[Prediction and preventive strategies for recurrence after surgery for hepatocellular carcinoma].

Many advances have been achieved in the clinical and basic studies on metastasis and recurrence of hepatocellular carcinoma (HCC) over the past 20 years. The achievements mainly include the following aspects: (1) a group of molecules related to metastasis and recurrence including osteopontin have been identified, and multi-molecular predictive models for metastasis have been established and optimized; (2) it has been found that the imbalance of immune response in tumor microenvironment is important in promoting metastasis and can be used to predict metastasis and recurrence; (3) it has been found and confirmed that interferon can prevent postoperative recurrence, and patients with a lower miR-26a expression level can achieve greater benefits; (4) the breakthroughs in liquid biopsy and immunotherapy bring a promising future for the prediction, prevention, and treatment of HCC metastasis and recurrence. However, these predictive models still need to be validated by multi-center studies, and the effects of adjuvant transarterial chemoembolization and targeted therapy with sorafenib still need further evaluation.

Value of alpha-fetoprotein and clinical characteristics in patients with liver neoplasm.

This article aimed to investigate the value of α-fetoprotein (AFP) for the diagnosis of hepatocellular carcinoma (HCC) and to evaluate the relationship between AFP and various clinical variables of HCC comprehensively. A retrospective study of postoperative patients diagnosed with liver neoplasm from two Chinese centers was enrolled in our study.A total of 3050 patients were included. The best cut-off point of AFP for the diagnosis of HCC was 20ng/ml with ideal sensitivity (69.74%), specificity (91.18%), LR (4.12) and YI (0.61). Non-HBV infection patients showed the highest specificity (94.44%) but lowest sensitivity (60.13%). In HBV infection. Patients, HBsAg, HBeAb, and HBcAb positive patients had the highest sensitivity (79.55%) and specificity (58.49%). AFP levels increased significantly in symptomatic patients (p=0.011). Those patients with tumor sizes ≥10cm had much higher serum AFP level compared with smaller tumors ones (p=0.014). AFP levels increased remarkably in patients with vascular invasion (p=0.015). Stepwise logistic regression showed tumor size (≥10cm) was an independent predictor of elevated AFP (OR=2.743, 95%CI: 1.167-6.447, P=0.021). The best discriminating AFP value for the diagnosis of HCC is 20ng/ml; HBsAg, HBeAb and HBcAb positive patients have the optimal sensitivity and specificity; tumor size ≥ 10cm is an independent predictor of elevated AFP.

Is FCGR2A a susceptibility gene to systemic lupus erythematosus in Chinese?

Recent genome-wide association scans and replication studies reinforce that FCGR2A is a susceptibility gene in systemic lupus erythematosus (SLE) in Caucasians. However, previous case control studies denied such conclusions in Chinese people. Besides genetic heterogeneity among different ethnicities, copy number variation (CNV), non-homogenous phenotypes and insufficient power may be confounders. We performed a case control study with 1066 Chinese (589 SLE patients and 477 healthy controls) and a meta-analysis based on 2328 SLE patients and 2313 healthy controls. FCGR2A CNV and FCGR2A131H/R [rs1801274] were detected by TaqMan assays. No variation of copy numbers of FCGR2A gene was found in Chinese. A further case control study suggested a dose-response character for FCGR2A131H/R and it affected disease activity, severity and prognosis. Finally, meta-analysis indicated FCGR2A that was a susceptibility gene to SLE in Chinese with an odds ratio of 1.094 and population attributable risk proportion of (PARP) 0.031. By an integrative strategy, we validate that FCGR2A bears no population-specific CNV. FCGR2A131H/R contributes to SLE susceptibility in Chinese, and affects disease activity, severity and prognosis. The undetected association in Chinese derives from under-power rather than any methodological obstacle due to CNV or population-specific genetic effect.

HTPAP gene on chromosome 8p is a candidate metastasis suppressor for human hepatocellular carcinoma.

