Quantitative analysis using ELISA of vascular endothelial growth factor and basic fibroblast growth factor in human colorectal cancer,liver metastasis of colorectal cancer and hepatocellular carcinoma

Angiogenesis consists of the sprouting of capillaries from pre-existing vessels. It is well-known that tumor growth is angiogenesis-dependent. Vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF） stimulated vascular endothelial cell proliferation and are involved in the neoplastic angiogenesis of several types of tumors including those of the intestinal tract. Authors usually investigated VEGF and using immunohistochemistry bFGF protein expressions or Western blotting and VEGF and bFGF transcripts using reverse transcriptase Dolymerase chain reaction （RT-PCR）.

Three-dimensional morphometric analysis for hepatectomy of centrally located hepatocellular carcinoma:A pilot study

AIM: To describe a three-dimensional model(3DM) to accurately reconstruct anatomic relationships of centrally located hepatocellular carcinomas(HCCs).METHODS: From March 2013 to July 2014, reconstructions and visual simulations of centrally located HCCs were performed in 39 patients using a 3D subject-based computed tomography(CT) model with customdeveloped software. CT images were used for the 3D reconstruction of Couinaud's pedicles and hepatic veins, and the calculation of corresponding tumor territories and hepatic segments was performed using Yorktal DMIT software. The respective volume, surgical margin, and simulated virtual resection of tumors were also estimated by this model preoperatively. All patients were treated surgically and the results were retrospectively assessed. Clinical characteristics, imaging data, procedure variables, pathologic features, and postoperative data were recorded and compared to determine the reliability of the model.RESULTS: 3D reconstruction allowed stereoscopic identification of the spatial relationships between physiologic and pathologic structures, and offered quantifiable liver resection proposals based on individualized liver anatomy. The predicted values were consistent with the actual values for tumor mass volume(82.4 ± 109.1 m L vs 84.1 ± 108.9 m L, P = 0.910), surgical margin(10.1 ± 6.2 mm vs 9.1 ± 5.9 mm, P = 0.488), and maximum tumor diameter(4.61 ± 2.16 cm vs 4.53 ± 2.14 cm, P = 0.871). In addition,the number and extent of portal venous ramifications, as well as their relation to hepatic veins, were visualized. Preoperative planning based on simulated resection facilitated complete resection of large tumors located in the confluence of major vessels. And most of the predicted data were correlated with intraoperative findings.CONCLUSION: This 3DM provides quantitative morphometry of tumor masses and a stereo-relationship with adjacent structures, thus providing a promising technique for the management of centrally located HCCs.

Elevated serum alpha fetoprotein levels promote pathological progression of hepatocellular carcinoma

AIM:To investigate the biological role of alpha fetoprotein (AFP) and its clinical signif icance in carcinogenesis of hepatocellular carcinoma (HCC).METHODS:Clinical analysis of HCC patients and im-munohistochemical examination were conducted to evaluate the relationship between serum AFP level and patient mortality. Confocal microscopy,Western blotting, dimethylthiahzolyl-2,5-diphenyl-tetrazolium bromide,Cell Counting Kit-8 assays and flow cytometry were performed to explore the possible mechanism.RESULTS: Among the 160 HCC patients enrolled in this study,130 patients survived 2 years (81.25%),with a survival rate of 86.8% in AFP < 2 0 μg/L group,88.9% in AFP 20-250 μg/L group,and 69.6% in AFP > 250 μg/L group, demonstrating a higher mortality rate in HCC patients with higher AFP levels. Surgical treatment was benef icial only in patients with low AFP levels.The mortality rate of HCC patients with high AFP levels who were treated surgically was apparently higher than those treated with conservative management.The results of immunohistochemistry showed that AFP and AFP receptor were merely expressed in tissues of HCC patients with positive serum AFP.Consistently,in vitro analysis showed that AFP and AFPS were expressed in HepG2 but not in HLE cells. AFP showed a capability to promote cell growth,and this was more apparent in HepG2 cells,in which the proliferation was increased by 3.5 folds. Cell cycle analysis showed that the percent-age of HepG2 cells in S phase after exposure to AFP was modestly increased.CONCLUSION:HCC patients with higher AFP levels show a higher mortality rate,which appears to be attributable to the growth promoting properties of AFP.

