Potential ultrastructure predicting factors for hepatocellular carcinoma in HCV infected patients

Hepatitis C virus represents one of the rising causes of hepatocellular carcinoma (HCC). Although the early diagnosis of HCC is vital for successful curative treatment, the majority of lesions are diagnosed in an irredeemable phase. This work deals with a comparative ultrastructural study of experimentally gradually induced HCC, surgically resected HCC, and potential premalignant lesions from HCV-infected patients, with the prospect to detect cellular criteria denoting premalignant transformation. Among the main detected pathological changes which are postulated to precede frank HCC: failure of normal hepatocyte regeneration with star shape clonal fragmentation, frequent elucidation of hepatic progenitor cells and Hering canals, hepatocytes of different electron density loaded with small sized rounded monotonous mitochondria, increase junctional complexes bordering bile canaliculi and in between hepatocyte membranes, abundant cellular proteinaceous material with hypertrophied or vesiculated rough endoplasmic reticulum (RER), sequestrated nucleus with proteinaceous granular material or hypertrophied RER, formation of lipolysosomes, large autophagosomes, and micro-vesicular fat deposition.

In conclusion, the present work has visualized new hepatocytic division or regenerative process that mimic splitting or clonal fragmentation that occurs in primitive creature. Also, new observations that may be of value or assist in predicting HCC and identifying the appropriate patient for surveillance have been reported. Moreover, it has pointed to the possible malignant potentiality of liver stem/progenitor cells.

For reliability, the results can be subjected to cohort longitudinal study.     

Expression of MTA2 and Ki-67 in hepatocellular carcinoma and their correlation with prognosis

The aim of this study was to investigate the relationship among MTA2, Ki-67 and HCC patient prognosis. Tissue microarray and immunohistochemistry were used to detect the expression of MTA2 and Ki-67 in HCC samples and corresponding adjacent samples. We found MTA2 and Ki-67 were both increased in HCC tissues than those in adjacent tissues and nuclear MTA2 was associated with Ki-67 (P = 0.019). Moreover, nuclear MTA2 was a risk factor of distant metastasis in patients with HCC andKi-67 showed a negative correlation with histological grade (P < 0.05, P < 0.01, respectively). Multivariate Cox model analysis revealed that Ki-67 expression was an independent prognosis factor in HCC patients (P = 0.020). These results indicated there might be a tight correlation among MTA2, Ki-67 and HCC prognosis. MTA2 combined with Ki-67 might be used to predict HCC patient prognosis.     

肝硬化组织中卵圆细胞的光镜、电镜与免疫电镜观察

目的：探讨人肝硬化肝组织是否存在卵圆细胞。方法：对30例人肝细胞肝癌手术标本的癌旁肝硬化组织行常规组织学观察,并用胆管上皮分化标记CK7和肝细胞分化标记白蛋白对组织作免疫组化染色,同时对其中10例作了超微结构,5例作免疫电镜标记和观察。另外对5例正常肝脏和5例肝炎后肝硬化组织同时作了光镜、免疫组化和电镜观察,对2例肝炎后肝硬化作了免疫电镜标记和观察。结果：癌旁肝硬化组织的30例光间和10例电镜观察以及5例肝炎肝硬化光、电镜观察,均可以再生的肝细胞结节边缘见到散在的小细胞和增生的小胆管样结构。这些小细胞和增生的小胆管内少数小细胞,为卵圆形,体积较小,核大,胸质少,胞质内含较多的游恼核糖体,仅含少量粗面内质网和线粒体,质内可见张力微丝结构,这类细胞与邻近细胞间均有细胞间连接。免疫电镜示,卵圆细胞均表达CK7和白蛋白,但有些细胞内表达CK7多些,有些细胞则表达白蛋白多些。在正常肝组织内未见到类似细胞。结论：与动物实验性肝癌模型肝组织一样,人类肝硬化肝组织中出存在同样细胞形态和免疫表型特点的卵圆细胞。结果支持卵圆细胞可能为肝前体细胞或干细胞的假设。     

Colorectal cancer and noncancer patients have similar labeling indices by microscopy and computed image analysis

