TP53 R249S mutation detected in circulating tumour DNA is associated with Prognosis of hepatocellular carcinoma patients with or without hepatectomy.

BACKGROUND AND AIMS: Somatic mutation R249S in TP53 is highly common in hepatocellular carcinoma (HCC). We aim to investigate the effects of R249S in ctDNA on the prognosis of HCC. METHODS: We analysed three cohorts including 895 HCC patients. TP53 mutation spectrum was examined by direct sequencing of genomic DNA from tissue specimens in HCC patients with hepatectomy (Cohort 1, N = 260). R249S and other recurrent missense mutations were assessed for their biological functions and associations with overall survival (OS) and progression-free survival (PFS) of HCC patients in Cohort 1. R249S within circulating tumour DNA (ctDNA) was detected through droplet digital polymerase chain reaction (ddPCR) and its association with OS and PRS was analysed in HCC patients with (Cohort 2, N = 275) or without (Cohort 3, N = 360) hepatectomy. RESULTS: In Cohort 1, R249S occupied 60.28% of all TP53 mutations. Overexpression of R249S induced more serious malignant phenotypes than those of the other three identified TP53 recurrent missense mutations. Additionally, R249S, but not other missense mutations, was significantly associated with worse OS (P = .006) and PFS (P = .01) of HCC patients. Consistent with the results in Cohort 1, HCC patients in Cohorts 2 and 3 with R249S had worse OS (P = 8.291 × 10-7 and 2.608 × 10-7 in Cohorts 2 and 3, respectively) and PFS (P = 5.115 × 10-7 and 5.900 × 10-13 in Cohorts 2 and 3, respectively) compared to those without this mutation. CONCLUSIONS: TP53 R249S mutation in ctDNA may serve as a promising prognosis biomarker for HCC patients with or without hepatectomy.

The transcription factor Ikaros inhibits cell proliferation by downregulating ANXA4 expression in hepatocellular carcinoma.

The occurrence and progression of hepatocellular carcinoma (HCC) are affected by complicated signal transduction factors. Our previous study identified Ikaros as a novel reactivated therapeutic target that acts as a transcriptional repressor and reactivates anticancer mechanisms in HCC therapy. Annexin A4 (ANXA4) is a member of the Annexin family that plays an essential role in several cancers, but it has not been investigated in HCC proliferation. Using cDNA microarrays, ANXA4 was shown to be associated with Ikaros in Ikaros-overexpressing cells. The aim of this work was to characterize the relationship between Ikaros and ANXA4 and the role of ANXA4 in HCC. The effect of Ikaros on ANXA4 was analyzed in HCC cell lines and HCC patient samples, and functional recovery experiments were performed between Ikaros and ANXA4. Furthermore, the effect of ANXA4 on cell proliferation in vitro was analyzed by MTT and colony formation assays in HCC cells. We used a subcutaneous xenograft model to elucidate the role of ANXA4 in vivo. We found that ANXA4 overexpression promotes HCC cell proliferation, but Ikaros can inhibit ANXA4 expression by repressing its promoter activity. Moreover, we demonstrated that downregulated expression of ANXA4 inhibited HCC cell proliferation and tumorigenesis in vitro and in vivo. Our findings indicate that ANXA4 may be a critical factor in HCC tumorigenesis. Ikaros is an attractive inhibitor of ANXA4 and may function as an anticancer agent in HCC.

Caspase polymorphisms and prognosis of hepatocellular carcinoma.

