Mutations In Hepatocellular Carcinoma Research Articles

Aflatoxin-Induced TP53 R249S Mutation in HepatoCellular Carcinoma in Thailand: Association with Tumors Developing in the Absence of Liver Cirrhosis

Primary Liver Cancer (PLC) is the leading cause of death by cancer among males in Thailand and the 3rd among females. Most cases are hepatocellular carcinoma (HCC) but cholangiocarcinomas represent between 4 and 80% of liver cancers depending upon geographic area. Most HCC are associated with chronic infection by Hepatitis B Virus while a G→T mutation at codon 249 of the TP53 gene, R249S, specific for exposure to aflatoxin, is detected in tumors for up to 30% of cases. We have used Short Oligonucleotide Mass Analysis (SOMA) to quantify free circulating R249S-mutated DNA in plasma using blood specimens collected in a hospital case:control study. Plasma R249S-mutated DNA was detectable at low concentrations (≥67 copies/mL) in 53 to 64% of patients with primary liver cancer or chronic liver disease and in 19% of controls. 44% of patients with HCC and no evidence of cirrhosis had plasma concentrations of R249S-mutated DNA ≥150 copies/mL, compared to 21% in patients with both HCC and cirrhosis, 22% in patients with cholangiocarcinoma, 12% in patients with non-cancer chronic liver disease and 3% of subjects in the reference group. Thus, plasma concentrations of R249S-mutated DNA ≥150 copies/mL tended to be more common in patients with HCC developing without pre-existing cirrhosis (p = 0.027). Overall, these results support the preferential occurrence of R249S-mutated DNA in HCC developing in the absence of cirrhosis in a context of HBV chronic infection.

Epidemiology of Viral Hepatitis and Hepatocellular Carcinoma

Most cases of hepatocellular carcinoma (HCC) are associated with cirrhosis related to chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Changes in the time trends of HCC and most variations in its age-, sex-, and race-specific rates among different regions are likely to be related to differences in hepatitis viruses that are most prevalent in a population, the timing of their spread, and the ages of the individuals the viruses infect. Environmental, host genetic, and viral factors can affect the risk of HCC in individuals with HBV or HCV infection. This review summarizes the risk factors for HCC among HBV- or HCV-infected individuals, based on findings from epidemiologic studies and meta-analyses, as well as determinants of patient outcome and the HCC disease burden, globally and in the United States.

Abstract 2109: PIK3CA mutations in hepatocellular carcinoma in Korea

Abstract PI3K/AKT pathway plays a pivotal role in HCC. Mutant PIK3CA, encoding the p110a catalytic subunit, stimulate the AKT pathway and promotes cell growth in various cancers. PIK3CA mutations have been researched in many cancer types and reported low frequency of PIK3CA mutation in HCC except Korea’ case. To investigated the frequency of PIK3CA mutations in Korean patients with HCC. We sequenced the exon2, 4, 5, 7, 8, 9, 10, 20 and 21 of PIK3CA in 268 HCC samples. In these experiments, the mutation were not detected in exon4, 7, 9, 20 and detected 1 of 268 at unknown SNP (G278A) in exon2 and at unknown SNP (C848G) in exon5, 1 of 265 at unknown SNP (C1306A) and 2 of 265 at unknown SNP (C1373A) in exon8, each of 262 at unknown SNP (T2970A) and SNP (G3026A) in exon21. However, exon10 were detected 1 of 266 at unknown SNP (C1629T), 1 of 268 at G1635T (E545D) and 266 of 266 at A1634C (E545A) in 268 HCC samples. Additional experiments through normal tissue and cloning experiments realized double peak at A1634C of exon10 is not mutation and maybe arose as a result gene duplication on 22q11 partial region because exon10 primer have higher efficiency. Our study suggests no clinical characteristic relationship between HCC and mutated of PIK3CA. In conclusion, our results with using large sample size suggest the mutations in PIK3CA gene of Korean patients’ frequency is low as like other countries have been previously reported. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2109. doi:1538-7445.AM2012-2109