Our previous studies suggested that chromosome 8p deletion is associated with metastasis of hepatocellular carcinoma (HCC), in which some novel metastasis suppressor genes might be harbored. The present study aimed to identify the metastatic suppressor gene(s). A cDNA chip was constructed with the expressed sequence tags (ESTs) from chromosome 8p and used to compare the difference of expression profiling between the MHCC97-H and MHCC97-L cell lines with different metastatic potentials and similar genetic backgrounds. In all, 10 ESTs were significantly downregulated in MHCC97-H cell line with higher metastatic potential. One full-length gene, HTPAP (phosphatidic acid phosphatase type 2 domain containing 1B), was identified at chromosome 8p12. Sequencing and bioinformatic analyses revealed that HTPAP has 826 bp and encodes a putative protein of 175 amino acids with a transmembrane segment at the NH2 terminus, two protein kinase C phosphorylation site and one tyrosine kinase phosphorylation site. Its expression level in metastatic tumor tissues was much lower than that of primary HCC tissues. Both in vitro and in vivo assays suggested that HTPAP could suppress the invasion and metastasis of HCC. These suggested that HTPAP is a novel metastatic suppressor gene for HCC. The mechanism of the effect of HTPAP on HCC metastasis is not clear yet and deserves further investigation.

The association of chromosome 8p deletion and tumor metastasis in human hepatocellular carcinoma.

To understand the genetic mechanisms underlying the progression of hepatocellular carcinoma (HCC) metastasis, differences of genomic alterations between 10 pairs of primary HCC tumors and their matched metastatic lesions were analyzed by comparative genomic hybridization. Several chromosomal alterations including loss of 8p, 4q, 17p, and 19p, gain of 5p and high-level amplification of 1q12-q22 were detected in two or more cases. The most significant finding is the loss of 8p which was detected in 8 metastatic tumors but only in 3 corresponding primary tumors (P = 0.03). This result suggests that the deletion of chromosome 8p might contribute to the development of HCC metastasis. Another interesting result is the detection of a minimum high-level amplification region at 1q12-q22 in HCC. This result provides a candidate amplification region in HCC for further study to identify amplified oncogenes related to the development or progression of HCC. Finally, this study provides a practicable model to detect specific genetic alterations related to the tumor metastasis through comparing the primary tumor and its corresponding metastatic lesion using comparative genomic hybridization technique.

Integrative genomics identifies YY1AP1 as an oncogenic driver in EpCAM(+) AFP(+) hepatocellular carcinoma.

Identification of key drivers and new therapeutic targets is important given the poor prognosis for hepatocellular carcinoma (HCC) patients, particularly those ineligible for surgical resection or liver transplant. However, the approach to identify such driver genes is facing significant challenges due to the genomically heterogenous nature of HCC. Here we tested whether the integrative genomic profiling of a well-defined HCC subset that is classified by an extreme EpCAM(+) AFP(+) gene expression signature and associated with poor prognosis, all attributes of a stem cell-like phenotype, could uncover survival-related driver genes in HCC. Following transcriptomic analysis of the well-defined HCC cases, a Gene Set Enrichment Analysis coupled with genomic copy number alteration assessment revealed that YY1-associated protein 1 (YY1AP1) is a critical oncoprotein specifically activated in EpCAM(+) AFP(+) HCC. YY1AP1 silencing eliminates oncogene addiction by altering the chromatin landscape and triggering massive apoptosis in vitro and tumor suppression in vivo. YY1AP1 expression promotes HCC proliferation and is required for the maintenance of stem cell features. We revealed that YY1AP1 cooperates with YY1 to alter the chromatin landscape and activate transcription of stemness regulators. Thus YY1AP1 may serve as a key molecular target for EpCAM(+) AFP(+) HCC subtype. Our results demonstrate the feasibility and power of a new strategy by utilizing well-defined patient samples and integrative genomics to uncover critical pathways linked to HCC subtypes with prognostic impact.

Clustered genes within the genome of Arabidopsis thaliana encoding pectin methylesterase-like enzymes.

