invasion:Two case reports

BACKGROUND In a phase III trial of lenvatinib as first-line treatment for advanced unresectable hepatocellular carcinoma(uHCC),the drug proved non-inferior to sorafenib in terms of the overall survival,but offered better progression-free survival.However,the effects of lenvatinib in uHCC patients with a tumor thrombus in the main portal vein and/or a high tumor burden(tumor occupancy more than 50%of the total liver volume),remain unclear,because these were set as exclusion criteria in the aforementioned trial.CASE SUMMARY A 53-year-old man(case 1)and 66-year-old woman(case 2)with uHCC presented to us with a tumor thrombus in both the main portal vein and inferior vena cava,a high tumor burden accompanied by a tumor diameter greater than>100 mm,and distant metastasis,with the residual liver function classified as grade 2A according to the modified Albumin–Bilirubin grading.We started both patients on lenvatinib.The therapeutic effect,as evaluated by the modified Response Evaluation Criteria in Solid Tumors,was rated as partial response in both case 1 and case 2(at 8 wk and 4 wk after the start of lenvatinib administration,respectively).The therapeutic effect was sustained for 6 mo in case 1 and 20 mo in case 2.Fever occurred as an adverse event in both case 1 and 2,and hyperthyroidism and thrombocytopenia in only case 2,neither of which,however,necessitated treatment discontinuation.CONCLUSION Even in hepatocellular carcinoma patients with poor prognostic factors,if the liver function is well-preserved,lenvatinib is effective and safe.