Management of hepatitis B virus infection during treatment for hepatitis B virus-related hepatocellular carcinoma

Although liver resection is considered the most effective treatment for hepatocellular carcinoma(HCC), treatment outcomes are unsatisfactory because of the high rate of HCC recurrence. Since we reported hepatitis B e-antigen positivity and high serum hepatitis B virus(HBV) DNA concentrations are strong risk factors for HCC recurrence after curative resection of HBV-related HCC in the early 2000 s, many investigators have demonstrated the effects of viral status on HCC recurrence and post-treatment outcomes. These findings suggest controlling viral status is important to prevent HCC recurrence and improve survival after curative treatment for HBV-related HCC. Antiviral therapy after curative treatment aims to improve prognosis by preventing HCC recurrence and maintaining liver function. Therapy with interferon and nucleos(t)ide analogs may be useful for preventing HCC recurrence and improving overall survival in patients who have undergone curative resection for HBV-related HCC. In addition, reactivation of viral replication can occur after liver resection for HBV-related HCC. Antiviral therapy can be recommended for patients to prevent HBV reactivation. Nevertheless, further studies are required to establish treatment guidelines for patients with HBVrelated HCC.

Hepatitis B virus and micro RNAs:Complex interactions affecting hepatitis B virus replication and hepatitis B virusassociated diseases

Chronic infection with the hepatitis B virus(HBV) is the leading risk factor for the development of hepatocellular carcinoma(HCC). With nearly 750000 deaths yearly, hepatocellular carcinoma is the second highest cause of cancer-related death in the world. Unfortunately, the molecular mechanisms that contribute to the development of HBV-associated HCC remain incompletely understood. Recently, micro RNAs(mi RNAs), a family of small non-coding RNAs that play a role primarily in post-transcriptional gene regulation, have been recognized as important regulators of cellular homeostasis, and altered regulation of mi RNA expression has been suggested to play a significant role in virus-associated diseases and the development of many cancers. With this in mind, many groups have begun to investigate the relationship between mi RNAs and HBV replication and HBV-associated disease. Multiple findings suggest that some mi RNAs, such as mi R-122, and mi R-125 and mi R-199 family members, are playing a role in HBV replication and HBV-associated disease, including the development of HBV-associated HCC. In this review, we discuss the current state of our understanding of the relationship between HBV and mi RNAs, including how HBV affects cellular mi RNAs, how these mi RNAs impact HBV replication, and the relationship between HBV-mediated mi RNA regulation and HCC development. We also address the impact of challenges in studying HBV, such as the lack of an effective model system for infectivity and a reliance on transformed cell lines, on our understanding of the relationship between HBV and mi RNAs, and proposepotential applications of mi RNA-related techniques that could enhance our understanding of the role mi RNAs play in HBV replication and HBV-associated disease, ultimately leading to new therapeutic options and improved patient outcomes.

Neoplastic disease after liver transplantation: focus on de novo neoplasms

De novo neoplasms account for almost 30% of deaths 10 years after liver transplantation and are the most common cause of mortality in patients surviving at least 1 year after transplant. The risk of malignancy is two to four times higher in transplant recipients than in an age- and sex-matched population, and cancer is expected to surpass cardiovascular complications as the primary cause of death in transplanted patients within the next 2 decades. Since exposure to immunosuppression is associated with an increased frequency of developing neoplasm, long-term immunosuppression should be therefore minimized. Promising results in the prevention of hepatocellular carcinoma(HCC) recurrence have been reported with the use of m TOR inhibitors including everolimus and sirolimus and the ongoing open-label prospective randomized controlled SILVER. Study will provide more information on whether sirolimus-containing vs m TOR-inhibitorfree immunosuppression is more efficacious in reducing HCC recurrence.