We focused our attention on the isolation and regulation of genes encoding for pectin methylesterase (PME) isoenzymes in Arabidopsis thaliana (At). The present data reports the identification of two PME-like genes, named AtPME1 and AtPME2, that are closely linked in the At genome. These genes possess different structural organisations. While all higher plant PME characterised so far possess an intron at a similar location relative to their coding sequence, AtPME2 shows an additional intron whose presence within other higher plant PME-like genes has not been previously reported. Sequence comparison of the N-terminal region suggests that the secretory pathways of the putative AtPME1 and AtPME2 isoforms are different, and that this region may contribute to specify a biological function to each isoform. Moreover, phylogenetic analysis reflects the possible existence of functional groups of PME isoforms in higher plant species that seem to have been conserved during evolution.

Petunia p34cdc2 protein kinase activity in G2/M cells obtained with a reversible cell cycle inhibitor, mimosine.

Protoplasts isolated from petunia leaf mesophyll are non-cycling cells mostly with 2C content. Cells regenerating from protoplast culture enter mitosis after 48 h. This experimental model is used to relate p34cdc2 kinase activity to cell cycle phase. Our results show that the histone H1 phosphorylation, and hence p34cdc2 kinase activity, peaks with G2+early M cell cycle phase. However, a trace kinase activity was already present when most cells were entering S phase. To obtain a maximum of cells in G1+S phases, the protoplast culture was treated with the rare amino acid, mimosine. Mimosine blocked plant cells derived from protoplast culture both at G1 and in early and mid S phase. Despite the increased G1+S level, p34cdc2 kinase activity did not increase. This suggests that the trace activity appearing when the majority of cells are entering S does not correspond to any putative p34cdc2 activation at G1/S transition but to the activation of the minor 4C population initially present in the leaf: the hypothesis remains that p34cdc2 kinase activity is solely related to G2+M phase in petunia.

Molecular cloning and characterisation of a putative pectin methylesterase cDNA in Arabidopsis thaliana (L.).

Pectin methylesterase (PME) is a cell wall enzyme that catalyses the de-esterification of pectins leading to fundamental changes which confer new properties to the micro-environment of each cell. In order to elucidate the meaning of PME-mediated changes of pectin in the time course of cell differentiation, we attempted to study the regulation of PME genes in Arabidopsis thaliana. In this report, the first full cDNA sequence showing sequence similarities with other PME genes characterised so far in other plant species has been isolated from an Arabidopsis shoot cDNA library. This ATPMEl cDNA is 1,970 bp long and contains an open reading frame encoding a protein of 64.1 kDa and a basic pI of 8.7 as predicted from the nucleotide sequence. Northern blot analyses denoted changes in the expression level of the ATPMEl mRNA according to plant organs. High mRNA levels were found in young developing organs such as cauline leaves while they were significantly lower in rosette leaves, stems and inflorescences, and almost undetectable in roots. Beside this molecular approach, isoelectrofocusing analyses revealed the occurrence of three PME isoforms in Arabidopsis. Two PME isoforms with pI values of 4.9 and 9.1 were found throughout the plant, but at a higher level in the root, while an other PME isoform with a pI of 5.7 was essentially detected in the inflorescence. The relationship between our observations and the data reported for other plant species is discussed.

Olomoucine, an inhibitor of the cdc2/cdk2 kinases activity, blocks plant cells at the G1 to S and G2 to M cell cycle transitions.

The cdc2/cdk2 protein kinases play key roles in the cell cycle at two control points: the G1/S transition and the entry into mitosis. Olomoucine, a specific inhibitor of these kinases, was tested in two plant cell systems: Petunia mesophyll protoplasts induced to divide and Arabidopsis thaliana cell suspension cultures. The cell cycle status was analysed from DNA histograms or through continuous labelling of cells with 5-bromodeoxyuridine (BrdUrd) followed by double staining with bis-benzimide (Hoechst 33258) and propidium iodide (PI). Such analyses resolve cells from several generations according to the extent of their DNA replication. Olomoucine was shown to reversibly arrest differentiated Petunia cells induced to divide at G1 phase and cycling Arabidopsis cells in late G1 and G2. A comparison of the effects of aphidicolin, oryzalin and olomoucine suggests that in the Arabidopsis cell suspension culture, a cdc2/cdk2-like kinase is activated at a restriction point in late G1.