The aim of our study was to determine the impact of genetic polymorphisms in the caspase (CASP) genes on prognosis of hepatocellular carcinoma (HCC). We genotyped 7 potentially functional polymorphisms in CASP3, CASP7, CASP8, CASP9, CASP10 genes in 362 HCC patients of receiving surgical resection of HCC tumor. The associations of genotype and haplotype with overall survival (OS) and disease free survival (DFS) were analyzed by using the Cox proportional hazards model. We found that the CASP9 rs4645981 C allele was significantly associated with positive effect on DFS (P = 0.011 and 0.016 for CT+CC vs. TT in univariate and multivariate analysis, respectively), CT genotype was associated with a better OS of HCC than the TT genotype both in univariate and multivariate analysis (P = 0.048 and 0.041, respectively). Moreover, the CASP3 rs2705897 GT genotype showed marginally significant association with decreased OS and DFS, compared with the GG genotype. One haplotype TT/TG in CASP3 (constructed by rs12108497 T>C and rs2705897 T>G) was significantly associated with decreased OS and DFS, compared to the common haplotype TT/TT both in univariate analysis (P = 0.021 and 0.026, respectively) and multivariate analysis (P = 0.025 and 0.030, respectively). The haplotype GT/GT in CASP9 (constructed by rs4645978 A>G and rs4645981 C>T) was significantly associated with decreased DFS both in univariate and multivariate analysis (P = 0.012 and 0.010, respectively). In conclusion, the CASP9 rs4645981 polymorphism, CASP3 and CASP9 haplotypes may be useful prognosis markers for HCC patients with surgical resection of tumor.

Id4 promotes cell proliferation in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common malignant tumor in the world, especially in China. As a member of the inhibitor of differentiation (Id) family, Id4 has been reported to function in many cancer types, but relatively little is known about its role in HCC. The purpose of this study was to investigate the potential relationship between Id4 and HCC development and the underlying mechanism involving the function of Id4 in HCC. METHODS: We used quantitative real-time polymerase chain reaction and Western blotting to examine the RNA and protein expression of Id4. In addition, we used Cell Counting Kit-8 assay and colony formation assay to identify the function of Id4 in the regulation of cell proliferation in human HCC. RESULTS: We found that the expression of Id4 protein was up-regulated in tumor tissues from HCC patients. Overexpression of Id4 promoted HCC cell proliferation, clonogenicity in vitro, and tumorigenicity in vivo. Id4 knockdown experiments showed that silencing Id4 blocked the proliferation and colony formation ability of HCC cells in vitro. Furthermore, overexpression of CCAAT/enhancer-binding protein ß inhibited Id4 expression in HCC cells. CONCLUSION: Id4 may be developed as a potent therapeutic agent for the treatment of HCC, but more details about the underlying mechanisms of action are needed.

Nine susceptibility loci for hepatitis B virus-related hepatocellular carcinoma identified by a pilot two-stage genome-wide association study.

Previous studies have indicated that complex interactions among viral, environmental and genetic factors lead to hepatocellular carcinoma (HCC). To identify susceptibility alleles for hepatitis B virus (HBV)-related HCC, the present study conducted a pilot two-phase genome-wide association study (GWAS) in 660 Han Chinese individuals. In phase 1, a total of 500,447 single-nucleotide polymorphisms (SNPs) were genotyped in 50 HCC cases and 50 controls using Affymetrix GeneChip 500k Array Set. In phase 2, 1,152 SNPs were selected from phase 1 and genotyped in 282 cases and 278 controls using the Illumina GoldenGate platform. The prior probability of HCC in control subjects was assigned at 0.01, and false-positive report probability (FPRP) was utilized to evaluate the statistical significance. In phase 1, one SNP (rs2212522) showed a significant association with HCC (Pallele=5.23×10-8; ORallele=4.96; 95% CI, 2.72-9.03). In phase 2, among 27 SNPs with unadjusted Pallele<0.05, 9 SNPs were associated with HCC based on FPRP criteria (FPRP <0.20). The strongest statistical evidence for an association signal was with rs2120243 (combined ORallele=1.76; 95% CI, 1.39-2.22; P=2.00×10-6), which maps within the fourth intron of VEPH1. The second strongest statistical evidence for an association was identified for rs1350171 (combined ORallele=1.66; 95% CI, 1.33-2.07; P=6.48×10-6), which maps to the region downstream of the FZD4 gene. The other potential susceptibility genes included PCDH9, PRMT6, LHX1, KIF2B and L3MBTL4. In conclusion, this pilot two-phase GWAS provides the evidence for the existence of common susceptibility loci for HCC. These genes involved various signaling pathways, including those associated with transforming growth factor ß, insulin/phosphoinositide 3 kinase, Wnt and epidermal growth factor receptor. These associations must be replicated and validated in larger studies.

miR-492G>C polymorphism (rs2289030) is associated with overall survival of hepatocellular carcinoma patients.

