Recent topics on α‐fetoprotein

Zinc‐fingers and homeoboxes 2 (ZHX2) and zinc‐finger and BTB domain containing 20 (ZBTB20) repress the postnatal expression of α‐fetoprotein (AFP) by interacting with the AFP gene promoter regions. ZHX2 inhibits the expression of AFP and cyclins A and E. ZBTB20 is negatively regulated by CUX1, which promotes cell‐cycle progression, suggesting that AFP reactivation is closely linked to hepatocyte proliferation. A slight elevation in the serum AFP level often occurs in patients with chronic hepatitis C in the absence of hepatocellular carcinoma (HCC) and is an independent risk factor for HCC development to complement the fibrosis stage. In addition, the sustained elevation of AFP after interferon therapy is a risk factor of HCC development. AFP levels are clinically useful in predicting the outcomes of liver transplantation and sorafenib therapy for HCC patients. A low preoperative AFP level is a predictor of long‐term survival and is associated with a low recurrence rate of HCC after liver transplantation. AFP response (≥20% decrease in AFP during 6–8 weeks of treatment) rather than radiological outcomes is a significant prognostic factor for survival in sorafenib‐treated HCC patients. Highly sensitive Lens culinaris agglutinin‐reactive AFP (AFP‐L3) is 5–10 times more sensitive than conventional AFP‐L3, and useful for early detection of HCC in patients with total AFP below 20 ng/mL.     

Analysis of serum α‐fetoprotein concentration in patients with viral hepatitis

OBJECTIVE: To investigate serum α‐fetoprotein (AFP) concentrations in patients with viral hepatitis. METHODS: Serum concentrations of total bilirubin (TB), alanine aminotransferase (ALT), aspartate amino­transferase (AST), albumin, globulin, AFP and viral markers were determined in 310 patients with pathologically proven viral hepatitis. The relation between the concentration of AFP and clinical manifestation, pathology, family history of liver malignant disease and virus type was studied. RESULTS: Serum AFP concentrations were elevated in 115 of the 310 patients (37.1%). According to the pathological diagnosis, the lowest positive rate of AFP was in acute hepatitis (11.7%), the highest was in chronic severe hepatitis (66.7%), the second highest in liver cirrhosis (57.5%), and chronic hepatitis was intermediate (34.2%). If the diagnosis was based on the clinical manifestation, the highest positive rate was found in chronic severe hepatitis, the lowest in chronic hepatitis, and acute hepatitis was intermediate. The positive rate of serum AFP by virus type was 35.5% for hepatitis B (HBV), superinfected with HAV or with HEV was 62.8%, and with HCV was 27.3%. Only one in six patients with HCV infection and none with simple HAV or HEV infection were positive for AFP. In patients with a family history of liver cancer, the positive rate of AFP was higher than in those without such a history (57.9%vs 38.2%; P = 0.75). CONCLUSIONS: The results indicate that AFP positivity is not uncommon in patients with viral hepatitis and if the patient has an elevated concentration, it is highly likely to be HBV infection or HBV super­infected with HAV or HEV.     

Increasing hepatitis B viral load is associated with risk of significant liver fibrosis in HBeAg‐negative but not HBeAg‐positive chronic hepatitis B

Background/aims: To evaluate the association between demographical features, serum ALT and HBV DNA and the prevalence of significant fibrosis and inflammation on liver biopsy in patients with chronic hepatitis B. Methods: In this cross‐sectional study of patients on St Vincent's Hospital HBV database, patients were classified into three groups on the basis of HBeAg status and HBV DNA level and the prevalence of significant (F2/3/4) fibrosis and (A2/3) inflammation in each group was established. Patients were also divided into HBeAg‐positive and ‐negative groups and examined for the prevalence of significant fibrosis/inflammation in the strata of HBV DNA and ALT. Predictors of significant fibrosis and inflammation in HBeAg‐positive and ‐negative patients were examined by logistic regression. Results: Three hundred and ninety four patients (HBeAg positive=198; HBeAg negative=196) with liver biopsy were identified. Fifty‐eight percent of HBeAg‐negative patients with HBV DNA >25 000 IU/ml had F2/3/4 fibrosis. HBV DNA and F2/3/4 were positively correlated in HBeAg‐negative patients [odds ratio (OR) 1.42, P=0.001] but inversely correlated in HBeAg‐positive patients (OR 0.71, P=0.03). HBV DNA was an independent predictor of significant fibrosis in HBeAg negative (P=0.03) but not HBeAg‐positive patients. In HBeAg‐positive patients, age was the only predictor of significant fibrosis (P=0.001) and ALT the only predictor of significant inflammation (P=0.003). In the whole cohort there was a close positive association between inflammation and fibrosis. Conclusion: Increasing levels of HBV DNA are associated with increasing prevalence of significant fibrosis only in patients with HBeAg‐negative CHB.     