Chromosome 8p deletion is associated with metastasis of human hepatocellular carcinoma when high and low metastatic models are compared.

Recently, we found that chromosome 8p deletion might be associated with hepatocellular carcinoma (HCC) metastasis by analyzing the differences in chromosomal alterations between primary tumors and their matched metastatic lesions of HCC with comparative genomic hybridization (CGH) (Qin et al. 1999). To further confirm this interesting finding, the genomic changes of two models bearing human HCC with different metastatic potentials (LCI-D20 and LCI-D35), and the new human HCC cell line with high metastatic potential (MHCC97) were analyzed by CGH. Gains on 1q, 6q, 7p, and 8q, and losses on 13p, 14p, 19p, 21, and 22 were detected in both LCI-D20 and LCI-D35 models. However, high copy number amplification of a minimum region at 1q12-q22 and 12q, and deletions on 1p32-pter, 3p21-pter, 8p, 9p, 10q, 14q, and 15p were detected only in the LCI-D20 model. Gains on 1p21-p32, 2p13-p21, 6p12-pter, 9p, 15q, and 16q11-q21, and losses on 2p23-pter, 4q24-qter, 7q31-qter, 12q, 17p, and 18 were detected only in the LCI-D35 model. The chromosomal aberration patterns in the MHCC97 cell line were similar to its parent LCI-D20 model, except that gains on 19q and losses on 4, 5, 10q, and 13q were found only in the cell line. These results provide some indirect clues to the metastasis-related chromosomal aberrations of HCC and further support the finding that 8p deletion is associated with HCC metastasis. 1q12-22 and 12q might harbor a novel oncogene(s) that contributes to the development and progression of HCC. Amplification on 8q and deletions on 4q and 17p may be not necessary for HCC metastasis.

Metastatic human hepatocellular carcinoma models in nude mice and cell line with metastatic potential.

Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient-like metastatic model of human HCC in nude mice (LCI-D20) and a low metastatic model of human HCC in nude mice (LCI-D35) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metastasis to liver, lungs, lymph nodes and peritoneal seeding. Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-1 increased gradually following tumor progression in LCI-D20 model, and correlated with tumor size and AFP level. Phasic expression of tissue intercellular adhesion molecule-1 in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including anti-angiogenesis,antisense approach, metalloproteinase inhibitor, differentiation inducer, etc. It is concluded that the establishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vitro and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.

Phase I clinical trial of oral furtulon and combined hepatic arterial chemoembolization and radiotherapy in unresectable primary liver cancers, including clinicopathologic study.

Surgical resection has been accepted as the only curative therapy for primary liver cancer (PLC). Unfortunately, most patients are surgically unresectable when they seek treatment. An alternative therapeutic approach for some of these patients is transcatheter arterial chemoembolization. However, this is not curative by itself, and additional therapy is required to eradicate residual disease. This study investigates the approach of preoperative hepatic arterial chemoembolization followed by the combination of oral Furtulon (5'-deoxy-5-fluorouridine) as a radiosensitizer and external beam radiotherapy (RT). From July 1997 to December 1998, 25 patients with unresectable PLC were treated with hepatic arterial chemoembolization followed by limited-field radiotherapy plus oral Furtulon as a radiosensitizer. Hepatic arterial chemoembolization was performed with 5-fluorouracil 1 g, cisplatin 80 mg (DDP), mitomycin C (MMC) 10 mg, and arterial embolization with iodized oil-10 ml mixed with 10 mg MMC. Hepatic arterial chemoembolization was performed at regular intervals of 6 weeks, and the patients then received limited-field RT. Mean tumor dose was 4,600 cGy (range, 4,100-5,200 cGy) in daily 1.8-Gy fractions, 5 times a week. The toxicity and responses between RT and surgery were assessed. After surgical evaluation, resection was performed. The histopathologic study was also performed in the specimens of both normal and radiation-injured liver tissues from the patients who underwent resection. Seventeen of 25 patients (68%) showed an objective response. One patient with cholangiocarcinoma involving the portal lymph nodes attained a complete response. Eight patients (32%) underwent sequential resection. The most common toxicity was an increase in liver enzymes, which were less than twofold of the upper limit of normal. Follow-up computed tomography studies after treatment showed a low-attenuation area adjacent to the hepatic tumor in the target volume. On pathologic evaluation, the low-attenuation area revealed hyperemia, distended hepatic sinusoids packed with erythrocytes, and hepatic cell loss when examined with microscopy; "new-born" hepatocytes, hepatic cords in the process of forming, and endothelial cells have appeared on electronic microscopic examination. The combination of hepatic arterial chemoembolization and external radiotherapy is efficacious and a safe modality for unresectable primary liver cancers. Furtulon offers the potential for use as a clinical radiosensitizer. Radiation can significantly damage the liver tissue between 41 Gy and 52 Gy, but the new hepatocytes were forming within the radiation-injured liver after RT.

