OBJECTIVE: To investigate the expression of claudin 4 (CLDN4) in cervical tissues from patients with different cervical lesions, and to explore the value of combined detection of CLDN4 and high risk human papilloma virus (HR-HPV). METHODS: The cervical tissue specimens of low-grade squamous intraepithelial lesion (LSIL, =30), high-grade squamous intraepithelial lesion (HSIL, =30), squamous cell carcinoma (SCC, =30) as well as chronic cervicitis (control, =30) were collected from the Sir Run Run Shaw Hospital of Zhejiang University during June 2015 and December 2016. The expression of CLDN4 protein in tissue specimens was detected by immunohistochemistry, HR-HPV was detected by real-time quantitative PCR, and the cervical exfoliated cells were examined by thinprep cytologic test (TCT). The ROC curve was applied to analyze the diagnostic value of TCT combined with HR-HPV and CLDN4 combined with HR-HPV tests for HSIL and SCC of the cervix. RESULTS: With the increase of the severity of cervical lesions, the positive rate of CLDN4 expression rose (=0.832, <0.05). Positivity of both HR-HPV infection and CLDN4 expression was found mainly in the HSIL and SCC groups. The areas under curve (AUC) of TCT combined with HR-HPV and CLDN4 combined with HR-HPV tests for diagnosis of HSIL and SCC were 0.683 and 0.633, respectively; the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of TCT combined with HR-HPV test for diagnosis of HSIL and SCC were 100.0%, 36.7%, 61.2%, 100.0% and 46.7% respectively; those of CLDN4 combined with HR-HPV test were 96.7%, 30.0%, 58.0%, 90.0% and 55.0%, respectively. CONCLUSIONS CLDN4 expression may be related to the occurrence and development of cervical carcinoma and precancerous lesions. CLDN4 combined with HR-HPV test may be used for diagnosis of HSIL and SCC of the cervix clinically.
Carcinoma, Squamous Cell ; diagnosis ; virology ; Cervical Intraepithelial Neoplasia ; diagnosis ; virology ; Claudin-4 ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunochemistry ; Papillomaviridae ; isolation & purification ; Real-Time Polymerase Chain Reaction ; Squamous Intraepithelial Lesions of the Cervix ; virology ; Uterine Cervical Neoplasms ; diagnosis

BACKGROUND: The incidence and etiology of hepatocellular carcinoma (HCC) vary widely according to race and geographic regions. The insertional mutagenesis of adeno-associated virus 2 (AAV2) has recently been considered a new viral etiology of HCC. The aim of this study was to investigate the frequency and clinical characteristics of AAV2 in Korean patients with HCC. METHODS: A total of 289 unrelated Korean patients with HCC, including 159 Hepatitis-B-related cases, 16 Hepatitis-C-related cases, and 114 viral serology-negative cases, who underwent surgery at the Samsung Medical Center in Korea from 2009 to 2014 were enrolled in this study. The presence of AAV2 in fresh-frozen tumor tissues was investigated by DNA PCR and Sanger sequencing. The clinical and pathological characteristics of AAV2-associated HCC in these patients were compared with previous findings in French patients. RESULTS: The AAV2 detection rate in Korean patients (2/289) was very low compared with that in French patients (11/193). Similar to the French patients, the Korean patients with AAV2-related HCC showed no signs of liver cirrhosis. The Korean patients were younger than the French patients with the same AAV2-associated HCC; the ages at diagnosis of the two Korean patients were 47 and 39 yr, while the median age of the 11 French patients was 55 yr (range 43-90 yr). CONCLUSIONS: AAV2-associated HCC was very rare in Korean patients with HCC. Despite a limited number of cases, this study is the first to report the clinical characteristics of Korean patients with AAV2-associated HCC. These findings suggest epidemiologic differences in viral hepatocarcinogenesis between Korean and European patients.
Adult ; Asian Continental Ancestry Group ; Capsid Proteins/genetics ; Carcinoma, Hepatocellular/etiology/*pathology/virology ; DNA, Viral/chemistry/genetics/metabolism ; DNA-Binding Proteins/genetics ; Dependovirus/*genetics/isolation & purification/pathogenicity ; Female ; Humans ; Incidence ; Inverted Repeat Sequences/genetics ; Liver Neoplasms/etiology/*pathology/virology ; Male ; Middle Aged ; Parvoviridae Infections/complications/epidemiology ; Polymerase Chain Reaction ; Republic of Korea ; Sequence Analysis, DNA ; Viral Proteins/genetics

