Philip P.C. Ip

HBsAg Seroclearance in Chronic Hepatitis B in Asian Patients: Replicative Level and Risk of Hepatocellular Carcinoma

BACKGROUND & AIMS Our aims were to study the virologic, histologic, and clinical outcome in chronic hepatitis B (CHB) patients with hepatitis B surface antigen (HBsAg) seroclearance. METHODS We determined the age of HBsAg seroclearance that is associated with a lower risk for hepatocellular carcinoma (HCC) in 298 CHB patients (median follow-up, 108 months). The following virologic and histologic features were also determined: liver stiffness (n = 229), liver histology, serum HBV DNA levels over time (n = 265), intrahepatic HBV DNA with covalently closed circular DNA (cccDNA) levels, and messenger RNA (mRNA) expression. RESULTS The median age of HBsAg seroclearance was 49.6 years. Seven (2.4%) patients developed HCC. Cumulative risk for HCC was higher in patients with HBsAg seroclearance at ages >or=50 years compared with those with HBsAg seroclearance at ages <50 (P = .004) years. Of these 2 groups of patients, 29.5% and 7.9%, respectively, had significant fibrosis by liver stiffness measurement (P = .001), and 15.4% of patients had mild histologic fibrosis. Intrahepatic total HBV DNA and cccDNA were detected in 100% and 79.3% of patients, respectively. All patients had undetectable surface and precore/pregenomic RNA transcripts. One (9.1%) patient had X mRNA expression. Serum HBV DNA were detectable in 13.4%, 6.1%, and 3.7% of patients within 1 year and 5-10 and >10 years after HBsAg seroclearance, respectively, and 82.1% patients had persistently normal alanine aminotransferase levels. CONCLUSIONS HBV persisted at low replicative and transcriptional levels after HBsAg seroclearance. HBsAg seroclearance at age <50 years was associated with a lower risk for the development of HCC.

Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation

Abstract Aberrant activation of hedgehog (HH) pathway has been implicated in the development of human malignancies. This study aimed at investigating the role of HH molecules in human ovarian carcinogenesis. The expression profiles of HH molecules were examined in ovarian tumor samples and ovarian cancer cell lines and the in vitro effects of HH molecules on cell proliferation, apoptosis, migration, invasion and cell differentiation as well as related downstream target genes were assessed. Overexpression of Patched and Gli1 protein in ovarian cancers correlated with poor survival of the patients (P = 0.008; P = 0.004). Significantly elevated expression of Sonic hedgehog messenger RNA was observed in ovarian cancers compared with normal tissues and benign ovarian tumors and such differential expression was specific to histological types (P < 0.05). Ectopic Gli1 overexpression in ovarian cancer cells conferred increased cell proliferation, cell mobility, invasiveness and change in differentiation in association with increased expression of E-cadherin, vimentin, Bcl-2, caspases as well as β1 integrin, membrane type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF). Treatment with 3-keto-N-(aminoethyl-aminocaproyl-dihydrocinnamoyl)-cyclopamine induced cancer cell apoptosis, suppressed cell growth, mobility and invasiveness and induced cancer cell dedifferentiation with decreased expression of E-cadherin, cytokeratin 7, Snail, calretinin, vimentin, Bcl-2, caspases, β1 integrin, MT1-MMP and VEGF. Our data suggested that abnormal HH signaling activation plays important roles in the development and progression of ovarian cancers. Gli1 expression is an independent prognostic marker. Inhibition of the HH pathway molecules might be a valid therapeutic strategy for ovarian cancers.

Uterine Smooth Muscle Tumors of Uncertain Malignant Potential (STUMP): A clinicopathologic analysis of 16 cases