Rab23 is a potential biological target for treating hepatocellular carcinoma

AIM： To elucidate the role of Rab23 in hepatocellular carcinoma （HCC） by assessing the expression of Rab23 in HCC tissue and in HCC cell lines. METHODS： Primary tumors （n = 100） were stained with Rab23 antibodies using immunohistochemistry and in situ hybridization in tissue microarrays. Relationships between gene expression and pathology parameters were analysed. The biological significance of Rab23 in Hep-3B cells was examined by knocking down Rab23 gene expression. We designed a pair of doublestranded RNAs against human rab23 and transfected siRNA into Hep-3B cells. Rab23 expression in these cells was examined using RT-PCR and Western blots. We investigated cell growth by MTT assays and fluorescenceactivated cell sorting. RESULTS： High cytoplasmic and nuclear expression of Rab23 was found in 38 of 71 （53.5%） and in 49 of 68 HCC patients （72%） respectively, which correlated with tumor size. HCC cell lines expressed Rab23. In Hep3B cells, siRNA for Rab23 decreased Rab23 mRNA by 4.5-fold and protein expression by 2-fold. Survival rates at 24 and 48 h for Hep-3B cells tTansfected with siRNA were lower and about 30% Hep-3B cells were apoptotic. Knocking down rab23 suppressed Hep3B cell growth, suggesting that rab23 could play an important role in Hep3B cell growth. CONCLUSION： Rab23 is overexpressed and/or activatedin HCC. Rab23 may be both a HCC predictor and a target for treating HCC.

Association between hepatitis B and metabolic syndrome:Current state of the art

Chronic hepatitis B(CHB)is a global health issue that increases the risk of liver cirrhosis and hepatocellular carcinoma in infected patients.Metabolic syndrome(Met S)is a disease endemic mostly to the developed countries.It is associated with high cardiovascular mortality and morbidity,diabetes mellitus as well as cancer.In this manuscript,we systematically review the published data on the relationship between Met S and CHB infection.Multiple studies have described highly variable correlations between CHB on one hand and Met S,non-alcoholic fatty liver disease and dyslipidemia on the other.No association between CHB and diabetes mellitus or atherosclerosis has been described as of now.The presence of Met S in patients infected with hepatitis B virus increases the risk of fibrosis,cirrhosis and hepatocellular carcinoma.Appropriate lifestyle,but also pharmacological interventions are needed to prevent the development of these complications.

Potential roles of EZH2, Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma cell line Hep3B

AIM: To investigate the potential roles of enhancer of zeste homolog2(EZH2), Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma(HCC) cell line Hep3 B.METHODS: A total of 73 patients who underwent surgical resection at Fuzong Clinical Medical College of Fujian Medical University were enrolled in this study. Hep3 B cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37?℃. Vectors that containing c DNA of the EZH2 gene or mi R-203 targeted sh RNA plasmid were constructed, and then transfected into Hep3 B cells. The m RNA expression of mi R-203, EZH2, and Bmi-1 was analyzed using quantitative real-time polymerase chain reaction analysis, and the protein levels of EZH2 and Bmi-1 were detected by Western blot analysis. Effect of EZH2 or mi R-203 on cell proliferation was observed by methyl thiazolyl tetrazolium assay, and cell apoptosis was assessed using flow cytometry. Besides, effect of EZH2 or mi R-203 on tumor cell invasion was detected using Transwell assay.RESULTS: The m RNA levels of EZH2 and Bmi-1 in HCC tissues and in Hep3 B cells were significantly higher compared with those in normal samples(P < 0.01), while mi R-203 level was significantly lower in HCC tissues(P < 0.01). Hep3 B cells transfected with EZH2-sh RNA or mi R-203-sh RNA showed lower expression levels of EZH2 and Bmi-1(P < 0.05). Compared with controls, Hep3 B cells transfected with EZH2-sh RNA had relative slow cell proliferation, indicating that low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could inhibit Hep3 B cell proliferation(P < 0.05). The average apoptosis rate of Hep3 B cells transfected with EZH2-sh RNA vector was about 18.631%, while that of Hep3 B cells transfected with sh RNA vector was about 5.33%, suggesting that EZH2 was down-regulated by transfecting with EZH2-sh RNA, and the down-regulated EZH2 contributed to the cell apoptosis. Low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could reduce Hep3 B cell invasion(P < 0.05).CONCLUSION: Our study suggests that EZH2 and Bmi-1 are up-regulated while mi R-203 is downregulated in Hep3 B cells. Mi R-203 may contribute to the metastasis and enhance apoptosis of HCC cells by regulating EZH2 and Bmi-1. Our study may provide a theoretical basis for metastasis of HCC and targeted therapy of HCC.