The labeling index (LI), a microscopic measurement of proliferative activity in colonic crypts, is proposed as an indicator of colonic cancer risk. Computed image analysis of proliferative regions is less labor intensive and more objective than is direct microscopy but has not been validated for labeling indices by direct comparison. The authors compared colonic crypt proliferation in 26 cancer and 13 noncancer patients by using Ki-67 monoclonal antibody (McAb) labeling of flat mucosa obtained from surgically removed, frozen specimens. In cancer patients, the mucosa specimen was excised 10 cm away from the tumor, and the LI was determined microscopically for the whole crypt, the upper two thirds, and the upper one third of 15 crypts. Nuclear antigen levels of 15 whole crypts were determined by using the CAS-200 computed image analyzer (Cell Analysis Systems, Elmhurst, IL). Cancer and noncancer specimens were compared as were microscopically determined LI and stained nuclei specimens by using image analysis. No statistically significant difference in proliferative activity of whole crypts, or the upper two thirds of crypts, was observed between cancer specimens and noncancer specimens from using either technique. However, a significant correlation existed between microscopic analysis and computed image analysis of labeled nuclei. Computed image analysis using Ki-67 McAb labeling can be used instead of microscopy to determine crypt LI, but neither method can be used to distinguish cancer specimens from noncancer specimens.     

人肝癌与癌旁肝AFP阳性细胞形态的图象分析及增殖活性的研究

采用免疫组化、双标免疫组化及图象分析技术，研究了２７例人体肝细胞癌与癌旁肝甲胎蛋白（ＡＦＰ）阳性细胞的量化形态特征及以其增殖细胞核抗原（ＰＣＮＡ）标记率显示的增殖活性。主要结果显示：（１）ＡＦＰ阳性与阴性癌及癌旁近癌肝细胞间难以用量化形态标准区分（Ｐ＞０．０５）；核面积与核浆比提示癌旁ＡＦＰ阳性肝细胞可能是尚无表型转化的癌前细胞。（２）ＡＦＰ阳性癌细胞ＰＣＮＡ的平均标记率（１５．７０％±６．２５％）显著低于肝癌的平均标记率（Ｐ＜０．０１），表明大多数ＡＦＰ阳性癌细胞处于非增殖状态。（３）癌旁近癌肝可见ＡＦＰ与ＰＣＮＡ双标阳性肝细胞。     

Serotyping and genotyping of hepatitis C virus (HCV) strains in chronic HCV infection

Hepatitis C virus (HCV) genotypes can be established by methods based on PCR typing and serological typing. The accuracy of these methods depends on their sensitivity and specificity. These should be compared with the reference method, direct sequencing, and analysis of viral genomes. Among the serologic methods recently developed, the performance of a new serotyping assay (RIBA HCV 3.0 SIA, Chiron corporation, Emeryville) was assessed using a panel of 147 well-characterized French isolates from chronic hepatitis C patients. Definitive genotypes of the isolates were established by direct sequencing in 5′ NC and in some cases in NS-5B. HCV serotypes 1, 2, and 3 were determined by measuring type specific antibodies to core and NS-4 derived peptide antigens. Of the 147 sera, serotypic-specific antibodies were detected in 136 (sensitivity, 92.5%). The specificity of the RIBA SIA HCV serotyping assay was 92.6% (including samples with mixed results); without these, the specificity was 80.1%. Analysis of the 28 discrepant samples showed that (1) a different serotype was found in 18 samples including five for genotype 1, three for genotype 2, two for genotype 3, five for genotype 4, and three for genotype 5, and that (2) ten patients showed a reactivity with mixed serotypes, one had circulating antibodies to type 1 or 2, and nine had circulating antibodies to type 1 or 3. In summary, except for genotypes 4 and 5, the results of the test were well correlated (85.7%) with those of direct sequence genotyping. The former test is rapid and does not require the strict HCV RNA storage and preservation conditions of the latter. This new method may thus be considered as an alternative for HCV typing. However, although it is convenient, its lower sensitivity compared to the molecular typing method and the discrepant results limit its routine use in a clinical context. J. Med. Virol. 52:391–395, 1997. © 1997 Wiley-Liss, Inc.     