Expression of SLCO1B3 is associated with intratumoral cholestasis and CTNNB1 mutations in hepatocellular carcinoma

Recent studies have shown that intratumoral cholestasis is a hallmark of CTNNB1 mutations in hepatocellular carcinomas (HCC). Here, we analyzed the expressions of genes involved in bile acid and bilirubin metabolism and their correlation with the mutational status of CTNNB1 in a series of HCC. The expressions of CYP7A1 and CYP27A1, which encode rate-limiting enzymes in bile acid synthesis, were unaltered or only marginally increased in CTNNB1-mutated HCC compared with those in HCC with wild-type CTNNB1. Among the genes involved in bile acid and bilirubin transport, the expression of SLCO1B3 was significantly elevated in HCC with CTNNB1 mutations, whereas the expression of ABCC4 was elevated in HCC with wild-type CTNNB1. Immunohistochemistry confirmed the frequent expression of SLCO1B3 in CTNNB1-mutated HCC at the protein level, but not in most HCC with wild-type CTNNB1. Immunohistochemistry for MRP4 (encoded by ABCC4) partly agreed with ABCC4 expression, but most cases did not express detectable levels of MRP4. Notably, all HCC with bile accumulation, including those without CTNNB1 mutations, expressed SLCO1B3, suggesting that SLCO1B3 expression, rather than CTNNB1 mutation, is the critical determinant of intratumoral cholestasis. As SLCO1B3 is involved in the uptake of a number of chemotherapeutic and diagnostic agents, SLCO1B3 expression and the status of CTNNB1 mutation might need to be considered in the drug delivery to HCC.

Low Frequency of PIK3CA Gene Mutations in Hepatocellular Carcinoma in Chinese Population

PI3K/AKT constitutes an important pathway regulating the signaling of multiple biological processes and plays a critical role in carcinogenesis. PIK3CA gene missense mutations have been reported in many human cancer types. The mutation of it in hepatocellular carcinoma cases varies with different races and regions. In this study, we investigated PIK3CA mutation in Chinese hepatocellular carcinoma patients. A total 90 Chinese patients of hepatocellular carcinoma were recruited in this study. Exons 9 and 20 hotspots mutations of PIK3CA gene were detected by PCR-based DNA sequencing. Two point mutations (E542K and D549H) in exon 9 were found in only one patient (1/90; 1.11%), no mutation was found in exon 20 in any cases. 57 patients are associated with HBV infection (57/90; 63.3%), and 8 patients with HCV infection (8/90; 8.9%). The frequency of the PIK3CA mutations in hepatocellular carcinoma seems to be lower in Chinese hepatocellular carcinoma patients. These findings suggest that PI3K mutations may not play a major role in hepatic carcinogenesis in Chinese. HBV infection has close relationship with HCC in Chinese.

Overexpression of α-methylacyl-CoA racemase is associated with CTNNB1 mutations in hepatocellular carcinomas

α-Methylacyl-CoA racemase (AMACR) is expressed in the majority of hepatocellular carcinomas (HCCs) at variable levels, but the significance of AMACR overexpression remains elusive. The aim of this study was to investigate the relationship between AMACR expression and the presence of CTNNB1 mutations in HCCs. The expression of AMACR and GLUL, an established downstream target of β-catenin was examined in HCCs, by quantitative reverse transcription polymerase chain reaction (PCR), and the expression of their protein products by immunohistochemistry. The quantitative reverse transcription PCR analysis showed that the expression of AMACR was significantly higher in HCCs with CTNNB1 mutations than in mutation-negative HCCs or normal livers, like the expression of GLUL. Immunohistochemistry also showed that strong AMACR protein expression was closely correlated with the presence of CTNNB1 mutations. HCCs with CTNNB1 mutations and those with AMACR overexpression frequently exhibited bile production. The overexpression of AMACR was closely correlated with the presence of CTNNB1 mutations in HCCs. AMACR is a putative target of β-catenin as well as an excellent immunohistochemically detectable marker of HCCs with CTNNB1 mutations. As AMACR is physiologically involved in bile acid synthesis, the current observation implies a regulatory role of β-catenin in bile acid metabolism.

