Well-differentiated Hepatocellular Carcinoma Research Articles

Liver cancer cell lines distinctly mimic the metabolic gene expression pattern of the corresponding human tumours

BackgroundAlthough metabolism is profoundly altered in human liver cancer, the extent to which experimental models, e.g. cell lines, mimic those alterations is unresolved. Here, we aimed to determine the resemblance of hepatocellular carcinoma (HCC) cell lines to human liver tumours, specifically in the expression of deregulated metabolic targets in clinical tissue samples.MethodsWe compared the overall gene expression profile of poorly-differentiated (HLE, HLF, SNU-449) to well-differentiated (HUH7, HEPG2, HEP3B) HCC cell lines in three publicly available microarray datasets. Three thousand and eighty-five differentially expressed genes in ≥2 datasets (P < 0.05) were used for pathway enrichment and gene ontology (GO) analyses. Further, we compared the topmost gene expression, pathways, and GO from poorly differentiated cell lines to the pattern from four human HCC datasets (623 tumour tissues). In well- versus poorly differentiated cell lines, and in representative models HLE and HUH7 cells, we specifically assessed the expression pattern of 634 consistently deregulated metabolic genes in human HCC. These data were complemented by quantitative PCR, proteomics, metabolomics and assessment of response to thirteen metabolism-targeting compounds in HLE versus HUH7 cells.ResultsWe found that poorly-differentiated HCC cells display upregulated MAPK/RAS/NFkB signaling, focal adhesion, and downregulated complement/coagulation cascade, PPAR-signaling, among pathway alterations seen in clinical tumour datasets. In HLE cells, 148 downregulated metabolic genes in liver tumours also showed low gene/protein expression – notably in fatty acid β-oxidation (e.g. ACAA1/2, ACADSB, HADH), urea cycle (e.g. CPS1, ARG1, ASL), molecule transport (e.g. SLC2A2, SLC7A1, SLC25A15/20), and amino acid metabolism (e.g. PHGDH, PSAT1, GOT1, GLUD1). In contrast, HUH7 cells showed a higher expression of 98 metabolic targets upregulated in tumours (e.g. HK2, PKM, PSPH, GLUL, ASNS, and fatty acid synthesis enzymes ACLY, FASN). Metabolomics revealed that the genomic portrait of HLE cells co-exist with profound reliance on glutamine to fuel tricarboxylic acid cycle, whereas HUH7 cells use both glucose and glutamine. Targeting glutamine pathway selectively suppressed the proliferation of HLE cells.ConclusionsWe report a yet unappreciated distinct expression pattern of clinically-relevant metabolic genes in HCC cell lines, which could enable the identification and therapeutic targeting of metabolic vulnerabilities at various liver cancer stages.

Therapeutic Efficacy of Vitamin D in Experimental c-MET-β-Catenin-Driven Hepatocellular Cancer.

Aberrant activation of β-catenin signaling is frequently observed in hepatocellular cancer. Although Wnt/β-catenin signaling can be targeted by vitamin D, therapeutic use of vitamin D for this purpose is not currently established. We evaluated the therapeutic use of vitamin D or its analogs using a synthetic transgenic mouse of hepatocarcinogenesis induced by mutant β-catenin, and MET overexpression in which 75% of mice develop well-differentiated HCC within 8 weeks in the absence of fibrosis. Vitamin D receptor expression was similar in both tumoral and nontumoral tissue. There was no significant difference in overall survival, or in tumor progression assessed by imaging, biochemical, or tumor cell burden assessments in mice receiving a vitamin D-supplemented diet containing 12.0 IU VD/g (HVD) compared with a standard diet (SD) containing 2.3 IU VD/g. Furthermore, systemic treatment with calcitriol [vitamin D analog 1α,25(OH)₂D₃] or EB1089 (synthetic vitamin D analog) by intraperitoneal injection for 4 weeks prolonged median survival but did not increase overall survival compared with controls. Although tumor formation was delayed in males compared with that in females, there was no difference in overall survival between males and females. In conclusion, although 1α,25(OH)₂D₃ is reported to inhibit β-catenin signaling, as well as proliferation, migration, and differentiation in cancer cells, neither dietary supplementation with vitamin D nor treatment with vitamin D analogs altered the formation or growth of HCC associated with β-catenin activation. These results conclusively demonstrate the lack of utility of targeting vitamin D for therapy of HCC in this setting.