Background The current World Health Organization classification indicates that a uterine smooth muscle tumor that cannot be histologically diagnosed as unequivocally benign or malignant should be termed “smooth muscle tumor of uncertain malignant potential” (STUMP). STUMPs represent a heterogeneous group of rare tumors that have been the subject of only a few published studies, some of which lack detailed clinicopathologic details and/or follow-up data. More recently, it has been suggested that immunohistochemical staining may be helpful in the diagnosis of STUMPs. Design The clinicopathologic features of 16 cases of STUMP that exhibited usual smooth muscle differentiation, diagnosed between 1992 and 2006 from 11 hospitals, were studied and classified into 4 subgroups using terminology and criteria described by Stanford investigators. Immunohistochemical stains for p16, p53, MIB1 (ki-67), and estrogen and progesterone receptors were performed. The results were compared with those in the literature. Results The tumors were classified as follows: 6 as “atypical leiomyoma with limited experience”, 7 as “smooth muscle tumor of low malignant potential”, 2 as “atypical leiomyoma, low risk of recurrence,” and 1 as “mitotically active leiomyoma, limited experience.” Follow-up was 21 to 192 months (mean, 80.8 and median, 51.5). Only 2 tumors recurred, at 15 and 51 months, respectively; both were atypical leiomyoma with limited experience (multifocal moderate-to-severe atypia, no tumor cell necrosis, and mitotic counts of 4 and 5 mitotic figures /10 high-power fields, respectively). Both tumors had areas that were indistinguishable from benign leiomyoma and both had diffuse immunoreactivity for p16 and p53. Six other tumors that had focal staining for these markers all had a benign outcome. Both patients with recurrence were alive at last follow-up (at 40 and 74 mo). All the other patients were alive and disease-free. Conclusions This and other studies suggest that uterine tumors classified as STUMPs using criteria proposed by Stanford investigators are usually clinically benign but should be considered tumors of low malignant potential because they can occasionally recur, in some cases, years after hysterectomy. After a mean follow-up of 80.8 months, only 2 of 16 tumors in this study recurred. Both of the latter tumors fulfilled the criteria for atypical leiomyoma with limited experience. Notably, the 2 recurrent tumors were the only ones that were strongly immunoreactive for p16 and p53, supporting earlier observations that these markers may be helpful in the prediction of the behavior of STUMPs. Patients diagnosed with STUMPs should receive long-term surveillance.

Uterine smooth muscle tumors other than the ordinary leiomyomas and leiomyosarcomas: A review of selected variants with emphasis on recent advances and unusual morphology that may cause concern for malignancy

Uterine smooth muscle tumors are classified according to their morphologic features that include architecture, growth pattern, cellular characteristics and constituents of the intercellular stroma. While terminologies used for the pathologic diagnosis of various subtypes may be eloquent and histologically accurate, some of these are confusing for the clinician and may also be open to interpretation by different pathologists: the labeling of atypical leiomyomas epitomizes this intricate system. Clinically, it is probably more useful to classify them as either tumors with or tumors without recurrent and/or metastatic potential. The term “atypical leiomyoma” has been used to label tumors that have a low risk of recurrence and is synonymous with benign tumors. The latter are known variously as leiomyoma with bizarre nuclei, symplastic leiomyoma, or pleomorphic leiomyoma. Variants of benign uterine smooth muscle tumors, such as mitotically active leiomyoma, cellular and highly cellular leiomyoma, epithelioid leiomyoma, and myxoid leiomyoma each have distinctive hallmarks that enable subclassification. Nevertheless, they may occasionally possess one or more unusual features that are cause for alarm. Tumors that have a dissecting growth pattern, with or without extrauterine extension, may mimic malignancy both grossly and microscopically. The current review discusses the pathologic diagnosis of and terminology applied to selected variants of uterine smooth muscle tumors other than the ordinary leiomyomas and leiomyosarcomas with emphasis on unusual reported features that may indicate malignancy. This includes an update on uterine smooth muscle tumor of uncertain malignant potential (STUMP), intravenous leiomyomatosis, benign metastasizing leiomyoma, and diffuse leiomyomatosis. Their clinicopathologic features, differential diagnoses, and management options based on findings in the previously reported cases will also be reviewed.

Reduction of hepatitis B surface antigen and covalently closed circular DNA by nucleos(t)ide analogues of different potency.

BACKGROUND & AIMS Few studies have investigated the effects of different nucleos(t)ide analogues against hepatitis B virus (HBV) on levels of covalently closed circular DNA (cccDNA) and hepatitis B surface antigen (HBsAg) in patients. We measured the magnitude of reduction of cccDNA and HBsAg by nucleos(t)ide analogue therapy and assessed the correlation between their reductions. METHODS We recruited 124 patients who were treated with 1 of the 5 nucleos(t)ide analogues (lamivudine, adefovir, entecavir, telbivudine, or clevudine). All patients had undergone liver biopsy when treatment began (baseline) and 1 year later. The cccDNA and HBsAg levels were measured by real-time polymerase chain reaction and the Elecsys II HBsAg Assay, respectively. RESULTS After 1 year of treatment, HBV in 7 patients had become resistant to the nucleos(t)ide analogue. The remaining 117 patients had an average reduction of approximately 0.2 log10 IU/mL in HBsAg, 5 log10 IU/mL in serum level of HBV DNA, 2 log10 copies/cell in intrahepatic total HBV DNA, and 1 log10 copies/cell in cccDNA. Although 88 of 117 patients (75%) had undetectable serum levels of HBV DNA (<12 IU/mL), all had detectable levels of HBsAg, and only 5 (4%) had undetectable levels of cccDNA. On treatment with nucleos(t)ide analogues, patients with greater reductions in levels of cccDNA had greater reductions in HBsAg, but these reductions did not reach statistically significant correlations. CONCLUSIONS Although nucleos(t)ide analogues potently reduced serum levels of HBV DNA, the reduction of HBsAg and cccDNA was small. In short-term therapy, the magnitude of HBsAg reduction does not correlate with that of cccDNA reduction.