Mitochondrial mutations in hepatocellular carcinomas and fibrolamellar carcinomas

Mitochondrial mutations are well documented in hepatocellular carcinoma, but their role in carcinogenesis remains unclear. To clarify their significance, a comprehensive analysis was performed of hepatocellular carcinomas (N=24), including quantifying the total mitochondrial DNA levels, quantifying the levels of mitochondrial DNA with the common deletion, and complete sequencing of the mitochondrial control region. In addition, these studies were expanded and reinforced by analysis of fibrolamellar carcinomas (N=15), a unique type of liver carcinoma that has increased numbers of mitochondria on electron microscopy. Overall, approximately 50% of hepatocellular carcinomas had lower levels of total mitochondrial DNA than paired non-neoplastic tissues. Interestingly, despite their increased numbers of mitochondria, primary fibrolamellar carcinomas had lower levels of total mitochondrial DNA. In contrast, metastatic fibrolamellar carcinomas had greatly increased mitochondrial DNA levels. Overall, deletions in the control region were associated with lower total DNA levels in typical hepatocellular carcinoma, but somatic single base pair mutations were not. In fact, almost all single base pair mutations were either reversions to the wild-type sequence or known population polymorphisms, strongly suggesting they are not directly oncogenic. Complete sequencing of the entire mitochondrial genome in fibrolamellar carcinomas identified several somatic mutations, but no consistent pattern of mutations was found. Overall, the levels of the common deletion were highest in tissues with lower total mitochondrial DNA. In conclusion, control region deletions, but not somatic mutations, may influence total DNA copy numbers. Somatic control region mutations in hepatocellular carcinoma are not directly oncogenic but instead may be adaptive.

Abstract 1983: TP53 mutations in hepatocellular carcinoma: Association with survival and stem cell-like gene expression traits

Abstract PURPOSE Mutations of TP53, a tumor suppressor gene, are associated with prognosis of many cancers. However, the prognostic value of TP53 mutations in hepatocellular carcinoma (HCC) is still unclear, largely because of the enormous heterogeneity in the geographic and etiological backgrounds of the HCC patients. Here, we analyzed the prognostic values of TP53 mutations. Gene expression profiling analysis was performed to evaluate the effect of TP53 mutations at the molecular level. PATIENTS AND METHODS TP53 mutations were investigated in a total of 409 HCC patients including Chinese (n=336) and Caucasian (n=73) patients using direct sequencing method. RESULTS A total of 125 TP53 mutations were found in Chinese HCC patients (37.2 %). HCC patients harboring TP53 mutations showed shorter overall survival compared to patients with wild-type TP53 (Hazard ratio [HR], 1.86; 95% confidence interval [CI], 1.37-2.52; P<0.001). The hotspot mutations at R249S and V157F were significantly associated with worse prognosis in both univariate (HR, 2.11; 95% CI, 1.51-2.94; P<0.001) and multivariate analyses (HR, 1.79; 95% CI, 1.29-2.51; P<0.001). In addition, gene expression profile analysis revealed the expression of stem cell-like traits in TP53 mutated tumors. This finding was validated in breast and lung cancers with TP53 mutations. CONCLUSION TP53 mutations, particularly hotspot mutations, are associated with poor prognosis in HCC. The acquisition of stem cell-like gene expression traits may contribute to the aggressive behavior of the TP53 mutated tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1983.