Utility of rapid on-site evaluation for needle core biopsies and fine-needle aspiration cytology done for diagnosis of mass lesions of the liver

Fine-needle aspiration (FNA) and core biopsy (CB) are used to diagnose liver lesions. Rapid onsite evaluation (ROSE) can improve the adequacy of the procedures and help triage diagnostic material appropriately. There are very few studies evaluating the role of ROSE for CB and FNA of mass lesions of the liver. Liver cases with ROSE material from 2007 to 2017 were retrieved and reviewed. The ROSE material was re-evaluated by 2 cytopathologists who were blinded to the final diagnosis. Data including age, number of lesions, number of passes, adequacy assessed at time of procedure, and diagnosis made by cytopathologist on ROSE material at time of re-review was compiled. A total of 82 cases were identified; 33 were primary lesions (group A) and 49 were metastatic lesions (group B). ROSE done by cytotechnologist at time of procedure showed an adequacy rate of 84%. During re-review of ROSE material by cytopathologists, the overall adequacy rates were similar, although the adequacy rates in group B increased (to 100% from 92%) and it dropped in group A (from 73% to 52%). The overall accuracy rate was 90%. Hepatocellular adenoma, regenerative nodules, well-differentiated hepatocellular carcinoma, and angiosarcoma were not possible to diagnose on smears alone during ROSE. ROSE for liver lesions is useful for assessing adequacy. Certain lesions cannot be accurately diagnosed on ROSE alone. ROSE material when assessed by cytopathologist can improve adequacy rate and possibly decrease number of nondiagnostic specimens in group A, though the cost effectiveness needs to be assessed.

Atypical Hepatocellular Neoplasms: Review of Clinical, Morphologic, Immunohistochemical, Molecular, and Cytogenetic Features.

The distinction of hepatocellular adenoma from well-differentiated hepatocellular carcinoma (HCC) can be difficult in some cases, especially on biopsy specimens. These borderline cases often occur in men or older patients and may have β-catenin activation or focal atypical morphologic features (such as small cell change, prominent pseudoacinar formation, cytologic atypia, focally thick plates, and/or focal reticulin loss) that are insufficient for an unequivocal diagnosis of HCC. The term "atypical hepatocellular neoplasm" has been advocated for these tumors, but a number of other terms, including "atypical adenoma," "hepatocellular neoplasm of uncertain malignant potential," and "well-differentiated hepatocellular neoplasm with atypical or borderline features" have also been proposed. This review proposes guidelines for designating tumors as atypical hepatocellular neoplasm and describes clinical, morphologic, immunohistochemical, molecular, and cytogenetic features that distinguish these tumors from typical hepatocellular adenoma and HCC.

Abstract 4029: The Wnt target gene CLAUDIN-2 regulates tumorigenesis and stemness in human liver cancer cells

Abstract Background: In our previous studies on T-cell factor (TCF)-4 isoforms, we identified a unique target gene CLAUDIN-2, which was upregulated by lack of SxxSS motif in exon 9 of TCF-4 (Exp Cell Res 2011, Liver Int 2013). This type of TCF-4 (TCF-4J) conferred high tumorigenic ability and hypoxia resistance to hepatocellular carcinoma (HCC) cells (PLoS One 2012). Of interest, the TCF-4J isoform-overexpressing HCC cells (J cells) strongly expressed the leaky tight junction protein CLAUDIN-2 under sphere-forming conditions, suggesting that J cells have cancer stem cell (CSC)-like features involving CLAUDIN-2 upregulation. Thus, the Aim of this study was to assess expression profile of CLAUDIN-2 and the CSC-relevant function of the molecule, such as tumorigenicity, in liver cancer cells. Methods: The human liver cancer cell lines used in this study were as follows: HAK-1A, HAK-1B (Hepatology 1993), Huh-7, HepG2, Hep3B, HLF, KMCH-1 (Hepatology 1987), KMCH-2 (J Hepatol 1996), KYN-1, KYN-2, and HAK-5. KMCH-1 and -2 are unique combined hepatocellular-cholangiocarcinoma cell lines, and HAK-5 is a sarcomatous HCC cell line. Lentivirus-mediated delivery of siRNA for CLAUDIN-2 (Santa Cruz) and plasmid-mediated CLAUDIN-2 cDNA (Origene) were utilized to generate stable knock-down (KD) and overexpressing cells, respectively. Results: CLAUDIN-2 was exclusively expressed in both KMCH-1 and KMCH-2 cells, showing 240~370-fold mRNA level of that in the well-differentiated HCC line HAK-1A. The protein expression was strongly observed at cytoplasmic membrane and cytoplasm in KMCH-1 and KMCH-2 cells, but not in the others examined. Gene silencing of CLAUDIN-2 resulted in slower cell proliferation. In xenograft experiment in nude mice, CLAUDIN-2-KD KMCH-2 cells formed much smaller tumors in size than the control cells. Of interest, overexpression of CLAUDIN-2 in constitutively low expressing cell lines, such as Hep3B and HLF cells, lead to high expression of Nanog, a stemness gene product. Conclusion: As previously shown in transformed HCC cells by the Wnt transcription factor TCF-4J, CLAUDIN-2 was a key molecule regulating cell proliferation and tumorigenesis in non-gene-engineered human combined hepatocellular-cholangiocarcinoma cells. To elucidate precise mechanism of exclusively high expression of this molecule in such difficult-to-treat type of liver cancer may provide insights into generating novel therapeutic approaches. Citation Format: Hironori Koga, Fumitaka Wada, Mitsuhiko Abe, Hideki Iwamoto, Toru Nakamura, Takahiko Sakaue, Atsutaka Masuda, Toshimitsu Tanaka, Hirohisa Yano, Takuji Torimura. The Wnt target gene CLAUDIN-2 regulates tumorigenesis and stemness in human liver cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4029.