PURPOSE: The role of IL28B gene variants and expression in hepatitis B virus (HBV) infections are not well understood. Here, we evaluated whether IL28B gene expression and rs12979860 variations are associated with HBV outcomes. MATERIALS AND METHODS: IL28B genetic variations (rs12979860) were genotyped by pyrosequencing of DNA samples from 137 individuals with chronic HBV infection [50 inactive carriers (IC), 34 chronic hepatitis B (CHB), 27 cirrhosis, 26 hepatocellular carcinoma (HCC)], and 19 healthy controls. IL28A/B mRNA expression in peripheral blood mononuclear cells was determined by qRT-PCR, and serum IL28B protein was measured by ELISA. RESULTS: Patients with IL28B C/C genotype had greater IL28A/B mRNA expression and higher IL28B protein levels than C/T patients. Within the various disease stages, compared to IC and healthy controls, IL28B expression was reduced in the CHB, cirrhosis, and HCC cohorts (CHB vs. IC, p=0.02; cirrhosis vs. IC, p=0.01; HCC vs. IC, p=0.001; CHB vs. controls, p<0.01; cirrhosis vs. controls, p<0.01; HCC vs. controls, p<0.01). When stratified with respect to serum HBV markers in the IC and CHB cohorts, IL28B mRNA and protein levels were higher in HBeAg-positive than negative individuals (p=0.01). Logistic regression analysis revealed that factors associated with high IL28B protein levels were C/C versus C/T genotype [p=0.016, odds ratio (OR)=0.25, 95% confidence interval (CI)=0.08-0.78], high alanine aminotransferase values (p<0.001, OR=8.02, 95% CI=2.64-24.4), and the IC stage of HBV infection (p<0.001). CONCLUSION: Our data suggest that IL28B genetic variations may play an important role in long-term development of disease in chronic HBV infections.
Adult ; Aged ; Alanine Transaminase/blood ; Asian Continental Ancestry Group/*genetics ; Biological Markers/blood ; Carcinoma, Hepatocellular/genetics ; Case-Control Studies ; China ; DNA, Viral/blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Genotype ; Hepatitis B virus/genetics ; Hepatitis B, Chronic/ethnology/*genetics/immunology/*virology ; Humans ; Interleukins/blood/*genetics/metabolism ; Leukocytes, Mononuclear ; Liver Cirrhosis/blood ; Liver Neoplasms/genetics ; Male ; Middle Aged ; RNA, Messenger/*genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the expression of cyclin D1 in cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma and its relationship with human papillomavirus 16 (HPV16) E7 gene expression.

METHODSBoth SiHa and Hcc94 cell lines were obtained from cervical epithelial cells of squamous cell carcinoma. E6/E7 gene was silent in Hcc94 cell line.Expression levels of cyclin D1 mRNA and protein in CIN and squamous cell carcinoma were detected by QT-PCR and immunohistochemistry (IHC) respectively. SiRNA was constructed for targeting the promoter of HPV16 E7 and then transfected into SiHa cells to establish cm-16 line with stable silencing of E7. Control cell line B3 was obtained by blank plasmid transfection into SiHa cells. RT-PCR and Western blot were used to detect cyclin D1 mRNA and protein expression in the SiHa, B3, and cm-16 cells, respectively.

RESULTSCyclin D1 was expressed in the basal cells of normal cervical squamous epithelia and the expression gradually decreased in the progression from CIN1 to CIN3. Squamous cell carcinoma showed negative or scattered expression of cyclin D1 (P<0.05). Both mRNA and protein of cyclin D1 in E7(+) SiHa cells were lower than those in cm-16 and Hcc94 cells.

CONCLUSIONSquamous cell carcinoma with high HPV E7 expression shows low level of cyclin D1, suggesting that HPV16 E7 gene inhibits the expression of cyclin D1.