Mdm2 Snp309 G allele displays high frequency and inverse correlation with somatic P53 mutations in hepatocellular carcinoma

Loss of function of the p53 protein, which may occur through a range of molecular events, is critical in hepatocellular carcinoma (HCC) evolution. MDM2, an oncogene, acts as a major regulator of the p53 protein. A polymorphism in the MDM2 promoter, SNP309 (T/G), has been shown to alter protein expression and may thus play a role in carcinogenesis. MDM2 SNP309 is also associated with HCC. However, the role of SNP309 in hepatocarcinogenesis with respect to TP53 mutations is unknown. In this study, we investigated the distribution of the MDM2 SNP309 genotype and somatic TP53 (the p53 tumor suppressor gene) mutations in 99 human HCC samples from Africa, Europe, China and Japan. Samples exhibited striking geographical differences in their distribution of SNP309 genotypes. The frequency and spectrum of p53 mutations also varied geographically; TP53 mutations were frequent in Africa, where the SNP309 T/T genotype predominated but were rare in Europe and Japan, where the SNP309 G allele was present more frequently. TP53 mutations were detected in 18% (4/22) of SNP309 T/G and G/G and 82% (18/22) of SNP309 T/T genotype holders; this difference was statistically highly significant ( P-value = 0.0006). Our results indicated that the presence of the SNP309 G allele is inversely associated with the presence of somatic TP53 mutations because they only coincided in 4% of HCC cases. This finding suggests that the SNP309 G allele may functionally replace p53 mutations, and in addition to known etiological factors, may be partly responsible for differential HCC prevalence.

Lack of somatic ErbB2 tyrosine kinase domain mutations in hepatocellular carcinoma

Small molecules targeting the epidermal growth factor receptor (EGFR) intracellular tyrosine kinase domain have shown promising activity in cancer therapeutics. Recent reports suggest activity of erlotinib, an ErbB1 inhibitor, and lapatinib, a dual inhibitor of ErbB1 and ErbB2, in hepatocellular carcinoma (HCC). Activating ErbB1 somatic mutations may predict treatment responses. We have previously reported ErbB1 tyrosine kinase domain mutations to be rare or absent in HCC, but data on the frequency of ErbB2 tyrosine kinase domain mutations in HCC is currently limited, apart from reports of a missense mutation identified in 11% of a small Caucasian sample. We studied exons 18-23 of the ErbB2 gene from tumor DNA of 100 Asian human HCC and found no exonic mutations of potential significance. Alternative mechanisms may be responsible for the observed therapeutic efficacy of ErbB1 and ErbB2 tyrosine kinase inhibitors.

Screen p53 mutations in hepatocellular carcinoma by FASAY: A novel splicing mutation

To establish a routine procedure for the detection of p53 mutations in hepatocellular carcinoma (HCC) surgical resections using the FASAY (functional analysis of separated alleles of p53 on yeast) procedure. p53 status was analyzed by FASAY and cDNA sequencing in 50 cases of HCC. After the extraction of RNA from the frozen tumor and corresponding normal tissues, reverse transcription RT-PCR was carried out using these samples. The assay can detect mutations of p53 mRNA between codons 67 and 347 by the DNA-binding activity of the protein and reveal them as red colonies. Of the 50 specimens, 29 (58%) were positive (mutant) by FASAY. Sequencing analysis confirmed that all 29 FASAY positive tumors harbored mutations, and that no mutations were detectable in any FASAY negative tumors. In 29 p53 mutations, 22 mutations were point missense mutation, 5 were deletions and 2 were splicing mutations. A novel splice mutation on splice donor of intron 6 was reported, which could produce two different mRNAs, respectively using the nearest upstream and downstream recessive splice donor sites. FASAY is a sensitive method for detecting the various types of p53 mutations in HCC, suggesting that the yeast functional assay for the detection of p53 mutations may be essential for elucidating their clinical significance.