Hepatocellular carcinoma and hepatitis B surface protein

The tumorigenesis of hepatitis B virus(HBV)-associated hepatocellular carcinoma(HCC) has been widely studied. HBV envelope proteins are important for the structure and life cycle of HBV, and these proteins are useful for judging the natural disease course and guiding treatment. Truncated and mutated pre S/S are produced by integrated viral sequences that are defective for replication. The pre S/S mutants are considered "precursor lesions" of HCC. Different pre S/S mutants induce various mechanisms of tumorigenesis, such as transactivation of transcription factors and an immune inflammatory response, thereby contributing to HCC. The pre S2 mutants and type Ⅱ "Ground Glass" hepatocytes represent novel biomarkers of HBVassociated HCC. The pre S mutants may induce the unfolded protein response and endoplasmic reticulum stress-dependent and stress-independent pathways. Treatments to inhibit hepatitis B surface antigen(HBs Ag) and damage secondary to HBs Ag or the pre S/S mutants include antivirals and antioxidants, such as silymarin, resveratrol, and glycyrrhizin acid. Methods for the prevention and treatment of HCC should be comprehensive.

Long noncoding RNAs in hepatitis B virus-related hepatocellular carcinoma

AIM: To study the expression of long noncoding RNAs(lncRNAs) in hepatitis B virus(HBV)-related hepatocellular carcinoma(HCC).METHODS: The lncRNA profiles between HBV-related HCC tissues and corresponding normal liver tissues were generated using microarray analysis. Datasets were analyzed using multiple algorithms to depict alterations in gene expression on the basis of gene ontology(GO), pathway analysis, and lncRNA levels.RESULTS: The microarray revealed that 1772 lncRNAs and 2508 mRNAs were differently expressed. The pathway analysis demonstrated that the cell cycle, cytokinecytokine receptor interaction, chemokine signaling pathway, and phosphoinositide 3-kinase-protein kinase B signaling pathway may play important roles in HCC.Several GO terms, such as cell cycle, DNA replication,immune response, and signal transduction, were enriched in gene lists, suggesting a potential correlation with HBVrelated HCC. The upregulated large intergenic noncoding RNA ULK4P2 was physically combined with enhancer of zeste homolog 2. Therefore, the lncRNAs may participate in regulating HBV-related HCC.CONCLUSION: lncRNAs play important roles in HCC,future studies should verify whether large intergenic noncoding ULK4P2 functions by combining with enhancer of zeste homolog 2 in HCC.

Key role of hepatitis B virus mutation in chronic hepatitis B development to hepatocellular carcinoma

Chronic hepatitis B virus(HBV) infection is a major risk factor for hepatocellular carcinoma(HCC). The HBV mutations, which include point mutation, deletion,insertion and truncation mutation of HBV gene in 4 open reading frames(S, C, P, X), are closely associated with HCC pathogenesis. Some mutations accumulated during chronic HBV infection could be regarded as a biomarker to predict the occurrence of HCC. The detection of the mutations in clinical practice could be helpful for defining better preventive and therapeutic strategies and, moreover, predicting the progression of liver disease.

Expression of phosphatase and tensin homolog deleted on chromosome ten in liver of athymic mice with hepatocellular carcinoma and the effect of Fuzheng Jiedu Decoction