Single-nucleotide polymorphisms (SNPs) of microRNAs (miRNAs) are considered potential markers of cancer risk and prognosis in various cancers. In the current study, the primary aim is to determine whether the miR-492G>C polymorphism (rs2289030) altered hepatocellular carcinoma (HCC) prognosis. The SNP rs2289030 of miR-492 was genotyped using DNA from blood samples of 362 HCC patients that had undergone surgical resection of a HCC tumor. The associations between overall survival and demographic characteristics, clinical features, and the SNP rs2289030 were estimated using the Cox proportional hazards model. Results showed that patients who carried the CG genotype (P = 0.015, hazard ratio [HR] = 0.704, 95 % confidence interval [CI] 0.530-0.934) and CG+GG genotype (P = 0.011, HR = 0.703, 95 % CI 0.536-0.924) had significantly decreased risk of death compared to those with the CC genotype. Similar results were found in the multivariate analysis adjusted by tumor size and venous invasion. Further stratification analysis indicated that the effect of rs2289030 had more prominence in patients ≤50 years old and that reported ever using alcohol, male gender, a family history of HCC, being HbsAg or alpha fetoprotein (AFP) positive, differentiation I + II, presence of venous invasion or cirrhosis, multiple tumors, and pTNM stage I + II. Results from this study illustrate the potential use of miR-492 rs2289030 as a prognostic marker for HCC patients that have undergone a surgical resection of the tumor.

MiR-608 rs4919510 is associated with prognosis of hepatocellular carcinoma.

Single nucleotide polymorphisms (SNPs) within microRNAs (miRNAs) are considered potential markers for risk and prognosis of various cancers. In the current study, we aimed to determine whether miR-608 rs4919510 affected hepatocellular carcinoma (HCC) prognosis. We genotyped rs4919510 using DNA from blood samples of 362 HCC patients receiving surgical resection of HCC tumor. Associations between rs4919510 and overall survival (OS) and demographic characteristics and clinical features were estimated using the Cox proportional hazards model. Results showed that HCC patients who carried the rs4919510 CC genotype had a significantly longer OS compared to those who carried the GG genotype (P = 0.013, hazard ratio [HR] = 0.600, 95 % confidence interval [CI] 0.402-0.897) and the CG + GG genotype (P = 0.033, HR = 0.681, 95 % CI 0.479-0.970) in univariate analysis. Similar results were obtained in multivariate analysis. Further stratification analysis indicated that rs4919510 was significantly associated with OS in patients who were satisfied with one of the following criteria: male gender, HbsAg-positive, α-fetoprotein (AFP)-positive, tumor size >5 cm, cirrhosis, solitary tumor, I + II pTNM stage, or no tumor capsule. Finally, a significantly higher frequency of rs4919510 CC genotype was observed in patients with cirrhosis (22.9 %, 55/240) than those without cirrhosis (14.0 %, 17/121) (P = 0.047). In conclusion, our results illustrated the potential role of miR-608 rs4919510 as a prognostic marker for HCC patients undergoing surgical resection of the tumor.

Genetic polymorphisms in apoptosis-related genes and the prognosis of hepatocellular carcinoma.

The apoptotic pathway is important in the control of vital processes of hepatocellular carcinoma (HCC). In the current study, we aimed to determine whether apoptotic gene-related polymorphisms modified HCC prognosis. We genotyped 16 single nucleotide polymorphisms (SNPs) in 10 core genes (TP53, TP53INP1, TP53BP1, CDKN2A, CDKN1A, CDKN1B, MDM2, BAX, CCDN1 and BCL2) in the apoptotic pathway by using DNA from blood samples of 362 HCC patients receiving surgical resection of HCC tumor. The associations between genotypes/haplotypes of the 10 genes and overall survival (OS) of HCC patients were assessed using the Cox proportional hazards model. We found one CDKN1B haplotype CCT/ACT (constructed by rs36228499 C>A, rs34330 C>T and rs2066827 T>G) significantly associated with decreased OS of HCC patients, compared to the common haplotype ACT/CTT both in univariate analysis (P=0.013, HR=1.198, 95% CI: 1.039-1.381) and multivariate analysis (P=0.006, HR=1.224, 95% CI: 1.059-1.413). We also find two SNPs (rs560191 G>C and rs2602141 T>G) in TP53BP1 shown to be marginally significantly associated with decreased OS of HCC patients. However, none of the other SNPs or haplotypes were significantly associated with HCC OS. Our results illustrated the potential use of CDKN1B haplotype as a prognostic marker for HCC patients with surgical resection of tumor.