Carcinoma, Squamous Cell ; metabolism ; virology ; Cell Line, Tumor ; Cervical Intraepithelial Neoplasia ; metabolism ; virology ; Cyclin D1 ; genetics ; metabolism ; Female ; Human papillomavirus 16 ; Humans ; Immunohistochemistry ; Papillomavirus E7 Proteins ; genetics ; Promoter Regions, Genetic ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; Transfection ; Uterine Cervical Neoplasms ; metabolism ; virology

Hepatitis C virus (HCV) is a leading etiology of hepatocellular carcinoma (HCC). The interaction of HCV with its human host is complex and multilayered; stemming in part from the fact that HCV is a RNA virus with no ability to integrate in the host's genome. Direct and indirect mechanisms of HCV-induced HCC include activation of multiple host pathways such as liver fibrogenic pathways, cellular and survival pathways, interaction with the immune and metabolic systems. Host factors also play a major role in HCV-induced HCC as evidenced by genomic studies identifying polymorphisms in immune, metabolic, and growth signaling systems associated with increased risk of HCC. Despite highly effective direct-acting antiviral agents, the morbidity and incidence of liver-related complications of HCV, including HCC, is likely to persist in the near future. Clinical markers to selectively identify HCV subjects at higher risk of developing HCC have been reported however they require further validation, especially in subjects who have experienced sustained virological response. Molecular biomarkers allowing further refinement of HCC risk are starting to be implemented in clinical platforms, allowing objective stratification of risk and leading to individualized therapy and surveillance for HCV individuals. Another role for molecular biomarker-based stratification could be enrichment of HCC chemoprevention clinical trials leading to smaller sample size, shorter trial duration, and reduced costs.
Biomarkers, Tumor/genetics/metabolism ; Carcinoma, Hepatocellular/*etiology ; Hepacivirus/genetics/*pathogenicity ; Hepatitis C/complications/pathology/virology ; Humans ; Liver Neoplasms/*etiology ; Risk

OBJECTIVE: To explore the differential expression of microRNA (miRNA) in HPV16-positive squamous carcinoma of the cervix in the Uygur of southern Xinjiang and to predict the target genes of the miRNAs.
 METHODS: Samples of HPV16-positive squamous carcinoma of the cervix from 5 Uygurs were collected for miRNA microarray assay. The differentially expressed miRNAs were selected for further verification by real-time quantitative RT-PCR. The software, including targetscan, miRwalk, miRanda and pictar, were used to predict the target genes of the verified miRNAs.
 RESULTS: Eighteen differentially expressed miRNAs were identified by miRNA microarray assay. The significantly differentially expressed miRNA-138 and miRNA-720 were verified by real-time quantitative RT-PCR. According to the prediction, the target genes for miRNA-138 were EZH2, LYPLA1, ARHGEF3, CLNS1A, EIF4EBP1, GNAI2, LIMK1, RHOC, ROCK2, SLC20A1, TERT, and H2AFX, while for miRNA-720 were EZH2, AGAP2, SPOCK2, FGF14, HNRNPA2B1, QKI, FOXG1, ACVR1B, DNMT3A, EPHB2, LATS2, KRAS, CCND2, NBN, ENAM, AMELX, PRNP, and CALB1.
 CONCLUSION miR-138 and miR-720 are the down-regulated target miRNAs in HPV16-positive squamous carcinoma of the cervix in the Uygur of southern Xinjiang. The common target gene for miR-138 and miR-720 is EZH2, which might be related to cervical squamous carcinoma invasion and metastasis.
Carcinoma, Squamous Cell ; metabolism ; virology ; China ; Down-Regulation ; Female ; Human papillomavirus 16 ; isolation & purification ; Humans ; MicroRNAs ; metabolism ; Papillomavirus Infections ; metabolism ; Real-Time Polymerase Chain Reaction ; Uterine Cervical Neoplasms ; metabolism ; virology

MicroRNA polymorphisms may be associated with carcinogenesis or immunopathogenesis of infection. We evaluated whether the mircoRNA-604 (miR-604) polymorphism can affect the persistence of hepatitis B virus (HBV) infection, and the development to hepatocellular carcinoma (HCC) in patients with chronic HBV infection. A total of 1,439 subjects, who have either past or present HBV infection, were enrolled and divided into four groups (spontaneous recovery, chronic HBV carrier without cirrhosis, liver cirrhosis and HCC). We genotyped the precursor miR-604 genome region polymorphism. The CC genotype of miR-604 rs2368392 was most frequently observed and T allele frequency was 0.326 in all study subjects. The HBV persistence after infection was higher in those subjects with miR-604 T allele (P=0.05 in a co-dominant and dominant model), which implied that the patients with miR-604 T allele may have a higher risk for HBV chronicity. In contrast, there was a higher rate of the miR-604 T allele in the chronic carrier without HCC patients, compared to those of the HCC patients (P=0.03 in a co-dominant model, P=0.02 in a recessive model). The T allele at miR-604 rs2368392 may be a risk allele for the chronicity of HBV infection, but may be a protective allele for the progression to HCC in chronic HBV carriers.
Adult ; Aged ; Aged, 80 and over ; Base Sequence ; Carcinoma, Hepatocellular/etiology/*genetics/pathology ; Case-Control Studies ; Demography ; Female ; Gene Frequency ; *Genetic Predisposition to Disease ; Genotype ; Hepatitis B Antibodies/blood ; Hepatitis B Surface Antigens/blood ; Hepatitis B e Antigens/blood ; Hepatitis B virus/metabolism ; Hepatitis B, Chronic/complications/*genetics/virology ; Humans ; Liver Neoplasms/etiology/*genetics/pathology ; Male ; MicroRNAs/*genetics/metabolism ; Middle Aged ; Polymorphism, Single Nucleotide ; Risk Factors