AIM： To explore the expression of phosphatase and tensin homolog deleted on chromosome ten （PTEN） in liver of athymic mice with hepatocellular carcinoma （HCC） and the effect of Fuzheng Jiedu Decoction （FJD）. METHODS： Forty eight male BALB/c athymic mice models were built by Bel-7402 with an indirect method. After 24 h of postoperation, the 48 athymic mice were distributed randomly into 4 groups： A, B, C, D, each group had 12 athymic mice. Group A were were treated by intragastric administration with FT207 （Tegafur） for 4 wk. Group B, C and D were treated by intragastric administration with FJD （complex prescription of Chinese crude drug） that had been delegated into 3 kinds of density as the low, middle, and high for 4 wk. At last, athymic mice were put to death, live time, volume of tumors, exponent of tumors and the tumor metastasis in livers were observed; and PTEN was detected in hepatic tissue, latero-cancer tissue and cancer tissue by immunohistochemistry. RESULTS： Four weeks later, the total survival rate in treatment group （A ＋ B ＋ C） was 50% and higher than the control group （0%） treated by FT207, （P 〈 0.01）. The survival rate in group A, B, C was higher than in group D, and except group A with D, there was significant differentces （Fisher＇s Exact Test P = 0.05 or 0.01）. And no differences were observed between the treatment groups and the control group in volume of tumors and exponent of tumors （P 〉 0.05）. Tumor metastasis in livers of the treatment group was less than the controls （Fisher＇s Exact Test, P = 0.021）. The result of immunohistochemistry showed that the intensity of PTEN in latero-cancer tissue was the highest, and then the hepatic tissue, the lowest was cancer tissue （Kruskal- Wallis test, X^2 = 60.67, P = 0.000）. It also showed that the intensity of PTEN in treatment groups （A, B, C） was higher than the control group （D） （F = 5.90, P = 0.002 in hepatic tissue and F = 15.99, P = 0.000 in latero-cancer tissue and X^2 = 26.08, P = 0.000 in cancer tissue）, and group B is the highest in the treatment groups （P 〈 0.05, r = 0.01. respectively）. However, there was no significant statistic difference between group A and group C （P 〉 0.05）. CONCLUSION： FJD can prolong the survival time and decrease tumor metastasis in livers of these experimental mice. Mechanisms of FJD healing HCC may partially be explained by enhancing the expression of PTEN in liver.

Induction of apoptosis and cell cycle arrest in human HCC MHCC97H cells with Chrysanthemum indicum extract

AIM： To investigate the effects of Chrysanthemum indicum extract （CIE） on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma （HCC） MHCC97H cell line. METHODS： Viable rat hepatocytes and human endothelial ECV304 cells were examined by trypan blue exclusion and MTT assay, respectively, as normal controls. The proliferation of MHCC97H cells was determined by MTT assay. The cellular morphology of MHCC97H cells was observed by phase contrast microscopy. Flow cytometry was performed to analyze cell apoptosis with annexin V/propidium iodide （PI）, mitochondrial membrane potential with rhodamine 123 and cell cycle with PI in MHCC97H cells. Apoptotic proteins such as cytochrome C, caspase-9, caspase-3 and cell cycle proteins, including P21 and CDK4, were measured by Western blotting. RESULTS： CIE inhibited proliferation of MHCC97H cells in a timeand dose-dependent manner without cytotoxicity in rat hepatocytes and human endothelial ceils. CIE induced apoptosis of MHCC97H cells in a concentration-dependent manner, as determined by flow cytometry. The apoptosis was accompanied by a decrease in mitochondrial membrane potential, release of cytochrome C and activation of caspase-9 and caspase-3. CIE arrested the cell cycle in the S phase by increasing P21 and decreasing CDK4 protein expression. CONCLUSION： CIE exerted a significant apoptotic effect through a mitochondrial pathway and arrested the cell cycle by regulation of cell cycle-related proteins in MHCC97H cells without an effect on normal cells. The cancer-specific selectivity shown in this study suggests that the plant extract could be a promising novel treatment for human cancer.

Scattered and rapid intrahepatic recurrences after radio frequency ablation for hepatocellular carcinoma