The Role of HSP70, CD34 and Ki 67 Expression in Liver Cirrhosis and Hepatocellular Carcinoma, Immunohistochemical Study

Background: Liver cirrhosis is an important risk factor for hepatocellular carcinoma (HCC). Aim: To evaluate the expression of HSP70, CD34, and Ki 67 in liver cirrhosis and early HCC. Methods: 60 liver biopsies were classified into 3 groups;Group 1(Control; 10 biopsies), Group 2 (Liver Cirrhosis; 25 cases), and Group 3 (HCC; 25 cases). All biopsies were stained by (HE low expressed (+) in 5 cases (5 out of 5 well-differentiated HCC) (20%);, intermediate expressed (++) in 13 cases(11 out of 11 moderately differentiated HCC and 2 out of 9 poorly differentiated HCC) (52%), and high expressed (+++) in 7 cases (7 out of 9 poorly differentiated HCC) (28%). CD34 was negative in all control cases, while in cirrhotic cases, it is minimally expressed in 5 cases (20%) and diffuse in 6 cases (24%) and positive in all cases of HCC; focal in one case (4%) and diffuse in 24 cases (96%). KI67 was negative in all control cases, isolated positively in one cirrhotic case (4%) while in HCC, it was negative in 2 cases (8%), isolated positive in 9 cases (36%), focal positive in 10 cases (40%) and diffusely positive in 4 cases (16%). Conclusion; there is a role of HSP70, CD34, and Ki-67 in differentiation between non-neoplastic cirrhotic lesions and early HCC.

The Impact of Tumor Differentiation on the Prognosis of HBV-Associated Solitary Hepatocellular Carcinoma Following Hepatectomy: A Propensity Score Matching Analysis.

The role of tumor differentiation in the prognosis of hepatocellular carcinoma (HCC) after hepatectomy remains controversial. The present study aimed to classify the impact of tumor differentiation on solitary hepatitis B viral (HBV)-associated HCC using propensity score matching analysis. Between January 2009 and March 2015, the data of 721 HCC patients in West China Hospital were prospectively collected and analyzed. Propensity matching analysis was applied to overcome the imbalance in baseline characteristics. Survival analysis was performed using the Kaplan-Meier method. Risk factors were identified by the Cox proportional hazards model. All HCC patients were classified into the moderately well-differentiated HCCs group (group A, n = 442, 61.3%) or poorly differentiated HCCs group (group B, n = 279, 38.7%). Patients with poorly differentiated HCCs commonly had a larger tumor size, more advanced tumors, and a higher alpha-fetoprotein (AFP) level. Patients with poorly differentiated HCCs had a poorer recurrence-free survival and overall survival before and after propensity score matching analysis. Poorly differentiated tumors, positive serum hepatitis B viral e antigen, positive hepatitis B virus deoxyribonucleic acid load, tumor size, microvascular invasion, and AFP > 400ng/ml were risk factors of a poor outcome. Our propensity model provided strong evidence that a poorly differentiated tumor had a negative impact on the recurrence and long-term survival of solitary HBV-associated HCCs after curative hepatectomy. Antiviral therapy might improve their prognosis.

Hepatocellular Carcinoma: Retrospective Evaluation of the Correlation Between Gadobenate Dimeglumine-Enhanced Magnetic Resonance Imaging and Pathologic Grade.