A Dose-Dependent Decrease in the Fraction of Cases Harboring M6P/IGF2R Mutations in Hepatocellular Carcinomas from the Atomic Bomb Survivors

The risk for hepatocellular carcinoma (HCC) development is significantly heightened in the atomic bomb survivors, but the mechanism is unclear. We have previously reported finding a radiation dose-dependent increase in HCCs with TP53 mutations from the survivors. We now show that, in the same HCC samples, the frequency of 3'-untranslated region (3'UTR) mutations in M6P/IGF2R, a candidate HCC tumor suppressor gene, decreases with dose (P = 0.0091), implying a radiation dose-dependent negative selection of cells harboring such mutations. The fact that they were in the 3'UTR implicates changes in transcript stability rather than in protein function as the mechanism. Moreover, these M6P/IGF2R 3'UTR mutations and the TP53 mutations detected previously were mutually exclusive in most of the tumors, suggesting two independent pathways to HCC development, with the TP53 pathway being more favored with increasing radiation dose than the M6P/IGF2R pathway. These results suggest that tumors attributable to radiation may be genotypically different from tumors of other etiologies and hence may provide a way of distinguishing radiation-induced cancers from "background" cancers--a shift from the current paradigm.

Mutations of tumor suppressor gene PTEN mutations in hepatocellular carcinoma and its implications in tumor proliferation and apoptosis

To study mutations of tumor suppressor gene PTEN in human hepatocellular carcinomas and its effects on the proliferation and apoptosis of hepatocellular carcinoma cell line HHCC. (1) PCR-SSCP and sequence analysis were used to detect the mutations of the 5th and 8th exon of PTEN in 42 cases of human primary hepatocellular carcinoma. (2) Eukaryotic expression vectors of the wild-type (pEGFP-wt-PTEN) and the mutant type (pEGFP-PTEN, G129R) of PTEN were constructed. Lipofectamine 2000 mediated gene transfection was used to transfect hepatocellular carcinoma cell line HHCC, in which the PTEN protein is not expressed. Culture medium containing G418 was used to select stable transfectants. MTT colorimetry was used to analyze the proliferation ability of selected cell lines. Naive HHCC cells and HHCC cells transfected with empty vector (pEGFP-C1) served as controls. (3) TNF-alpha was used to induce apoptosis of selected cell clones. (1) Point mutation involving the 5th exon of PTEN was detected in 4 of 42 primary hepatocellular carcinomas. (2) Compared with the control groups, the proliferation of hepatocellular carcinoma cells was significantly inhibited by the transfection of wild-type PTEN gene, while the transfection with mutant PTEN construct did not significantly change the proliferation. (3) The apoptosis indices of cells transfected with the wild-type and the mutant PTEN genes were 13.8% and 8.1% respectively. Compared with the control, the apoptosis index of HHCC cell transfected by the wild type PTEN was significantly lower (P < 0.05). There were no significant differences between HHCC cells transfected with mutated PTEN gene and the control (P > 0.05). The expression of internal 473-phosphorylated Akt of HHCC was weak, but was enhanced when the cells treated with TNF-alpha. However, it was down regulated by the wild type PTEN. (1) First time report that PTEN mutations can be found in 9.5% human primary hepatocellular carcinomas. (2) The expression of the wild-type PTEN can suppress the proliferation of HHCC cells, and such suppression was lost when PTEN gene was mutated. (3) PTEN inhibition of the proliferation and the enhancement of apoptosis of hepatocellular carcinoma cells is likely related to a down-regulation of the TNF-alpha induced activation of protein kinase Akt pathway.