Serum vascular endothelial growth factor is a predictor of invasion and metastasis in hepatocellular carcinoma.

Tumor progression is angiogenesis dependent, and vascular endothelial growth factor (VEGF) is a key growth factor in this process. sVEGF concentrations in 44 patients with hepatocellular carcinoma (HCC) and 5 with benign liver lesions were determined with an enzyme-link immunoadsorbent assay system (ELISA). Reverse transcript-polymerase chain reaction (RT-PCR) was carried out on surgical specimens of 51 patients with HCC. The relative levels of VEGF mRNA expression were measured by determining a ratio between PCR products of VEGF and the endogenous internal standard gene b-actin. UEA-1 was histochemically used to count microvascularity in tumor tissue. Elevated sVEGF concentrations were found in patients with HCC (172.84+/-111.75 pg/ml) as compared to individuals with benign liver lesions (95.74+/-36.20 pg/ml, P<.05). Of 44 cases with HCC, sVEGF concentrations in the patients with PV-emboli or with poor-encapsulated tumors were significantly higher than in those without PV-emboli or with well-encapsulated tumors (P<0.05). The expression levels of VEGF mRNA in tumors with PV-emboli and in poor-encapsulated tumors were higher than in those without PV-emboli and in well-encapsulated tumors (P<0.05). Microvascular density in HCC tissues was significantly correlated with the expression levels of VEGF mRNA (P<0.01; r=0.7). Circulating VEGF was derived from HCC tissue. sVEGF concentrations could be a new marker for predicting the angiogenesis, invasion and metastasis of HCC.

Effect of antiangiogenic agents on experimental animal models of hepatocellular carcinoma.

A new therapeutic strategy for treating metastasis in hepatocellular carcinoma (HCC) has entailed the use of antiangiogenic agents such as suramin, BB-94 (Batimastat), TNP-470, and carboxyamido-triazole (CAI, a synthetic inhibitor of non-excitable calcium channels that reversibly inhibits angiogenesis). These agents have been used to treat metastatic model of HCC in nude mouse (LCI-D20 mouse model). The results of these studies are summarized in this paper with emphasis on the inhibitory effects of the drugs on tumour growth, angiogenesis, invasion and metastasis in LCI-D20 mouse models. The results suggest that all of the agents used can significantly inhibit tumour growth, angiogenesis, invasion and metastasis of human HCC in nude mouse models, and may be candidates for the control of recurrence and metastasis after HCC resection.

Mutations in isocitrate dehydrogenase 1 and 2 occur frequently in intrahepatic cholangiocarcinomas and share hypermethylation targets with glioblastomas.