Hepatitis C virus protein interaction network for HCV clearance and association of DAA to HCC occurrence via data mining approach: A systematic review and critical analysis

HCV has been associated with a pro‐inflammatory state, which predisposes to hepatocellular carcinoma (HCC). However, the different molecular mechanisms underlying the effect of HCV infection on HCC progression remain unclear. Although HCV infection illustrates the potential role of host genetics in the outcome of infectious diseases, there is no clear overview of some single nucleotide polymorphisms (SNPs) influencing spontaneous or treatment‐induced HCV eradication. We studied the possible role of HCV infection in the processes of HCC initiation and performed a systematic analysis using data mining approaches to identify host polymorphisms associated with treatment response and HCC development using topological analysis of protein‐proteins interactions (PPI) networks. On the basis of our analysis performed, we identified key hub proteins related to HCV‐treatment response infection and to HCC development. Host genetic polymorphisms, such as inosine triphosphatase (ITPA), interferon, lambda 3 (IFNL3), Q5 interferon, lambda 4 (IFNL4), toll‐like receptors (TLRs) and interferon‐stimulated gene 15 (ISG‐15), were identified as key genes for treatment prediction and HCC evolution. By comparing unique genes for HCV‐treatment response and genes particular to HCV‐HCC development, we found a common PPI network that may participate in more extensive signalling processes during anti‐HCV treatment, which can play important roles in modulating the immune response to the occurrence of HCC. Data mining is an effective tool for identifying potential regulatory pathways involved in treatment response and HCC development. Our study may contribute to a better understanding of HCV immunopathogenesis and highlights the complex role of host genetics in HCV clearance.     

肝细胞肝癌中卵圆细胞的组织学与超微结构研究

目的：探讨人肝癌肝组织存在卵圆细胞的可能性。方法：对２０例人肝细胞肝癌的手术标本行常规组织学和超微结构观察，并用胆管上皮分化标记ＣＫ７和肝细胞分化标记白蛋白对以上组织作免疫组化染色，同时对其中５例作免疫电镜标记。结果：光镜下，１４／２０例癌肿边缘常可见到增生的小胆管样结构。电镜下，１４／２０例可找到三型卵圆细胞。其中Ⅰ型细胞体积较小、核大、胞质少，此为较为原始的卵圆细胞。Ⅱ型细胞体积稍大，胞质稍多     

Small epithelial cells in extrahepatic biliary atresia: electron microscopic and immunoelectron microscopic findings suggest a close relationship to liver progenitor cells

AIMS: It is still unclear whether hepatic stem cells that give rise to both biliary epithelial cells and hepatocytes exist in the human liver. The aim of this study was to investigate whether cells with ultrastructural and immunophenotypical features similar to those of the oval cells of rodents or the small epithelial cells (SEC) described recently in hepatoblastoma, i.e. putative hepatic progenitor cells, are found in the liver of patients with extrahepatic biliary atresia. METHODS AND RESULTS: Liver biopsies from 10 infants with extrahepatic biliary atresia were investigated by transmission electron microscopy. Single and double immunolabelling for cytokeratin 7, a marker of biliary differentiation, and albumin, a marker of hepatocytic differentiation, was investigated by immunoelectron microscopy. Electron microscopy revealed SEC that were ultrastructurally similar to the oval cells and coexpressed albumin and cytokeratin 7. The SEC exhibited a spectrum of differentiation that, in addition to relatively undifferentiated cells, included cells that exhibited morphological and immunophenotypical signs of differentiation towards biliary epithelial cells and hepatocytes. CONCLUSIONS: The findings demonstrate that SEC with morphological and immunophenotypical features of the oval cells of rodents and the SEC described in hepatoblastoma are found in the liver of patients with extrahepatic biliary atresia. The data further support the hypothesis that the SEC represent possible candidates for hepatic progenitor cells.     

Small epithelial cells in human liver cirrhosis exhibit features of hepatic stem-like cells: immunohistochemical,electron microscopic and immunoelectron microscopic findings