Serum DLK1 is a potential prognostic biomarker in patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the third most frequent cause of cancer-related death in developing countries, especially in East Asia and South Africa, and the identification of new biomarkers for early diagnosis and prognosis is needed. Delta-like 1 homologue (Drosophila) (DLK1) is expressed in malignancies and promotes cancer cell stemness and tumourigenicity, which makes this molecule a potential target for therapies directed against cancer stem/progenitor cells. Here, we aimed to assess the predictive value of DLK1 as a diagnostic and prognostic biomarker in HCC. With this purpose, serum DLK1 levels were detected using an enzyme-linked immunosorbent assay (ELISA) in serum specimens from 397 HCC patients, 114 healthy individuals, 43 patients with chronic hepatitis B virus (HBV) infection and 24 cirrhotic liver patients with HBV infection, and the correlation between DLK1 levels and clinical features was evaluated. Our data showed that the serum DLK1 level was significantly higher in HCC patients than in healthy individuals or patients with chronic HBV infection (HBV carriers) (P < 0.05). Moreover, the serum DLK1 levels were positively correlated with tumour size and α-fetoprotein (AFP) levels, but not with gender, age, histological grade, HBV infection, intrahepatic metastasis or cirrhosis in HCC patients. Kaplan-Meier analysis indicated that higher DLK1 levels were associated with shorter survival in HCC patients. These results suggest that the serum levels of DLK1 may serve as a prognostic biomarker for HCC patients.

Genetic variants in five novel loci including CFB and CD40 predispose to chronic hepatitis B.

UNLABELLED: Hepatitis B virus affects more than 2 billion people worldwide, 350 million of which have developed chronic hepatitis B (CHB). The genetic factors that confer CHB risk are still largely unknown. We sought to identify genetic variants for CHB susceptibility in the Chinese population. We undertook a genome-wide association study (GWAS) in 2,514 CHB cases and 1,130 normal controls from eastern China. We replicated 33 of the most promising signals and eight previously reported CHB risk loci through a two-stage validation totaling 6,600 CHB cases and 8,127 controls in four independent populations, of which two populations were recruited from eastern China, one from northern China and one from southern China. The joint analyses of 9,114 CHB cases and 9,257 controls revealed significant association of CHB risk with five novel loci. Four loci are located in the human leukocyte antigen (HLA) region at 6p21.3, including two nonsynonymous variants (rs12614 [R32W] in complement factor B [CFB], Pmeta =1.28 × 10(-34) ; and rs422951 [T320A] in NOTCH4, Pmeta = 5.33 × 10(-16) ); one synonymous variant (rs378352 in HLA-DOA corresponding to HLA-DOA*010101, Pmeta = 1.04 × 10(-23) ); and one noncoding variant (rs2853953 near HLA-C, Pmeta = 5.06 × 10(-20) ). Another locus is located at 20q13.1 (rs1883832 in the Kozak sequence of CD40, Pmeta = 2.95 × 10(-15) ). Additionally, we validated seven of eight previously reported CHB susceptibility loci (rs3130542 at HLA-C, rs1419881 at TCF19, rs652888 at EHMT2, rs2856718 at HLA-DQB1, rs7453920 at HLA-DQB2, rs3077 at HLA-DPA1, and rs9277535 at HLA-DPA2, which are all located in the HLA region, 9.84 × 10(-71) ≤ Pmeta ≤ 9.92 × 10(-7) ). CONCLUSION: Our GWAS identified five novel susceptibility loci for CHB. These findings improve the understanding of CHB etiology and may provide new targets for prevention and treatment of this disease.

Epidermal growth factor receptor pathway polymorphisms and the prognosis of hepatocellular carcinoma.