Absence of PIK3CA hotspot mutations in hepatocellular carcinoma in Japanese patients

A recent study revealed that the p110alpha (PIK3CA), catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is somatically mutated in many types of cancer. For example, PIK3CA is mutated in an estimated 35.6% of hepatocellular carcinoma (HCC) cases. To measure the frequency of PIK3CA hotspot mutations in Japanese HCC patients, exons 9 and 20 of the PIK3CA gene were sequenced in 47 clinical HCC samples. Contrary to expectations, no hotspot mutations were found any of the HCC samples. In addition, we found abnormally migrating waves near the end of exon 9 in the PCR chromatograms from 13 of the 47 samples. PCR amplification and subsequent cloning and sequencing revealed that these chromatograms contained two distinct sequences, the wild-type p110alpha sequence and a different sequence found on human chromosome 22q11.2, the Cat Eye Syndrome region, which contains a putative pseudogene of PIK3CA. These abnormally migrating waves were also found in noncancerous liver tissue, indicating that this was not a result of HCC-associated mutations. Therefore, it is likely that the percentage of hotspot mutations in the PIK3CA gene of Japanese HCC patients is lower than was previously reported.

MYH Y165C and G382D mutations in hepatocellular carcinoma and cholangiocarcinoma patients

Production of reactive oxygen species (ROS) during chronic inflammation has been implicated in the progression of liver diseases and carcinogenesis. Subjects with inflammatory liver disease and one non-functional allele of the base excision repair gene, MYH, may be more susceptible to progression to cancer due to MYH haploinsufficiency in repairing oxidative damage caused by ROS. Here, we investigated the association of two common germline MYH mutations in patients with hepatocellular carcinoma (HCC) and cholangiocarcinoma. DNA from patients with HCC (n=48) or cholangiocarcinoma (n=84) compared to non-cancerous controls (n=308) were genotyped for the Y165C and G382D mutations in MYH. There was no significant difference in MYH mutation carrier status between patients with HCC (1/48), cholangiocarcinoma (3/84), and non-cancerous controls (4/308). Patients with HCC or cholangiocarcinoma do not have an increased incidence of monoallelic MYH mutations pre-disposing them to disease.

Absence of epidermal growth factor receptor exon 18–21 mutation in hepatocellular carcinoma

The epidermal growth factor receptor (EGFR) is overexpressed in many epithelial tumors and plays an important role in the tumorigenesis of these tumors. Inhibitors of EGFR reduce the proliferation rate of cancers and are promising therapeutic agents of cancers. Recently, two studies have identified somatic mutations in the exons 18–21 of EGFR that were strongly correlated with robust clinical response to gefitinib treatment in patients with non-small cell lung cancer. To investigate whether EGFR mutation is involved in the tumorigenesis of hepatocellular carcinoma (HCC), we performed direct sequencing of exons 18–21 on 89 HCCs. No mutations causing amino acid changes or deletions were identified. The results indicate mutation of the kinase domain of EGFR does not play a significant role in the tumorigenesis of HCC and gefitinib is unlikely to be used as single-drug therapy for HCC.

Correlation between clinical characteristics and mitochondrial D-loop DNA mutations in hepatocellular carcinoma

Mitochondrial DNA (mtDNA) mutations are found in many kinds of human cancer. The aim of this study was to evaluate the relationship between mtDNA mutations in the liver and human hepatocarcinogenesis. Direct sequencing of mtDNA was done in 54 hepatocellular carcinomas (HCCs) and 47 surrounding liver tissue samples, obtained from 54 patients with HCC, and in 5 liver samples without inflammation, obtained from 5 patients with metastatic liver tumors. We also examined p53 mutations in the 54 HCCs to examine the correlation between nuclear DNA mutations and mtDNA mutations. Mutations of mtDNA in the D-loop region were found in both HCC and noncancerous liver tissue. In normal liver without chronic inflammation, no mtDNA mutation was detected. In every case, the number of mtDNA mutations in HCC correlated with that in noncancerous liver tissue. Twelve of 52 mutation sites in the D-loop region of mtDNA were specific for HCC. The mean number of mtDNA mutations was 1.7 in well-differentiated HCC, as compared with 4.5 in moderately differentiated HCC and 4.6 in poorly differentiated HCC. The frequency of mtDNA mutations was thus higher in less differentiated HCC. We detected p53 mutations in 15 (28%) of 54 HCCs. The mean number of mtDNA mutations was 5.3 in HCC with p53 mutations and 3.8 in HCC with wild-type p53 (P = 0.024). A higher frequency of mtDNA mutations was found in less differentiated HCCs, and it is also possible that mtDNA mutations are related to nuclear DNA mutations in HCC. The accumulation of mtDNA mutations is a useful predictor of hepatocarcinogenesis.