OBJECTIVETo investigate the clinical value of serum anti-Ku86 in early detection of hepatocellular carcinoma (HCC).

METHODSExpression levels of Ku86 protein in HCC and adjacent normal liver tissues were detected by Western blotting. Serum anti-Ku86 level in 83 patients with early HCC and 124 patients with liver cirrhosis were detected by enzyme-linked immunosorbent assay (ELISA). Chemiluminescence was used to measure the serum level of α-fetoprotein (AFP).

RESULTSExpression of Ku86 protein in HCC was increased when compared with the adjacent normal liver tissues (0.21 ± 0.05 vs. 0.08 ± 0.02, P < 0.01). Serum anti-Ku86 level was significantly elevated in HCC patients compared with that in liver cirrhosis patients (0.47 ± 0.22 vs. 0.22 ± 0.06 Abs at 450 nm, P < 0.01), but there was no significant difference between HBV infection and HCV infection in HCC patients (0.51 ± 0.19 vs. 0.47 ± 0.24, P = 0.267). Of note, serum anti-Ku86 level was significantly decreased after surgical resection of the tumors in the 30 HCC cases tested (P < 0.01). The results of ROC analysis indicated a better performance of anti-Ku86 (0.857) than AFP (0.739) for early detection of HCC. In 83 HCC patients, the positive rate of anti-Ku86 was 61.4% (51/83), significantly higher than that of the AFP positive rate (27.7%, 23/83). The anti-Ku86 level was positive in 37 of 60 HCC cases with negative AFP. Combination assay of AFP and anti-Ku86 could detect 60 of 83 HCC cases (72.3%, 60/83). There was no significant correlation of anti-Ku86 and AFP (r = 0.156, P = 0.161).

CONCLUSIONSSerum anti-Ku86 level is significantly elevated and is not related to HBV and HCV infection in HCC patients. Serum anti-Ku86 antibody may be a potential biomarker for early detection of HCC, and can be used in combination with AFP in clinics.

Adult ; Aged ; Antigens, Nuclear ; immunology ; Autoantibodies ; blood ; Biomarkers, Tumor ; blood ; Carcinoma, Hepatocellular ; blood ; diagnosis ; virology ; DNA-Binding Proteins ; immunology ; Early Detection of Cancer ; Female ; Hepatitis B ; blood ; Hepatitis C ; blood ; Humans ; Ku Autoantigen ; Liver Cirrhosis ; blood ; Liver Neoplasms ; blood ; diagnosis ; virology ; Male ; Middle Aged ; ROC Curve ; alpha-Fetoproteins ; metabolism

OBJECTIVETo detect the infection of human papillomavirus (HPV) 6/11, 16/18, 31/33 in patients with head and neck squamous cell carcinoma and explore the relationship between HPV infection and expression of p16 and EGFR in the tumor tissue and their clinical significance.

METHODSThe infection of HPV6/11, 16/18, 31/33 was detected by in situ hybridization (ISH), and expression of p16 and EGFR was assessed by immunohistochemistry in biopsy or surgical specimens of 43 cases of head and neck squamous cell carcinoma, and analyzed its impact on the prognosis. Spearman rank correlation method was used for analysis of the relationship. Overall survival rate of the patients was estimated by Kaplan-Meier analysis. Cox regression model was used for multivariate analysis.