The aim of this study was to evaluate the usefulness of gadobenate dimeglumine-enhanced magnetic resonance imaging in characterizing the grade of hepatocellular carcinoma (HCC) using the signal intensity (SI) of the erector spinae as internal reference. Clinical data of 40 patients (a total of 44 lesions) confirmed by pathology for HCC were retrospectively reviewed. Gadobenate dimeglumine-enhanced magnetic resonance imaging was performed in all patients, and SI of lesions (SIles), liver parenchyma around the lesions (SIhep), erector spinae (SImus) and standard deviation of SI of the surrounding noise (SDnoi) on nonenhanced T2WI, nonenhanced T1WI, and contrast-enhanced T1WI (in both arterial and hepatobiliary phase [AP and HBP]) were measured, respectively. Contrast-to-noise ratio (CNR) were separately defined as CNR1 ([SIles - SIhep]/SDnoi) and CNR2 ([SIles - SImus]/SDnoi). Statistical analyses were performed using one-way analysis of variance, least significant difference test, logistic regression analysis, Spearman rank correlation, and receiver operating characteristic curves analysis. Forty-four HCCs, including 3 well-differentiated HCCs, 26 moderately differentiated HCCs, and 15 poorly differentiated (PD) HCCs, were confirmed. On logistic regression analysis, only CNR2 in the HBP was predictor of PD HCCs (P = 0.015, odds ratio = 1.040). The size of lesions, CNR1 in the AP, CNR2 in the AP, and CNR2 in the HBP, showed significant correlations with the degree of differentiation (correlation coefficients = -0.371, 0.435, 0.503, and 0.512, respectively; P = 0.013, 0.003, 0.001, and 0.000, respectively). Contrast-to-noise ratio 2 in the HBP with the cutoff of less than 4.56 could distinguish moderately differentiated HCCs from PD HCC with the sensitivity and specificity of 84.6% and 60.0%, respectively. Relatively low arterial enhancement and low CNR2 value in the HBP are predictive for poor histological grade of HCCs.

Establishment and characterization of an immortalized human hepatocyte line for the development of bioartificial liver system.

For further research and application of Bioartificial liver systems (BAL), active proliferation capacity and full hepatic functionality of the biomaterials is mandatory. However, there are still no suitable cell lines meeting the requirements for an ideal cell source in BAL development that makes it necessary to explore other sources. Here, we constructed a new cell line derived from well-differentiated hepatocellular carcinoma tissues designated NHBL2. Immunol staining showed that NHBL2 possessed the capacity of synthesizing albumin and CYP2E1 and quantitative analysis showed that the albumin synthesis ability of NHBL2 was comparable to C3A while urea production was highly abundant of NHBL2 compared with that of C3A. Using gene expression microarray analysis, we found that the expression levels of a set of genes encoding Phase I and Phase II metabolizing enzymes as well as many others related to common bioconversion and metabolic processes were significantly higher in NHBL2 cell line than that in C3A. Moreover, functional optimization assay in Matrigel showed obvious improvements of liver-related function level and a low malignance of this cell line. These findings indicated that NHBL2 possessed relatively attractive and full hepatic functionality that might be a potential cell line for BAL development.

Primary Liver Cancers-Part 1: Histopathology, Differential Diagnoses, and Risk Stratification.

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are the 2 most common primary malignant liver tumors, with hepatocellular and bile ductular differentiation, respectively. This article reviews the key histopathological findings of these 2 primary liver cancers and includes a review of the role of ancillary testing for differential diagnosis, risk stratification according to the American Joint Committee on Cancer (AJCC) staging recommendation, and a review of precancerous lesions. A literature review was conducted to identify articles with information relevant to precancerous precursors, current histopathological classification, ancillary testing, and risk stratification of primary malignant liver tumors. The histomorphology of normal liver, preinvasive precursors, primary malignancies, and morphological variants, and the utilization of ancillary tests for the pathological diagnosis are described. Dysplastic nodules are the preinvasive precursors of HCC, and intraductal papillary neoplasms of bile ducts and biliary intraepithelial neoplasia are the preinvasive precursors of CC. Benign liver nodules including focal nodular hyperplasia and adenomas are included in this review, since some forms of adenomas progress to HCC and often they have to be differentiated from well-differentiated HCC. A number of morphological variants of HCC have been described in the literature, and it is necessary to be aware of them in order to render the correct diagnosis. Risk stratification is still dependent on the AJCC staging system. The diagnosis of primary liver carcinomas is usually straightforward. Application of the appropriate ancillary studies aids in the differential diagnosis of difficult cases. The understanding of the carcinogenesis of these malignancies has improved with the standardization of the pathological classification of preinvasive precursors and studies of the molecular pathogenesis. Risk stratification still depends on pathological staging.

EUS-guided radiofrequency ablation: an option for the extrapancreatic region.