The EGFR signaling pathway is important in the control of vital processes in the carcinogenesis of hepatocellular carcinoma (HCC), including cell survival, cell cycle progression, tumor invasion and angiogenesis. In the current study, we aim to assess if genetic variants in the genes of the EGFR signaling pathway are associated with the prognosis of HCC. We genotyped 36 single nucleotide polymorphisms (SNP) in four core genes (EGF, EGFR, VEGF, and VEGFR2) by using DNA from blood samples of 363 HCC patients with surgical resection. The associations between genotypes and overall survival (OS) and disease-free survival (DFS) were estimated using the Kaplan-Meier method. Hazard ratios (HRs) and 95% confident intervals (CIs) were estimated for the multivariate survival analyses by Cox proportional hazards regression models, adjusting for age, gender, family history, HBsAg and AFP. We found that five SNPs in the VEGFR2 gene were significantly associated with clinical outcomes of HCC patients. Among them, four SNPs (rs7692791, rs2305948, rs13109660, rs6838752) were associated with OS (p=0.035, 0.038, 0.029 and 0.028, respectively), and two SNPs (rs7692791 and rs2034965) were associated with DFS (p=0.039 and 0.017, respectively). Particularly, rs7692791 TT genotype was associated with both reduced OS (p=0.037) and DFS (p=0.043). However, only one SNP rs2034965 with the AA genotype was shown to be an independent effect on DFS (p=0.009) in the multivariate analysis. None of the other 31 polymorphisms or 9 haplotypes attained from the four genes was significantly associated with OS or DFS. Our results illustrated the potential use of VEGFR2 polymorphisms as prognostic markers for HCC patients.

Novel approach to identifying the hepatitis B virus pre-S deletions associated with hepatocellular carcinoma.

AIM: To develop a novel non-sequencing method for the detection of hepatitis B virus (HBV) pre-S deletion mutants in HBV carriers. METHODS: The entire region of HBV pre-S1 and pre-S2 was amplified by polymerase chain reaction (PCR). The size of PCR products was subsequently determined by capillary gel electrophoresis (CGE). CGE were carried out in a PACE-MDQ instrument equipped with a UV detector set at 254 nm. The samples were separated in 50 µm ID eCAP Neutral Coated Capillaries using a voltage of 6 kV for 30 min. Data acquisition and analysis were performed using the 32 Karat Software. A total of 114 DNA clones containing different sizes of the HBV pre-S gene were used to determine the accuracy of the CGE method. One hundred and fifty seven hepatocellular carcinoma (HCC) and 160 non-HCC patients were recruited into the study to assess the association between HBV pre-S deletion and HCC by using the newly-established CGE method. Nine HCC cases with HBV pre-S deletion at the diagnosis year were selected to conduct a longitudinal observation using serial serum samples collected 2-9 years prior to HCC diagnosis. RESULTS: CGE allowed the separation of PCR products differing in size > 3 bp and was able to identify 10% of the deleted DNA in a background of wild-type DNA. The accuracy rate of CGE-based analysis was 99.1% compared with the clone sequencing results. Using this assay, pre-S deletion was more frequently found in HCC patients than in non-HCC controls (47.1% vs 28.1%, P < 0.001). Interestingly, the increased risk of HCC was mainly contributed by the short deletion of pre-S. While the deletion ≤ 99 bp was associated with a 2.971-fold increased risk of HCC (95%CI: 1.723-5.122, P < 0.001), large deletion (> 99 bp) did not show any association with HCC (P = 0.918, OR = 0.966, 95%CI: 0.501-1.863). Of the 9 patients who carried pre-S deletions at the stage of HCC, 88.9% (8/9) had deletions 2-5 years prior to HCC, while only 44.4%4 (4/9) contained such deletions 6-9 years prior to HCC. CONCLUSION: CGE is a sensitive approach for HBV pre-S deletion analysis. Pre-S deletion, especially for short DNA fragment deletion, is a useful predictive marker for HCC.

Inhibitory effects of transcription factor Ikaros on the expression of liver cancer stem cell marker CD133 in hepatocellular carcinoma.