Somatic mutations in the BRCA1 gene in Chinese sporadic breast and ovarian cancer

Inherited mutations in the BRCA1 gene confer increased susceptibility to breast and ovarian cancer. Its role in sporadic carcinogenesis is not well defined. Somatic mutations in breast cancers have not been reported and to date there are only three reports of somatic mutations in sporadic ovarian cancers. To investigate the contribution of BRCA1 mutations to sporadic breast and ovarian cancer in the Chinese population, we analysed 62 samples from Chinese women using the protein truncation test. There were 40 cases of breast cancer under age 50 and 22 cases of ovarian cancer, all unselected for family history. There was no age selection for the ovarian cancers. We found two somatic BRCA1 mutations in exon 11, one in a breast cancer and the other in an ovarian cancer, both of which result in truncated proteins. Our results indicate that somatic BRCA1 mutations, like somatic mutations in the BRCA2 gene, though very rare, can be found in both breast and ovarian cancers and support a tumor suppressor function for BRCA1 in sporadic tumors.

Tumor suppressor effect of follistatin-like 1 in ovarian and endometrial carcinogenesis—a differential expression and functional analysis

Endometrial and ovarian cancers are the most common and the most lethal gynecologic malignancies worldwide, respectively. By performing differential expression analysis using annealing control primer-based reverse transcription (RT)-polymerase chain reaction (PCR) on pooled complementary DNA (cDNA) from 45 endometrial and 36 ovarian cancers and their non-tumor samples, reduced expression of the follistatin-like 1 (FSTL1) was identified. Downregulation of FSTL1 was further confirmed on individual samples and cell lines by quantitative real-time RT-PCR and western blotting. For in vitro functional study, full-length cDNA of FSTL1 was cloned and transiently transfected into the ovarian cancer cell line Ovca420 and endometrial cancer cell line AN3CA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and cell count demonstrated significantly slower proliferation rate. By terminal uridine deoxynucleotidyl transferase dUTP nick end labeling and flow cytometric analysis, higher apoptotic activity and a remarkable increase in sub-G(1) cell population were observed in transfected cells, suggesting that FSTL1 induced apoptosis in cancer cells. Subsequent messenger RNA and protein expression analysis on downstream apoptotic molecules revealed upregulation and/or activation of FAS, FASLG, TRADD, Caspase-3, Caspase-7 and PARP by FSTL1 transfection, suggesting that FSTL1-induced apoptosis may be initiated mainly by FAS/FASLG death receptor-ligand binding. Cell migration and invasion assays demonstrated a remarkably lower cell migration and invasion capability in FSTL1-transfected cells in relation to downregulation of matrix metallopeptidase-2. Our findings suggested that a tumor suppressor role of FSTL1 may be important in ovarian and endometrial carcinogenesis.

A New Model Using Routinely Available Clinical Parameters to Predict Significant Liver Fibrosis in Chronic Hepatitis B

Objective We developed a predictive model for significant fibrosis in chronic hepatitis B (CHB) based on routinely available clinical parameters. Methods 237 treatment-naïve CHB patients [58.4% hepatitis B e antigen (HBeAg)-positive] who had undergone liver biopsy were randomly divided into two cohorts: training group (n = 108) and validation group (n = 129). Liver histology was assessed for fibrosis. All common demographics, viral serology, viral load and liver biochemistry were analyzed. Results Based on 12 available clinical parameters (age, sex, HBeAg status, HBV DNA, platelet, albumin, bilirubin, ALT, AST, ALP, GGT and AFP), a model to predict significant liver fibrosis (Ishak fibrosis score ≥3) was derived using the five best parameters (age, ALP, AST, AFP and platelet). Using the formula log(index+1) = 0.025+0.0031(age)+0.1483 log(ALP)+0.004 log(AST)+0.0908 log(AFP+1)−0.028 log(platelet), the PAPAS (Platelet/Age/Phosphatase/AFP/AST) index predicts significant fibrosis with an area under the receiving operating characteristics (AUROC) curve of 0.776 [0.797 for patients with ALT <2×upper limit of normal (ULN)] The negative predictive value to exclude significant fibrosis was 88.4%. This predictive power is superior to other non-invasive models using common parameters, including the AST/platelet/GGT/AFP (APGA) index, AST/platelet ratio index (APRI), and the FIB-4 index (AUROC of 0.757, 0.708 and 0.723 respectively). Using the PAPAS index, 67.5% of liver biopsies for patients being considered for treatment with ALT <2×ULN could be avoided. Conclusion The PAPAS index can predict and exclude significant fibrosis, and may reduce the need for liver biopsy in CHB patients.