AIM： To evaluate a series of patients with hepatocellular carcinoma （HCC） treated with several different protocols and devices. METHODS： We treated 138 patients [chronic hepatitis/ liver cirrhosis （Child-Pugh A/B/C）, 3/135 （107/25/3）] with two different devices and protocols： cool-tip needle [initial ablation at 60 W （standard method） （n = 37） or at 40 W （modified method） （n = 28）] or; ablation with a LeVeen needle using a standard single-step, full expansion （single-step） method （n = 39） or a multi-step, incremental expansion （multi-step） method. RESULTS： Eleven patients experienced rapid and scattered recurrences 1 to 7 mo after the ablation. Nine patients were treated by the cool-tip original protocol （60 W） （9/37 = 24%） and the other two by the LeVeen single-step method （2/39 = 5%）. The location of the recurrence was surrounding and limited to the site of ablation segment in three cases, and spread over one Iobule or both Iobules in the other eight cases. There was no recurrence in the patients treated with the modified cool-tip modified method （40 W） or the LeVeen multi-step method. CONCLUSION： There is a risk of rapid and scattered recurrence after RFA, especially when the standard cool- tip procedure is used. Because such recurrence would worsen the prognosis, we recommend that modified protocols for the cool-tip and LeVeen needle methods should be used in clinical practice.

Chemiluminescence enzyme immunoassay based on magnetic nanoparticles for detection of hepatocellular carcinoma marker glypican-3

Glypican-3 （GPC3） is reported as a great promising tumor marker for hepatocellular carcinoma （HCC） diagnosis. Highly sensitive and accurate analysis of serum GPC3 （sGPC3）, in combination with or instead of traditional HCC marker alpha-fetoprotein （AFP）, is essential for early diagnosis of I-ICC. Biomaterial-functionalized magnetic particles have been utilized as solid supports with good biological compatibility for sensitive immunoassay. Here, the magnetic nanoparticles （MnPs） and magnetic microparticles （MmPs） with carboxyl groups were further modified with streptavidin, and applied for the development of chemiluminescence enzyme immunoassay （CLEIA）. After comparing between MnPs- and MmPs-based CLEIA, MnPs-based CLEIA was proved to be a better method with less assay time, greater sensitivity, better linearity and longer chemiluminescence platform. MnPs-based CLEIA was applied for detection of sGPC3 in normal liver, hepatocirrhosis, secondary liver cancer and HCC serum samples. The results indicated that sGPC3 was effective in diagnosis of HCC with high performance.

Assessment of KL-6 as a tumor marker in patients with hepatocellular carcinoma

AIM： To investigate the clinical significance of KL-6 as a tumor marker of HCC in two different ethnic groups with chronic liver disease consecutively encountered at outpatient clinics. METHODS： Serum KL-6 was measured by the sandwich enzyme immunoassay method using the KL-6 antibody （Ab） as both the capture and tracer Ab according to the manufacturer＇s instructions （Eisai, Tokyo, Japan）. Assessment of alpha fetoprotein （AFP） and protein induced vitamin K deficiency or absence （PIVKA-II） was performed in both groups using commercially available kits. RESULTS： A significantly higher mean serum KL-6 （556±467 U/L） was found in HCC in comparison with non-HCC groups either with （391±176 U/L; P〈0.001） or without （361±161 U/L; P〈0.001） liver cirrhosis （LC）. Serum KL-6 level did not correlate with either AFP or PIVKA-II serU/Levels. Using rec：eiver operating curve analysis for KL-6 as a predictor for HCC showed that the area under the curve was 0.574 （95%CI = 0.50-0.64） and the KL-6 level that gave the best sensitivity （61%） was found to be 334 U/L but according to the manufacturer＇s instructions; a cut-off point of 500 U/L was used that showed the highest specificity （80%） in comparison with AFP and PIVKA-II （78% vs 72% respectively）. Combining the values of the three markersimproved specificity of AFP for HCC diagnosis from 78% for AFP alone; 93% for AFP plus PIVKA-II to 99% for both plus KL-6 value （P〈0.001）. Mean serum alkaline phosphatase level was significantly higher in KL-6 positive （564＋475） in comparison with KL-6 negative （505＋469） HCC patients （P = 0.021）, but such a difference was not found among non-HCC corresponding groups. CONCLUSION： KL-6 is suggested as a tumor for HCC. Its positivity may reflect HCC-associated cholestasis and/ or local tumor invasion.