Radiofrequency ablation (RFA) achieves tumor necrosis by cell protein denaturation, induced by tissue heating above 45°C to obtain irreversible cellular injury. Energy is supplied by a generator connected to an active electrode tip inserted into the tumor. RFA was developed in the early nineties for percutaneous or intraoperative treatment of hepatocellular carcinoma (HCC). It is recommended by the EASL–EORTC Clinical Practice Guidelines for early-stage disease (not eligible for surgery).[1] Today, RFA is widely used in the treatment of HCC and has been given attention as a potential treatment for many tumors (e.g., kidney, lung, and bone). Recently, new devices have been used to perform RFA through linear echoendoscopes, for targeting focal pancreatic lesions (neuroendocrine tumors, adenocarcinomas, and pancreatic cysts).[234] Moreover, the left lobe of the liver can be well visualized using endoscopic ultrasound (EUS), making it amenable to EUS-guided interventional procedures, including ablations.[5] A 76-year-old HBV/HCV-cirrhotic female presenting with portal hypertension and ascites had been diagnosed with HCC on radiological imaging (Barcelona Clinic Liver Cancer Stage A: Child–Pugh B8/Performance status: 1), but she was deemed unfit for surgery. She was, therefore, referred for EUS-guided sampling of hilar lymph nodes, in view of further management of a suspicious HCC in the fourth segment of her liver [Figure 1a–c]. Sampling of the hepatic nodule and the lymph nodes resulted in a diagnosis of well-differentiated HCC and reactive adenopathies [Figure 2].Figure 1: Abdominal computed tomography shows a 30-mm nodule with diffuse hyperenhancement in the arterial phase, and slow wash out in the portal and late phases (a-c). After 1 month, an abdominal computed tomography scan shows a large ablated area in the arterial, portal, and late phases (d-f)Figure 2: High-power images show pseudoglandular aggregates of atypical epithelial cells (a) (H and E, ×400). Gomori's staining (b) highlights the presence of neoplastic pseudoglandular structures (×400). CD34 immunostaining demonstrates sinusoidal capillarization (c), a typical change that characterizes the endothelial cells of hepatocellular carcinoma (×200)Considering the easy approach provided by EUS and obstacles to the percutaneous route (large umbilical hernia), the nodule was ablated with EUS guidance (GIF-160 Olympus, Tokyo, Japan) using a 20-mm active tip water-cooled electrode needle (EUSRA™ RF Electrode-VIVA RF Generator, STARmed, Seoul Korea). The procedure required three passes at five positions, using a multi-pass technique that allowed a larger ablation. Video 1 shows the insertion of the needle inside the HCC and the development of air bubbles near the electrode tip (first pass). Later, a panoramic view shows a large hyperechoic halo occupying the nodule. This marked the conclusion of the procedure, after a total of 30 min of energy application (starting power 30 W). An ultrasound imaging contrast (Sonovue®, Bracco, Italy) was administered during the procedure for marking the ablated area (nonenhancing tissue) and targeting further insertions. No adverse event was observed, and the patient was discharged on the 3rd postoperative day. One month later, computed tomography scan showed a large ablated area [Figure 1d–f]. This case highlights the possibilities for an EUS-guided transluminal approach to perform RFA of extrapancreatic neoplasms, such as a liver lesion, when percutaneous or surgical treatments are technically challenging or prohibitive. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient has given her consent for her images and other clinical information to be reported in the journal. The patient understands that her name and initial will not be published and due efforts will be made to conceal her identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Association of Low Zinc Finger Antiviral Protein Expression with Progression and Poor Survival of Patients with Hepatocellular Carcinoma

Background/Aims: Zinc finger antiviral protein (ZAP) has been reported to be expressed in hepatocellular carcinoma (HCC), and ZAP expression is associated with apoptotic signaling in cancer cells. This study aimed at investigating the expression of ZAP in HCC cells and its significance in clinical pathology. Methods: Real-time quantitative PCR and western blot assays were employed to detect ZAP RNA and protein expression in normal human hepatocytes, HCC cells, and five primary HCC cell lines. Immunohistochemistry was performed to detect ZAP expression in 147 paraffin-embedded HCC tissues and adjacent normal tissues. The clinical significance of ZAP expression was analyzed in tissue samples from patients with or without infection by hepatitis B virus (HBV). Results: ZAP expression in HCC cells and human primary HCC cell lines was significantly lower than that of normal human hepatocytes. Among 147 HCC samples, ZAP expression was lower in HCC tissues than in adjacent normal tissues for 107 (77.0%) samples. In patients with HCC and HBV infection, ZAP expression was related to pathological grade (P < 0.05); in HBV-negative patients with HCC, ZAP expression was associated with tumor size (P < 0.05) and clinical stage (P < 0.05). The overall survival time in patients with low ZAP expression was significantly shorter than survival times of those with high ZAP expression (P < 0.05), especially for patients with moderately to well-differentiated HCC (Grade 1–2) and HCC at stage T1 and T2 (P < 0.05). Cox multivariate analysis showed that ZAP expression was an independent predictor of survival of patients with HCC (P < 0.01). Conclusion: Low ZAP expression is closely associated with disease progression and poor prognosis for patients with HCC.