Mutations in the genes encoding isocitrate dehydrogenase, IDH1 and IDH2, have been reported in gliomas, myeloid leukemias, chondrosarcomas and thyroid cancer. We discovered IDH1 and IDH2 mutations in 34 of 326 (10%) intrahepatic cholangiocarcinomas. Tumor with mutations in IDH1 or IDH2 had lower 5-hydroxymethylcytosine and higher 5-methylcytosine levels, as well as increased dimethylation of histone H3 lysine 79 (H3K79). Mutations in IDH1 or IDH2 were associated with longer overall survival (P=0.028) and were independently associated with a longer time to tumor recurrence after intrahepatic cholangiocarcinoma resection in multivariate analysis (P=0.021). IDH1 and IDH2 mutations were significantly associated with increased levels of p53 in intrahepatic cholangiocarcinomas, but no mutations in the p53 gene were found, suggesting that mutations in IDH1 and IDH2 may cause a stress that leads to p53 activation. We identified 2309 genes that were significantly hypermethylated in 19 cholangiocarcinomas with mutations in IDH1 or IDH2, compared with cholangiocarcinomas without these mutations. Hypermethylated CpG sites were significantly enriched in CpG shores and upstream of transcription start sites, suggesting a global regulation of transcriptional potential. Half of the hypermethylated genes overlapped with DNA hypermethylation in IDH1-mutant gliobastomas, suggesting the existence of a common set of genes whose expression may be affected by mutations in IDH1 or IDH2 in different types of tumors.

Randomized clinical trial of the effects of abdominal drainage after elective hepatectomy using the crushing clamp method.

BACKGROUND: Abdominal drainage is a standard procedure after hepatectomy, but this practice has been challenged recently. METHODS: Between September 2004 and March 2005, 120 consecutive patients who had undergone hepatic resection by the same surgical team were randomly allocated into drainage and no drainage groups (60 in each group). Patient characteristics, preoperative liver function, presence of cirrhosis, resection-related factors and postoperative complications were compared between the two groups. RESULTS: The groups were comparable in terms of demographics, indications for surgery, preoperative liver function test results, presence of cirrhosis, extent of hepatectomy, intraoperative blood loss and requirement for blood transfusion. Symptomatic subphrenic collection and pleural effusion occurred in four patients (7 per cent) who had abdominal drainage and three (5 per cent) who did not. Local wound complications occurred in 17 (28 per cent) and two (3 per cent) patients respectively (P < 0.001). The postoperative hospital stay was similar in the two groups. Multivariate analysis indicated that the presence of cirrhosis and abdominal drainage were independently related to the development of postoperative wound complications. CONCLUSION: Routine abdominal drainage is unnecessary after elective hepatectomy using the crushing clamp method.

Studies on monoclonal anti-idiotypic and anti-isotypic antibodies against leukemia and myeloma: III. Analysis of monoclonal antibodies recognizing the antigenic epitopes by use of ELISA additivity test and microcomputer grouping programme.

ELISA double antibodies additivity test was employed to identify the epitopes which can be recognized by monoclonal antibodies (McAbs) against IgM of a patient with B chronic lymphocytic leukemia (B-CLL). The computer grouping programme analysis showed that 4 anti-isotype McAbs could be divided into two groups and 10 anti-idiotype McAbs could be divided into four groups. The result was consistent with that of the indirect ELISA, ELISA sandwich as well as ELISA inhibition test. The above findings suggested that there are at least 6 distinct IgM epitopes which can specifically react with 14 McAbs. Our study indicated that the combination of the ELISA additivity test and the computer grouping programme analysis provides a helpful tool for researchers into the relationship between the structure and function of antigens.

A cdc2 gene of Petunia hybrida is differentially expressed in leaves, protoplasts and during various cell cycle phases.

Analysis of p34cdc2 kinase in higher eukaryotes has demonstrated that p34cdc2 function is conserved in all eukaryotic cells. The p34cdc2 kinase (the product of the cdc2 gene) is required during the G1 cell cycle phase at the initiation of DNA replication and also in G2-M phases for entry into mitosis. In this paper we report the isolation and characterization of a cdc2 Petunia hybrida PCR fragment (cdc2Pet). Using a DNA probe based on this fragment and a p34cdc2-specific antibody, cdc2Pet transcript and p34 protein levels were found to be constant both in 2C nuclei of highly proliferating mesophyll 2C cells derived from protoplasts and in 2C nuclei isolated directly from expanded petunia leaves. Both the cdc2Pet transcript and p34cdc2 protein levels were found to be higher in nuclei at 4C than in those at 2C, even when these 4C nuclei were from non-proliferating tissue. Thus cdc2Pet mRNA and protein levels measured in different tissues should not be interpreted to reflect exclusively the proliferative state of the tissue but also the frequency of G2 cells including those in the differentiated state.