AIMS: To investigate whether cells with features similar to those of the oval cells of rodents and the small epithelial cells (SEC) recently described in certain human liver diseases, i.e. hepatic progenitor cells, also occur in human liver cirrhosis. METHODS AND RESULTS: Surgical specimens from 35 cases of hepatitis B virus-positive cirrhosis (30 cases containing hepatocellular carcinoma) were investigated by immunohistochemical staining for cytokeratin 7 and albumin. Electron microscopic investigations, and immunoelectron microscopic investigations using the same antibodies and a double-labelling technique were performed in 15 and seven cases, respectively. SEC were observed in proliferated bile ductules, at the margins of regenerating nodules and in the fibrous septa in all cases of cirrhosis. The SEC were morphologically similar to the SEC described previously, and to the oval cells seen in experimental hepatocarcinogenesis. They were characterized by their small size, oval shape, scanty electron-dense or electron-lucent cytoplasm, a high nucleo-cytoplasmic ratio, tonofilaments and intercellular junctions. Immunoelectron microscopy revealed that the SEC co-expressed cytokeratin 7 and albumin. Both relatively undifferentiated SEC and SEC with morphological and immunophenotypical signs of differentiation towards biliary epithelial cells and hepatocytes were found. CONCLUSIONS: SEC that exhibit morphological and immunophenotypical features of the SEC seen in certain other liver diseases are found in cirrhosis. These findings further support the hypothesis that a bipotent hepatic stem cell that may give rise to biliary epithelial cells and hepatocytes exists in the human liver.     

影响肝细胞癌生物学行为及预后的相关因子分析

目的探讨影响肝癌生物学行为及预后的重要因素和寻求肝癌基因治疗的有效靶点。方法运用免疫组化方法检测Ki67、nm23H1、cMet及MT1MMP蛋白在93例肝细胞癌组织中的表达水平,并判断其与肝癌细胞恶性生物学行为及患者预后的关系。结果Ki67、nm23H1、cMet及MT1MMP蛋白表达水平与肝癌患者预后均具有显著相关性(P均<0.01);nm23H1的表达与肿瘤远处转移之间呈负相关关系(P=0.041);cMet、MT1MMP的表达与肝癌瘤旁浸润及远处转移之间均呈正相关关系(P<0.05)。结论nm23H1基因的失活或突变,伴随cMet以及MT1MMP蛋白的异常表达可能是导致肝癌浸润、转移的关键因素。     

Association of IL28B gene polymorphism with development of hepatocellular carcinoma in Japanese patients with chronic hepatitis C virus infection

IL28B single nucleotide polymorphisms (SNPs) are associated with spontaneous and treatment-induced elimination of hepatitis C virus (HCV). To assess whether the IL28B rs8099917 SNP also affects the progression of chronic HCV infection, we genotyped 511 Japanese HCV patients, including 69 with hepatocellular carcinoma (HCC). The T/T genotype of rs8099917 was not associated with the development of HCC (p = 0.623), although stepwise logistic regression analysis showed that liver cirrhosis, age greater than 68 years, and serum albumin <4.2 mg/dl were associated with HCC onset. It appears that the IL28B SNP does not directly influence hepatocarcinogenesis in chronic HCV infection.     

Immunohistochemical expression of CD95 (Fas), c-myc and epidermal growth factor receptor in hepatitis C virus infection, cirrhotic liver disease and hepatocellular carcinoma

Gene product expression in normal and chronic hepatitis C virus infection was determined in an attempt to improve our understanding of the molecular events leading to the development of cirrhosis and liver carcinoma. Activation of CD95 (Fas) causes apoptosis of cells and liver failure in mice and has been associated with human liver disorders. c-myc is involved in cell proliferation and EGFR in regeneration of cells. The material of the current study included 50 cases of chronic hepatitis C (CHC) (and negative hepatitis B virus infection), 29 cases of liver cirrhosis and HCV (LC), and 19 cases of hepatocellular carcinoma and HCV (HCC) admitted to the Theodor Bilharz Research Institute (TBRI) during the years 2003-2004. Ten wedge liver biopsies - taken during laparoscopic cholecystectomy - were included in the study as normal controls. Laboratory investigations, including liver function tests, serological markers for viral hepatitis and serum alpha fetoprotein level (alpha-FP), were determined for all cases. Histopathological study and immunohistochemistry using monoclonal antibodies for CD95, c-myc and EGFR were also done. In CHC cases, the histological activity index (HAI) revealed more expression of Fas antigen in liver tissues with active inflammation than in those without active inflammation (p < 0.01). EGFR and c-myc act synergistically in liver tumorigenesis. Upregulation of Fas in chronic hepatitis C infection and of c-myc & EGFR in malignant transformation was concluded from this study. c-myc expression may obstruct the induction of apoptosis of HCC cells and lead to uncontrolled cell growth.     