CD133 is a cellular surface glycoprotein that has been reported as a marker for the enrichment of cancer stem cells (CSCs). However, the regulatory mechanism of CD133 remains unknown. CSCs have been proposed to contribute to radioresistance and multi-drug resistance. The elucidation of key regulators of CD133 and CSCs is critical for the development of CSC-targeted therapy. In this study, we showed that Ikarosinhibited the expression of CD133 via direct binding to the CD133 P1 promoter and repressed the tumorigenic and self-renewal capacity of CD133(+) cancer stem-like cells in hepatocellular carcinoma (HCC). We found that Ikaros interacted with CtBP as a transcription repressor complex, which inhibited CD133 expression in HCC. We also demonstrated that Ikaros expression was up-regulated by ETS1 which activity was regulated by MAPKs pathway. Furthermore, decreased expression of Ikaroswas significantly associated with poor survival in HCC patients. Overall, our study identifies that Ikaros plays a role as a transcription repressor in HCC and is a new reactivated therapeutic target for the treatment of HCC. Meanwhile, our findings provide evidence that Ikaros could be an attractive inhibitor of the target gene CD133, which reactivates anticancer mechanisms in targeted CSC therapy.

Female breast cancer survival in Qidong, China, 1972-2011: a population-based study.

BACKGROUND: Based on data from the population-based Qidong Cancer Registry, we report a survival analysis for female breast cancer patients diagnosed during 1972-2011 in order to assess the long-term trends for the prognosis of this cancer. METHODS: The last follow-up for survival status of the 3,398 registered female breast cancer cases was April, 2012. Cumulative observed survival (OS) and relative survival (RS) rates were calculated using Hakulinen's method performed by the SURV3.01 Software developed at the Finnish Cancer Registry. RESULTS: The one-, three-, five-, ten-, fifteen-, twenty-, thirty-, and forty- year OS rates were 83.61%, 67.53%, 58.75%, 48.56%, 42.57%, 38.30%, 29.19%, 19.35%; and the RS rates were 84.76%, 70.45%, 63.12%, 56.81%, 55.26%, 56.36%, 62.59%, 84.00%, respectively. Five-year RS rates of age groups 15-34, 35-44, 45-54, 55-64, 65-74, and 75+ were 60.17%, 68.27%, 67.79%, 56.03%, 55.50%, and 57.28%; 10-year RS rates were 54.16%, 59.59%, 61.34%, 47.78%, 51.30%, and 59.28%, respectively. There were statistical differences among the age groups (RS: χ2 = 152.15, P = 0.000). Remarkable improvement could be seen for the 5-year RS rates from 52.08% in 1972 to 69.26% in 2003-2007, and the 10-year RS rates from 43.16% in 1972 to 60.85% in 1998-2002, respectively. CONCLUSIONS: Survival outcomes from Qidong registered cases with breast cancer have shown gradual progress during the past 40 years. The disparities between survival rates of this area and developed countries are getting narrower, but there is still great need for improving survival in Qidong.

Rapid and sustainable detoxication of airborne pollutants by broccoli sprout beverage: results of a randomized clinical trial in China.

Broccoli sprouts are a convenient and rich source of the glucosinolate, glucoraphanin, which can generate the chemopreventive agent, sulforaphane, an inducer of glutathione S-transferases (GST) and other cytoprotective enzymes. A broccoli sprout-derived beverage providing daily doses of 600 µmol glucoraphanin and 40 µmol sulforaphane was evaluated for magnitude and duration of pharmacodynamic action in a 12-week randomized clinical trial. Two hundred and ninety-one study participants were recruited from the rural He-He Township, Qidong, in the Yangtze River delta region of China, an area characterized by exposures to substantial levels of airborne pollutants. Exposure to air pollution has been associated with lung cancer and cardiopulmonary diseases. Urinary excretion of the mercapturic acids of the pollutants, benzene, acrolein, and crotonaldehyde, were measured before and during the intervention using liquid chromatography tandem mass spectrometry. Rapid and sustained, statistically significant (P ≤ 0.01) increases in the levels of excretion of the glutathione-derived conjugates of benzene (61%), acrolein (23%), but not crotonaldehyde, were found in those receiving broccoli sprout beverage compared with placebo. Excretion of the benzene-derived mercapturic acid was higher in participants who were GSTT1-positive than in the null genotype, irrespective of study arm assignment. Measures of sulforaphane metabolites in urine indicated that bioavailability did not decline over the 12-week daily dosing period. Thus, intervention with broccoli sprouts enhances the detoxication of some airborne pollutants and may provide a frugal means to attenuate their associated long-term health risks.

Association of hepatitis B virus pre-S deletions with the development of hepatocellular carcinoma in Qidong, China.