Signal Transducer and Activator of Transcription 3 for the Differentiation of Hepatocellular Carcinoma from Cirrhosis.

Background:Overexpression and constitutive activation of signal transducer and activator of transcription (STAT) 3 have been suggested in the tumorigenesis of many human cancers, including multiple carcinomas, melanoma, and lymphoma. The diagnosis of hepatocellular carcinoma (HCC) in lobectomy specimens is usually straightforward, but distinguishing cirrhosis from well-differentiated HCC can be challenging in core biopsies. Our aims were to investigate the expression level of STAT3 and phosphorylated STAT3 (pSTAT3) in HCC and cirrhosis, and the application of STAT3 in the differential diagnosis of HCC and cirrhosis.Methods:Sixty cases were divided into three groups: patients with HCC only (Group 1), HCC and cirrhosis (Group 2), and cirrhosis only (Group 3). Formalin-fixed and paraffin-embedded tissue sections were stained immunohistochemically for STAT3, pSTAT3, and CD163. The values obtained from the tissue sections of each group were compared in statistical analysis.Results:STAT3 showed a high level in HCC and was a significant marker for differentiating HCC from cirrhosis (P < 0.0001). The odds ratio between HCC and cirrhosis increased 34.4 times when the intensity of STAT3 increased by 1 level. Spearman's correlation and Chi-square tests also demonstrated that expression level of STAT3 did not correlate with age, gender, or the presence of a cirrhotic background.Conclusions:STAT3 staining differs significantly in HCC and cirrhosis. The findings reinforce the role of STAT3 in the tumorigenesis of HCC and provide a useful marker to differentiate HCC from cirrhosis in challenging liver biopsies.

Near-infrared photothermal/photodynamic therapy with indocyanine green induces apoptosis of hepatocellular carcinoma cells through oxidative stress

Indocyanine green (ICG) is a photothermal agent, photosensitizer, and fluorescence imaging probe which shows specific accumulation in hepatocellular carcinoma (HCC) cells. We recently developed a photodynamic therapy (PDT) using ICG and near-infrared (NIR) laser as a new anti-cancer treatment for HCC. However, the molecular mechanism underlying this effect needs to be elucidated. HuH-7 cells, a well-differentiated human HCC cell line, were transplanted subcutaneously into BALB/c-nu/nu mice for in vivo experiment. ICG was administered 24 h before NIR irradiation. The irradiation was performed at three tumor locations by 823-nm NIR laser on days 1 and 7. The temperature of HuH-7 xenografts increased to 48.5 °C 3 minutes after ICG-NIR irradiation start. Reactive oxygen species (ROS) production was detected after ICG-NIR irradiation both in vitro and in vivo. There was certain anti-tumor effect and ROS production even under cooling conditions. Repeated NIR irradiation increased the cell toxicity of ICG-NIR therapy; the mean tumor volume on day 9 was significantly smaller after ICG-NIR irradiation compared to tumor without irradiation (87 mm3 vs. 1332 mm3; p = 0.01) in HCC mice xenografts model. ICG-NIR therapy induced apoptosis in HCC cells via a photothermal effect and oxidative stress. Repeated ICG-NIR irradiation enhanced the anti-tumor effect.

A subset of well-differentiated hepatocellular carcinomas are Arginase-1 negative

Hepatocellular carcinoma (HCC) is a common malignancy both in the United States of America and worldwide. Despite the refinement of imaging techniques in at-risk populations, a needle biopsy diagnosis remains an important diagnostic tool for HCC in many cases. Various immunohistochemical markers have been developed to facilitate this diagnosis, such as HepPar-1, glypican-3 and, most recently, arginase-1. Amongst them, arginase-1 has been shown to have superior sensitivity and specificity than the others. Performance of arginase-1 has been reported to be excellent for diagnosis of well-differentiated HCCs, with some tail-off in sensitivity for poorly differentiated tumors. Our experience has suggested that a subset of well-differentiated HCCs can be negative for arginase-1. We examined 68 consecutive confirmed cases of well-differentiated HCC diagnosed on needle biopsy, and found 7 (10%) to be completely negative for arginase-1. This finding is of fundamental clinical importance in view of previous studies that have shown arginase-1 to be always positive in well-differentiated HCC.

Identification and Comparison of Differentiation-Related Proteins in Hepatocellular Carcinoma Tissues by Proteomics.