High levels of p53 protein expression do not correlate with p53 mutations in hepatocellular carcinoma

To determine the relationship between p53 altered expression and p53 mutations in hepatocellular carcinoma (HCC), we analysed p53 protein immunohistochemically and assessed the presence of mutations in exons 4-8 of the p53 gene using SSCP assay in 117 HCCs corresponding to 78 patients. We also determined the relationship of p53 expression with cellular proliferation by immunostaining with monoclonal antibodies to Ki-67. We found significant levels of p53 protein expression in 23.1% of the 117 cases studied, but identified mutations in only 12 cases (10.3%). Only four of the p53-positive cases had mutations in the regions analysed. Six of the cases that displayed mutations at p53 gene were negative for immunohistochemical analysis (IHC) and two cases showed positive immunoreactivity in the cytoplasm of the cell. In conclusion, strong IHC reactivity for p53 protein is not an indicator of the presence of p53 gene mutations at exons 4-8 in HCC. Thus, p53 loss of function in HCC should be evaluated both by p53 mutation analysis and p53 protein expression, as both give complementary information about p53 status.

Heparanase mRNA expression and point mutation in hepatocellular carcinoma.

To explore the expression of heparanase mRNA and point mutation in hepatocellular carcinoma (HCC). Reverse transcription polymerase chain reaction was used to measure the expression of heparanase mRNA in the primary tumor tissues and surrounding liver tissues of 33 HCC patients. T-A cloning and sequencing were used to detect whether there was any mutation in the amplified PCR products. The expression of heparanase mRNA was positive in 16 primary tumor tissues of HCC, and the positive rate was 48.5%, which was significantly higher than that in the surrounding liver parenchyma (P<0.01). The positive rate for heparanase gene in high-tendency to metastatic recurrence group (71.4%, 10/14) was obviously higher than that in low-tendency to metastatic recurrence group (31.6%, 6/19) (P = 0.023). The positive rate for heparanase gene in patients with metastatic recurrence during postoperative follow-up (78.6%, 11/14) was also significantly higher than that in those without metastatic recurrence (21.4%, 3/14) (P = 0.003). Sequence analysis of the HPA PCR products was made in 7 patients, and 2-point mutations were found in 4 patients, one of which was sense mutation, neither base insertion nor deletion was detected. The mutation rate was 57.1% (4/7). The expression rate of heparanase mRNA increases in HCC, and HPA mRNA may be one of the reliable markers for the metastatic activity gained by the liver tumor cells and could be used clinically in predicting metastatic recurrence of HCC. Point mutation may be one of the causes for enhanced heparanase mRNA expression.

Plasma 249Ser p53 mutation in patients with hepatocellular carcinoma residing in a high risk area.

To investigate plasma p53 mutation in hepatocellular carcinoma (HCC) patients from Qidong and to define its significance. Blood samples from 25 hepatocellular carcinoma patients, 20 cirrhotic patients and 30 healthy controls in Qidong area. DNA was extracted and purified from 200 microl of plasma from each sample. The 249(Ser) p53 mutation was detected by restriction digestion analysis and by direct sequencing of exon-7 PCR products. G-->T transversion at the third base of 249 codon resulting in 249(Arg)-->249(Ser) mutation in exon 7 of p53 gene were found in 11/25(44%) hepatocellular carcinoma cases, 4/20 (20%) cirrhotics, and 2/30 (7%) healthy controls (p<0.01). These data show that the 249(Ser) p53 mutation in plasma is strongly associated with hepatocellular carcinoma patients in Qidong area and the mutation should be screened as a new early diagnostic marker for HCC.