BACKGROUND/AIM: To investigate the roles of mutations in pre-S and S regions of hepatitis B virus (HBV) on the progression of hepatocellular carcinoma (HCC) in Qidong, China. METHODS: We conducted an age matched case-control study within a cohort of 2387 male HBV carriers who were recruited from August, 1996. The HBV DNA sequence in pre-S/S regions was successfully determined in 96 HCC cases and 97 control subjects. In addition, a consecutive series of samples from 11 HCC cases were employed to evaluate the pre-S deletion patterns before and after the occurrence of HCC. RESULTS: After adjustment for age, history of cigarette smoking and alcohol consumption, HBeAg positivity, pre-S deletions, pre-S2 start codon mutations, and T53C mutation were significantly associated with HCC, showing adjusted odds ratios (ORs) from 1.914 to 3.199. HCC patients also had a lower frequency of T31C mutation in pre-S2 gene, compared with control subjects (0.524; 95% CI 0.280-0.982). HBV pre-S deletions were clustered mainly in the 5' end of pre-S2 region. Multivariate analysis showed that pre-S deletions and pre-S2 start codon mutations were independent risk factors for HCC. The OR (95% CI) were 2.434 (1.063-5.573) and 3.065 (1.099-8.547), respectively. The longitudinal observation indicated that the pre-S deletion mutations were not acquired at the beginning of HBV infection, but that the mutations occurred during the long course of liver disease. CONCLUSION: Pre-S deletions and pre-S2 start codon mutations were independently associated with the development of HCC. The results also provided direct evidence that pre-S deletion mutations were not acquired from the beginning of infection but arose de novo during the progression of liver disease.

Effect of combined mutations in the enhancer II and basal core promoter of hepatitis B virus on development of hepatocellular carcinoma in Qidong, China.

AIM: To investigate the roles of mutations in enhancer II (Enh II), basal core promoter (BCP) and precore (PC) regions of hepatitis B virus (HBV) in the progression of hepatocellular carcinoma (HCC) in Qidong, China. METHODS: We conducted a case-control study within a cohort of 2387 male HBV carriers who were recruited between August and September 1996. The HBV DNA sequence was determined in 152 HCC and 131 chronic hepatitis patients. Mutation exchanges during follow up in 115 cases were compared with 108 controls with serum samples taken during a similar length of follow up. In addition, a longitudinal study was conducted in 22 cases in which serial serum samples were available before HCC. RESULTS: After adjustment for age, history of cigarette smoking and alcohol consumption, hepatitis B e-antigen positivity, T1653, V1753 and T1762/A1764 double mutations were associated with risk of HCC. Multivariate analysis showed that T1653, V1753 and T1762/A1764 double mutations were independent risk factors of HCC. Moreover, a significant biological gradient of HCC risk by number of mutations in Enh II/BCP regions was observed. Paired samples analysis indicated that the increased HCC risk for at-risk sequence mutations were attributable to the persistence of these mutations, but not a single time point mutation. The longitudinal observation demonstrated a gradual combination of mutations in Enh II/BCP regions accumulated during the development of HCC. CONCLUSION: T1653, V1753 and T1762/A1764 double mutations were independent risk factors of HCC. The effect of combined mutations in Enh II/BCP regions increased the risk and persistence of at-risk sequence mutations and was critical for HCC development.

[Retrospective observation of dynamic levels of serum Golgi protein 73 in patients prior to the onset of liver cancer].