Histological differentiation is a major pathological criterion indicating the risk of tumor invasion and metastasis in patients with hepatocellular carcinoma. The degree of tumor differentiation is controlled by a complex interacting network of associated proteins. The principal aim of the present study is to identify the possible differentiation-related proteins which may be used for early diagnosis and more effective therapies. We compared poorly differentiated and well-differentiated hepatocellular carcinoma tissues by using 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among the 11 identified protein spots, 6 were found to be upregulated in poorly differentiated hepatocellular carcinoma tissues and 5 were correspondingly downregulated. Immunohistochemistry was performed on 106 hepatocellular carcinoma tissues to confirm the results of the proteomic analysis. By using bioinformatic tools GO and STRING, these proteins were found to be related to catalytic activity, binding, and antioxidant activity. In particular, our data suggest that overexpression of peroxiredoxin-2, annexin A2, and heat shock protein β-1 was correlated with tumor invasion, metastasis, and poor prognosis, and therefore, these proteins may serve as potential diagnostic and therapeutic biomarkers.

A novel chromonyl thiohydantoin with anti-proliferative action on primary hepatocellular carcinoma cells

Chromone, imidazolidinedione, thiohydantoin and 2,4-thiazolidinedione structures are known to be cytotoxic to cancer cells. In this study, biological activities of previously synthesized 18 chromonyl-2,4-thiazolidinediones/imidazolidinediones/thiohydantoins were tested by sulforhodamine B assay in five liver and one breast cancer cell lines. It was shown that a hybrid chromonyl thiohydantoin derivative C9 was able to significantly suppress the proliferation of the liver cancer cell lines which are very resistant to anticancer drugs. According to 50% growth inhibition concentrations of C9, well differentiated hepatocellular carcinoma cell line Huh7 and breast adenocarcinoma cell line Mcf7 cells are found to be highly sensitive to C9 with IC50 values between 4.9–5.2 µM. Poorly differentiated human liver cancer cell line Snu449 is found to be the most resistant to C9. Further studies show that C9 causes morphological changes in Snu449 cells. However, it does not induce significant senescence response or cell cycle arrest. On the other hand, Huh7 cells are found to be arrested in G2/M phase of the cell cycle after 24 and 72 h treatment. In conclusion, a novel chromonyl thiohydantoin hybrid molecule effectively stops or slows down the proliferation of well-differentiated hepatocellular carcinoma cells that exist in primary tumors.

The stromal morphological changes for differential diagnosis of uninodular high-grade dysplastic nodule and well-differentiated small hepatocellular carcinoma.

AimThe stromal invasion has been regarded as the most valuable clue to distinguish high-grade dysplastic nodules (HGDNs) and well-differentiated small hepatocellular carcinomas (WD-SHCCs). The purposes of this study are to explore the stromal morphological changes for the differential diagnosis of these two equivocal lesions.ResultsBased on the systemic studies of histological characteristics of HGDNs and WD-SHCCs, the stromal morphological changes, including sinusoid capillarization, ductular reaction and solitary artery, were performed to make a differential diagnosis between them. Separately, the solitary artery had the best sensitivity (93.75%) and accuracy (88.89%), and the sinusoid capillarization had the best specificity of 90.32%. On the whole, when at least 2 stromal morphological changes were abnormal, no matter what combination, the diagnostic performance was favorable and optimal with the highest accuracy of 92.06%, balancing the sensitivity (93.75%) and specificity (90.32%). The diagnostic performances were prior to the classical immunohistochemical panel comprising heat shock protein 70, glypican 3 and glutamine synthetase with the best sensitivity, specificity and accuracy of 62.50%, 80.65% and 71.43%, respectively.Materials and MethodsA retrospective case-control study was conducted on 63 patients who underwent partial hepatectomy for uninodular HGDNs or WD-SHCCs at the Eastern Hepatobiliary Surgery Hospital from 2005 to 2015.ConclusionsThe stromal morphological changes, containing sinusoid capillarization, ductular reaction and solitary artery could provide a more considerable diagnostic and differential diagnostic performance between HGDNs and WD-SHCCs. And they should be the key points during the histopathological diagnosis.

Abstract 4797: Continuous hepatocyte apoptosis accelerates diethylnitrosamine-induced liver tumor development