Diagnostic and therapeutic application of noncoding RNAs for hepatocellular carcinoma

Micro RNAs(mi RNAs) are small,noncoding RNA molecules that regulate gene expression posttranscriptionally,targeting thousands of messenger RNAs. Long noncoding RNAs(lnc RNAs),another class of noncoding RNAs,have been determined to be also involved in transcription regulation and translation of target genes. Since deregulated expression levels or functions of miR NAs and lncR NAs in hepatocellular carcinoma(HCC) are frequently observed,clinical use of noncoding RNAs for novel diagnostic and therapeutic applications in the management of HCCs is highly and emergently e xpe c t e d. H e r e,we s ummar iz e r e c e nt f indings regarding deregulated mi RNAs and lnc RNAs for their potential clinical use as diagnostic and prognostic biomarkers of HCC. Specifically,we emphasize the deregulated expression levels of such noncoding RNAs in patients' sera as noninvasive biomarkers,a field that requires urgent improvement in the clinical surveillance of HCC. Since nucleotide-based strategies are being applied to clinical therapeutics,we further summarize clinical and preclinical trials using oligonucleotides involving the use of miR NAs and small interfering RNAs against HCC as novel therapeutics. Finally,we discuss current open questions,which must be clarified in the near future for realistic clinical applications of these new strategies.

Relationship between therapeutic efficacy of arterial infusion chemotherapy and expression of P-glycoprotein and p53 protein in advanced hepatocellular carcinoma

AIM： To investigate the relationship between the chemotherapeutic drug efficacy and the expression of P-glycoprotein （PGP） and p53 protein in advanced hepatocellular carcinoma （HCC）. METHODS： The study was conducted on 41 patients with advanced HCC who were treated by repeated arterial infusion chemotherapy. Biopsy specimens from the tumor were collected before the start of treatment in all the patients, and the specimens were stored frozen until immunohistochemical staining, which was performed after the start of treatment, to detect PGP and p53 protein expressions. Twenty of the fortyone patients were treated with an anthracycline drug （epirubicin hydrochloride; anthracycline group）, and the remaining 21 were treated with a non-anthracycline drug （mitoxantrone hydrochloride in 11 patients and carboplatin in 10 patients; non-anthracycline group）. The relationship between the chemotherapeutic efficacy and the results of immunostaining were compared between the two groups. RESULTS： Before the start of the treatment, PGPpositive rate was 90.2% （strongly-positive, 36.6%） and p53 protein-positive rate was 34.1% （strongly-positive, 19.5%）. In the anthracycline group, the response rate was 40.0%. The number of patients showing poor response to the treatment was significantly larger in the patients with strongly positive PGP expression （P= 0.005）, and their prognoses were poor （P= 0.001）. in the nonanthracycline group, the response rate was 42.9%,and there was no significant relationship between the chemotherapeutic drug efficacy and the PGP or p53 protein expression. When only the data from the 11 patients treated with anthraquinone drug, mitoxantrone, were analyzed, however, the number of patients who showed poor response to treatment was significantly higher among the p53-positive patients （P= 0.012）, irrespective of the survival outcome. CONCLUSION： The chemotherapeutic efficacy with an anthracycline drug for advanced HCC can be predicted by immunohistochemical analysis of PGP expression. Similarly, immunostaining to evaluate p53 protein may be useful to predict the response in patients treated with an anthraquinone drug.

Current concepts in the immunohistochemical evaluation of liver tumors

Immunohistochemistry often plays an important role in the evaluation of liver tumors. Recent advances have established a classification system for hepatocellular adenomas(HCAs) based on morphology,molecular alterations,and immunohistochemistry. Specifically,loss of liver fatty acid binding protein is seen in HNF1α-inactivated HCA,staining with serum amyloid A isseen in inflammatory HCA,and diffuse staining with glutamine synthetase(GS) is seen in β-catenin activated HCA. A panel of immunohistochemical stains including glypican-3(GPC-3),heat shock protein 70,and GS are useful in distinguishing HCC from non-malignant dysplastic nodules. Immunohistochemistry is also useful to determine whether a liver tumor is of primary hepatocellular or metastatic origin. Recently described markers useful for this purpose include arginase-1,GPC-3,and bile salt export pump. These newer markers may offer superior utility when compared to traditional markers of hepatocellular differentiation such as alpha-fetoprotein,hepatocyte paraffin-1,polyclonal carcinoembryonic antigen,and CD10. This paper will review recent advances in the immunohistochemical evaluation of liver tumors.