OBJECTIVE: To observe the dynamic levels of serum Golgi protein 73(GP73) in patients prior to and after the onset of liver cancer, and to explore the related factors. METHODS: From 2007 to 2012, a periodical screening program was carried out in a group of high risk population with positive Hepatitis B surface antigens (HBsAg) , twice a year. Their serum specimens from every screening time point were kept in Qidong Biobank until liver cancer was diagnosed. Thirty-nine patients with liver cancer were recruited for the study, each of them at least had three times of specimens collected as well as B ultrasound scan (BUS) exam results at onset of disease and within 30 months before diagnosed, amongst 6 time points. In total, there were 162 specimens collected to test GP73 by double-antibody sandwich enzyme-linked immuno-sorbent assay (ELISA). Statistical analyses of time series and differences among groups were performed by stata software 10. RESULTS: The average value of 39 patient's GP73 at the time point of liver cancer onset was (126.77 ± 73.73) µg/L, while the values at the other five time points prior to the onset were (128.32 ± 81.18) , (129.97 ± 83.62) , (127.38 ± 80.10) , (135.52 ± 97.88) and (138.24 ± 93.58) µg/L, respectively, with no significant difference (F = 0.07, P = 0.997). No obvious changing trends of GP73 were observed among the 39 liver cancer cases at the 6 time points. All 162 samples were divided into two groups: without hepatic cirrhosis (63 samples) and with cirrhosis (99 samples) according to findings of B-ultrasonic wave; whose average GP73 values were separately (97.16 ± 51.39) and (151.20 ± 91.68) µg/L. The difference showed statistical significance (F = 18.22, P < 0.01). Furthermore, if we grouped the samples by the average value of GP73 at 130.19 µg/L, then there were only 1/14 of the subjects without hepatic cirrhosis having higher GP73 values, but 12 of the 25 subjects with hepatic cirrhosis having higher GP73 values. The difference showed statistical significance (P = 0.013). The results of Linear regression model also showed that there was no correlation between GP73 and time series (t = 0.75, P = 0.455), but significant correlation between GP73 and hepatic cirrhosis (t = 4.30, P < 0.01). CONCLUSION: No significant changes of the dynamic levels of GP73 could be found among the liver cancer patients within 30 months prior to the onset of disease. GP73 values of the patients with liver cancer may depend on their background of hepatic diseases; and hepatic cirrhosis might be one of the main influencing factors or confounding factors.

Keap1-nrf2 signaling: a target for cancer prevention by sulforaphane.

Sulforaphane is a promising agent under preclinical evaluation in many models of disease prevention. This bioactive phytochemical affects many molecular targets in cellular and animal models; however, amongst the most sensitive is Keap1, a key sensor for the adaptive stress response system regulated through the transcription factor Nrf2. Keap1 is a sulfhydryl-rich protein that represses Nrf2 signaling by facilitating the polyubiquitination of Nrf2, thereby enabling its subsequent proteasomal degradation. Interaction of sulforaphane with Keap1 disrupts this function and allows for nuclear accumulation of Nrf2 and activation of its transcriptional program. Enhanced transcription of Nrf2 target genes provokes a strong cytoprotective response that enhances resistance to carcinogenesis and other diseases mediated by exposures to electrophiles and oxidants. Clinical evaluation of sulforaphane has been largely conducted by utilizing preparations of broccoli or broccoli sprouts rich in either sulforaphane or its precursor form in plants, a stable ß-thioglucose conjugate termed glucoraphanin. We have conducted a series of clinical trials in Qidong, China, a region where exposures to food- and air-borne carcinogens has been considerable, to evaluate the suitability of broccoli sprout beverages, rich in either glucoraphanin or sulforaphane or both, for their bioavailability, tolerability, and pharmacodynamic action in population-based interventions. Results from these clinical trials indicate that interventions with well characterized preparations of broccoli sprouts may enhance the detoxication of aflatoxins and air-borne toxins, which may in turn attenuate their associated health risks, including cancer, in exposed individuals.

Genetic variants in STAT4 and HLA-DQ genes confer risk of hepatitis B virus-related hepatocellular carcinoma.

To identify genetic susceptibility loci for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) in the Chinese population, we carried out a genome-wide association study (GWAS) in 2,514 chronic HBV carriers (1,161 HCC cases and 1,353 controls) followed by a 2-stage validation among 6 independent populations of chronic HBV carriers (4,319 cases and 4,966 controls). The joint analyses showed that HCC risk was significantly associated with two independent loci: rs7574865 at STAT4, P(meta) = 2.48 × 10(-10), odds ratio (OR) = 1.21; and rs9275319 at HLA-DQ, P(meta) = 2.72 × 10(-17), OR = 1.49. The risk allele G at rs7574865 was significantly associated with lower mRNA levels of STAT4 in both the HCC tissues and nontumor tissues of 155 individuals with HBV-related HCC (P(trend) = 0.0008 and 0.0002, respectively). We also found significantly lower mRNA expression of STAT4 in HCC tumor tissues compared with paired adjacent nontumor tissues (P = 2.33 × 10(-14)).