Abstract Background and Aim: Apoptosis serves as an important mechanism for removing DNA damaged-cells and is considered to inhibit carcinogenesis. Hepatocyte apoptosis is a key feature of chronic liver disease, as established precancerous condition for liver cancer. The present study examined the impact of continuous hepatocyte apoptosis on liver tumor development. Methods: We used male hepatocyte-specific knockout (KO) mice of Mcl-1, one of anti-apoptotic proteins, as a model of continuous hepatocyte apoptosis, and hepatocyte-specific single or double KO mice of Bak and Bax, proapoptotic proteins, as a model of apoptosis-resistant liver. To induce liver carcinogenesis those mice were intraperitoneally administered 20 mg/kg diethylnitrosamine (DEN) at 2 weeks. Results: Among control group treated with PBS, neither Mcl-1 KO nor wild-type (WT) mice developed macroscopically and microscopically any liver tumors at age of 6 months. On the other hand, while only 8% (2/26) of DEN-treated WT mice developed macroscopic liver tumors, 100% (15/15) of Mcl-1 KO mice developed (p&amp;lt;0.05). Microscopically, 57% (15/26) of DEN-treated WT mice developed liver tumors, 100% (15/15) of Mcl-1 KO mice developed (p&amp;lt;0.05). These tumors were histologically classified as well-differentiated HCC. The average of maximum tumor size (12.4 vs. 6.5 mm), tumor numbers (21.4 vs. 4.0) and liver weight to body weight ratio (12.3 vs. 4.2 %) in Mcl-1 KO mice were significantly higher than those in WT mice. At 6 weeks, serum ALT levels and caspase 3/7 activity of DEN-treated Mcl-1 KO mice were significantly higher than those of DEN-treated WT mice. Immunohistochemistry revealed that PCNA, Ki67, phospho-H2AX and 8-OHdG positive cells significantly increased in DEN-treated Mcl-1 KO mice compared with DEN-treated WT mice. The mRNA expression levels of TNF-alfa and Mcp-1 in DEN-treated Mcl-1 KO mice were significantly higher than those of DEN-treated WT mice.In contrast, macroscopic liver tumor incidence rate in WT, Bak KO, Bax KO and Bak/Bax double KO mice treated with DEN at 9 months was 46 (6/13), 40 (6/15), 35 (7/20) and 52% (11/21), respectively. Microscopic liver tumor incidence rate in WT, Bak KO, Bax KO and Bak/Bax double KO mice was 100 (13/13), 80 (12/15), 70 (14/20) and 81% (17/21), respectively. There was no statistically significant difference in incidence rate of both macroscopic and microscopic tumors among 4 groups. At age of 6 weeks, there was no significant difference in serum ALT levels and caspase 3/7 activity among 4 groups. Conclusion: Continuous hepatocyte apoptosis is suggested to promote DEN-induced liver tumor development. Increased liver damage, compensative hepatocyte proliferation, genomic instability and inflammation may accelerate the incidence of DEN-induced liver tumorigenesis. In contrast, inhibition of apoptosis does not make much impact on DEN-induced liver tumor development. Citation Format: Yasutoshi Nozaki, Hayato Hikita, Satoshi Tanaka, Yuta Myojin, Sadatsugu Sakane, Kazuhiro Murai, Yuto Shiode, Yugo Kai, Yuki Makino, Tasuku Nakabori, Yoshinobu Saito, Takahiro Kodama, Ryotaro Sakamori, Tomohide Tatsumi, Eiji Miyoshi, Tetsuo Takehara. Continuous hepatocyte apoptosis accelerates diethylnitrosamine-induced liver tumor development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4797. doi:10.1158/1538-7445.AM2017-4797

Heat shock protein 22 (HSPB8) reduces the migration of hepatocellular carcinoma cells through the suppression of the phosphoinositide 3-kinase (PI3K)/AKT pathway

Small heat shock proteins (HSPs) regulate a variety of cell functions. Among them, HSP22 and HSP20 are recognized to be ubiquitously expressed in various tissues. With regard to hepatocellular carcinoma (HCC) cells, we previously reported that phosphorylated HSP20 plays a suppressive role in transforming growth factor (TGF)-α-induced cell migration and invasion. In the present study, we investigated whether or not HSP22 is implicated in HCC cell migration. We detected HSP22 protein expression both in human HCC tumor (189.9±68.4ng/mg protein) and the adjacent non-tumor liver tissues (167.9±94.6ng/mg protein). The cases of low-quantity HSP22 protein level group (88.3≧ng/mg protein, the optimum cut-off value of HSP22) were increased in tumor tissues compared with the adjacent non-tumor tissues. The migration of human HCC-derived HuH-7 cells stimulated by TGF-α or hepatocyte growth factor (HGF) was significantly enhanced by the knockdown of HSP22 expression. Down-regulation of HSP22 protein in the cells markedly strengthened the AKT phosphorylation induced by TGF-α or HGF. Inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathway, which suppressed the TGF-α-induced migration, significantly reduced the amplification by HSP22 knockdown. PI3K but not AKT was coimmunoprecipitated with HSP22 in HuH-7 cells. In addition, in human HCC tissues, a significantly lower HSP22 protein level in tumor tissues than in adjacent non-tumor tissues was observed more frequently in cases of moderately or poorly differentiated HCC than well-differentiated HCC. Taken together, our results strongly suggest that HSP22 represses HCC progression, especially HCC cell migration, by the down-regulation of the PI3K/AKT signaling pathway.