Promoter Methylation and Differential Expression of π-Class Glutathione S-Transferase in Endometrial Carcinoma

Pi-class glutathione S-transferase (GSTP1), located on chromosome 11q13, codes for a phase II metabolic enzyme that detoxifies reactive electrophilic intermediates. The protein also interacts with steroid hormones in the human body. The role of GSTP1 in endometrial carcinoma has not been reported. In this study, we aimed at determining the expression of GSTP1 in relation to the epigenetic and genetic changes of the gene in endometrial carcinoma. The GSTP1 protein and mRNA expression was assessed by immunohistochemistry on tissue microarray and quantitative real-time reverse transcriptase-polymerase chain reaction, respectively. Its methylation status was studied by methylation-specific polymerase chain reaction and bisulfite sequencing. Possible mutations in coding region of GSTP1 were assessed by cDNA sequencing. Ninety-seven cases of endometrial carcinoma with available tissue blocks and clinical data were studied. Our results showed that 68.0% (66 of 97) of the cases showed reduced protein expression while 64% (16 of 25) showed reduced mRNA expression; 30.9% (30 of 97) of the cases demonstrated methylated alleles in at least one of the six methylation-specific polymerase chain reaction reactions. The methylation status significantly correlated with reduced protein expression (P = 0.008) and reduced mRNA expression (P = 0.003). Methylation at non-CpG sites including CpCpG trinucleotides and CpT dinucleotides were also observed. cDNA sequencing did not reveal genetic alterations in coding region of the gene. The extent of myometrial invasion was found to be significantly correlated with both the methylation status (P = 0.009) and the protein expression (P = 0.036) of the GSTP1 gene. We postulated that hypermethylation of the GSTP1 gene promoter region may act as a dynamic regulation mechanism contributing to reduced GSTP1 expression, which is associated with myometrial invasion potential of the endometrial carcinoma.

High hepatitis B surface antigen levels predict insignificant fibrosis in hepatitis B e antigen positive chronic hepatitis B.

Introduction There is no data on the relationship between hepatitis B surface antigen (HBsAg) levels and liver fibrosis in hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B (CHB). Methods Serum HBsAg and HBV DNA levels in HBeAg-positive CHB patients with liver biopsies were analyzed. The upper limit of normal (ULN) of alanine aminotransferase (ALT) was 30 and 19 U/L for men and women respectively. Histologic assessment was based on Ishak fibrosis staging for fibrosis and Knodell histologic activity index (HAI) for necroinflammation. Results 140 patients (65% male, median age 32.7 years) were recruited. 56 (40%) had ALT ≤2×ULN. 72 (51.4%) and 42 (30%) had fibrosis score ≤1 and necroinflammation grading ≤4 respectively. Patients with fibrosis score ≤1, when compared to patients with fibrosis score >1, had significantly higher median HBsAg levels (50,320 and 7,820 IU/mL respectively, p<0.001). Among patients with ALT ≤2×ULN, serum HBsAg levels achieved an area under receiver operating characteristic curve of 0.869 in predicting fibrosis score ≤1. HBsAg levels did not accurately predict necroinflammation score. HBsAg ≥25,000 IU/mL was independently associated with fibrosis score ≤1 (p = 0.025, odds ratio 9.042).Using this cut-off HBsAg level in patients with ALT ≤2×ULN, positive and negative predictive values for predicting fibrosis score ≤1 were 92.7% and 60.0% respectively. HBV DNA levels had no association with liver histology. Conclusion Among HBeAg-positive patients with ALT ≤2×ULN, high serum HBsAg levels can accurately predict fibrosis score ≤1, and could potentially influence decisions concerning treatment commencement and reduce the need for liver biopsy.