Immunohistochemical staining of cancer stem cell markers in hepatocellular carcinoma

Background

Cancer stem cells (CSCs) are thought to be a critical subpopulation in tumor development, progression, metastasis and recurrence, and the identification of these cells is an initial step in understanding their role in oncogenesis and in seeking valuable markers for diagnosis or development of targeting therapeutics.

Aims

To identify CSCs in hepatocellular carcinoma (HCC) specimens and define their tissue specificity.

Methods

Immunohistochemical staining of CSC markers: CD44, CD90, CD133 and aldehyde dehydrogenase (ALDH) was performed in 25 HCC specimens, 4 hepatoblastomas, 8 peri-malignant tissues, and 19 cases of viral hepatitis.

Results

The positivity of CD44 staining in HCC specimens was significantly lower than in viral hepatitis specimens. The positive rate of CD133 in HCC was similar to viral hepatitis specimens. CD133⁺ cells were largely localized to ALDH-positive cells in HCC as revealed by confocal microscopy. In contrast, the co-expression of both markers was visualized within vessels or in the portal areas in viral hepatitis. Moreover, among 7 liver specimens adjacent to HCC tissue, 3-6 samples were positive for CD44, CD90, CD133 and ALDH, especially in dysplastic cells. One of 4 hepatoblastoma cases was positive for all these markers; whereas, the other three specimens were negative for all these CSC markers.

Conclusions

In HCC and dysplastic tissues, clusters of CD133⁺/ALDH^high cells were identified. The use of cancer stem cell markers to screen tissues with chronic liver diseases provides limited guidance in the identification of malignant cells.     

High frequency of hepatocellular carcinoma in Mongolia; association with mono-, or co-infection with hepatitis C, B, and delta viruses

To investigate the association between viral infection pattern and hepatocellular carcinoma (HCC), 292 chronic hepatitis patients, including 108 with developed HCC were screened using serological and molecular genetics methods. Viral etiology was established in 267 (91.4%), anti-HCV detected in 198 (67.8%), and HBsAg in 124 (42.5%) including 93 (74.4%) cases with HDV co-infection. HCV mono-infection predominated in both, "non-HCC" and "HCC" groups (54% and 39%, respectively) with higher frequency in the first group (P = 0.011), whereas HBV in co-infection with HDV was more frequent in HCC group (14% vs 25%, P = 0.017). Patients with HCV mono-infection were older than those with co-infection (P<0.02), had higher frequency of HCV-viraemia (82% vs 7%, P < 0.0001), and yet had significantly lower prevalence of HCC (29.6% vs. 49.1%, P = 0.003). Alpha-fetoprotein (AFP) and protein induced by vitamin K antagonist-II (PIVKA-II) were specifically elevated in 71% of HCC patients. In conclusion, although HCV monoinfection pattern predominates in Mongolia, co-infection with HBV and HDV had stronger association with HCC development at younger age. Liver tumor markers; AFP and PIVKA-II are useful tools for complex HCC-screening and clinical follow-up for chronic hepatitis patients in Mongolia.     

Case report: a case of light chain deposition disease involving liver and stomach with chronic hepatitis C virus infection and hepatocellular carcinoma

Light chain deposition disease (LCDD) is a rare, plasma cell proliferative disorder characterized by mainly abnormal light chain deposition in various organs. Hepatitis C virus (HCV) is a hepatotrophic and lymphotrophic virus and significantly related to B-cell proliferation. This is a case report of systemic LCDD involving liver, stomach, bone marrow, and probably kidney, in a patient with HCV-related hepatocellular carcinoma (HCC). A 62-year-old man with chronic HCV infection who presented with a small HCC in segment 8 of the liver and nephrotic syndrome showed kappa typed immunoglobulin light chain depositions in biopsy specimens of bone marrow, stomach, and non-tumorous liver parenchyma. After treatment of the HCC with transarterial chemoembolization, antiviral therapy for chronic hepatitis C was started. The patient showed early virologic response at 12 weeks of treatment; however, antiviral therapy was discontinued due to adverse effects and he was lost to follow-up. This is the first case of LCDD involving the liver and stomach in a patient with chronic HCV infection and HCC, which may represent LCDD as a rare HCV-associated B cell proliferative disease.