RESULTSHPV6/11, 16/18, 31/33 were detected in 25.6% (11/43) of this group of patients, among them, HPV16/18 accounted for 63.6%, HPV31/33 accounted for 27.3%, and HPV6/11 accounted for 0. EGFR was expressed in 69.8% and p16 was expressed in 53.5% of the patients. The difference was statistically significant between the HPV-positive and HPV-negative groups in ethnicity, smoking, alcohol consumption (P = 0.045, 0.040, 0.011, respectively). HPV infection was found to be positively correlated with p16 expression and inversely correlated with EGFR expression (P = 0.029, P = 0.009). The expression of p16 protein was negatively correlated with EGFR protein expression (r = -0.447, P = 0.003). The 3-year overall survival rate was 60.0% in the HPV-positive group and 59.7% in the negative group (P = 0.789); 72.2% in the p16-positive patients and 43.9% in the p16-negative patients (P = 0.012); 48.8% in the EGFR-positive patients and 81.8% in the EGFR-negative patients (P = 0.037).

CONCLUSIONSThe results of our study suggest that the HPV infection rate, HPV subtypes and clinicopathological features of HPV-positive SCCHN are in accordance with those reported in Western literatures. There may be differences between the HPV infections in Uygur and Han nationalities. HPV infection is positively correlated with p16 and negatively correlated with EGFR expressions. The prognosis of p16-positive patients is significantly better than that of negative cases, and p16 is an independent prognostic factor for head and neck squamous cell carcinoma.

Carcinoma, Squamous Cell ; mortality ; virology ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Head and Neck Neoplasms ; mortality ; virology ; Human papillomavirus 16 ; Humans ; Papillomavirus Infections ; metabolism ; Prognosis ; Receptor, Epidermal Growth Factor ; metabolism ; Survival Analysis

OBJECTIVETo investigate the role of absent ductular reaction (DR) at hepatocellular-stromal boundaries in early stage hepatocellular carcinoma (HCC) with cirrhosis in patients with chronic hepatitis B.

METHODSCytokeratin (CK)7 and CK19 expression was detected by the SP immunohistochemistry method in 112 hepatic nodules taken from 20 cases of early HCC, 26 cases of HCC with nodules more than 3 cm, 20 cases of high-grade dysplastic nodule (HGDN), 26 cases of low-grade dysplastic nodule (LGDN), and 20 cases of cirrhosis (CIR). DR/CK7 and DR/CK19 were assessed separately on a semi-quantitative scale and statistically analyzed.

RESULTSThe mean age of the patients in the study was 53.71 years-old, and the study population consisted of 73 males and 39 females. The follow-up time ranged from 3 to 90 months. Positive CK7 and CK19 staining was detected in the cytoplasm of DR-positive hepatobiliary cells, interlobular bile duct, and a portion of hepatic cells. All of the DR/CK7- and DR/CK19-positive cells were localized around the non-invasive nodules. Specimens with focal or diffuse DR/CK7- and DR/CK19-loss had more robust stromal invasion. Specimens from early HCC cases showed greater DR/CK19 loss than specimens from HGDN cases, LGDN cases and CIR cases (all P less than 0.01). DR/CK7 loss of early HCC was less than HCC with nodules more than 3 cm (P less than 0.05), and more than LGDN cases and CIR cases (both P less than 0.01).The area under the receiver operating characteristic curve of DR/CK7 was very similar to that of DR/CK19 (P more than 0.05). Pearson's correlation analysis indicated that DR/CK7 and DR/CK19 were positively correlated with tumor-free time (P less than 0.01) and negatively correlated with early recurrence time as well as death rate (both P less than 0.01). Furthermore, cases showing DR/CK7 or DR/CK19 loss had lower overall survival rate and tumor-free survival rate (P less than 0.01) and higher early recurrence rate (P less than 0.01).

CONCLUSIONDR/CK7 and DR/CK19 immunostaining may help to distinguish non-invasive HGDNs from both minimally-invasive and overtly-invasive HCCs by identifying small foci of invasion and predicting increased risk of invasiveness.

Adult ; Aged ; Aged, 80 and over ; Bile Ducts, Intrahepatic ; pathology ; Carcinoma, Hepatocellular ; diagnosis ; pathology ; virology ; Early Diagnosis ; Female ; Hepatitis B, Chronic ; pathology ; Humans ; Immunohistochemistry ; Keratin-19 ; metabolism ; Keratin-7 ; metabolism ; Liver Neoplasms ; diagnosis ; pathology ; virology ; Male ; Middle Aged