Interferon‐α treatment in children and young adults with chronic hepatitis B: a long‐term follow‐up study in Taiwan

Background/Aims: The short‐ and long‐term benefits of interferon (IFN)‐α therapy in young patients with chronic hepatitis B (CHB) acquiring infection perinatally or during early childhood have been questioned. Methods: Twenty‐one Taiwanese hepatitis B envelope antigen (HBeAg)‐positive CHB patients aged 1.8–21.8 years (median 14.0 years) with alanine aminotransferase (ALT)>80 IU/L at entry were enrolled for IFN‐α therapy. They received IFN‐α therapy with a dose of 3 MU/m²/day three times a week for 24 weeks. A control group included untreated 21 CHB patients closely matched for gender, age, duration of ALT >80 IU/L and HBeAg status. All 42 patients were prospectively followed for 6.5–12.5 years after the end of therapy. Results: The cumulative rate of virological response [anti‐HBe seroconversion and serum hepatitis B virus (HBV)‐DNA <10⁵ copies/ml] was not different between the IFN‐treated patients and control patients at 1 year (41 vs 44%) and at 6 years (88 vs 89%) after stopping treatment. Serum hepatitis B surface antigen loss occurred in two (9.5%) treated patients and in one (4.8%) control patient. Patients with a successful treatment response (anti‐HBe seroconversion, HBV‐DNA <10² copies/ml and ALT normalization at 1 year after stopping treatment) were younger than those without a successful response (P=0.03). A lower pretreatment serum HBV‐DNA level (<2 × 10⁸ copies/ml) is not only a significant factor to predict successful treatment response (P=0.008) but also has a beneficial effect on the long‐term cumulative rate of virological response in IFN‐treated patients (P=0.021), but not in control patients. Genotype difference or emergence of a precore stop codon mutant before treatment was not predictive for HBeAg clearance. Conclusion: For young CHB patients in Taiwan with infection occurring perinatally or in early childhood, the real advantage of IFN‐α therapy was not observed. IFN‐α therapy showed a beneficial effect on short‐ and long‐term virological outcomes only in those with a lower pretreatment serum HBV‐DNA level.     

Serum hepatitis B core‐related antigen is an effective tool to categorize patients with HBeAg‐negative chronic hepatitis B

The discrimination between active chronic hepatitis B (CHB) and the clinically quiescent infection (CIB) is not always easy, as a significant portion of patients falls in a “grey” zone. Hepatitis B core‐related antigen (HBcrAg) is a now quantifiable serological marker with potential applications in diagnosis and therapy monitoring. The aim of the present study was to evaluate the HBcrAg serum levels in HBeAg‐negative HBV infection, and its ability in identifying the clinical profile, in comparison with HBsAg serum levels. HBcrAg was retrospectively assessed on serum samples from a population of treatment‐naive HBeAg‐negative patients by ChemiLuminescent Enzyme Immunoassay (CLEIA). HBsAg and HBV‐DNA data were collected. Serological data were associated to clinical profile, defined in the subsequent follow‐up of at least 1 year. In the overall population of 160 HBeAg‐negative patients, HBcrAg results weakly correlated with qHBsAg levels (Spearman r = 0.471, P < 0.0001) and correlated closely with HBV‐DNA (Spearman r = 0.746, P < 0.0001). HBcrAg levels were significantly higher in 85 CHB patients relative to 75 CIB carriers. A value of 2.5 logU/mL produced the optimal cut‐off to identify CIB patients, with diagnostic accuracy comparable to HBsAg levels. In long‐term clinical evaluation, a single measurement of HBcrAg at the established cut‐off was optimally consistent with clinical outcome. Conversely, the HBsAg cut‐off performed well in the true quiescent phase and less in more difficult‐to‐categorize patients. In conclusion, single‐point use of HBcrAg serum levels provides an accurate identification of CIB and represents a useful tool for patient classification.     

Clinical symptoms and pedigree analysis in two families with benign familial persistently elevated α‐fetoprotein

OBJECTIVE : To evaluate the nature of persistently elevated α‐fetoprotein (AFP) in a family and the importance of correct recognition and diagnosis of familial AFP elevation. METHODS : In 1984 and 1997, a series of AFP radio‐immunoassays from two families with persistently elevated AFP were investigated and the family pedigrees were subanalyzed. RESULTS : Of the 29 members in the two families, 15 were examined. The AFP level of 10 people was persistently elevated, two of these had been misdiagnosed with primary hepatocellular carcinoma. CONCLUSIONS : Familial elevation of AFP is benign in nature. It should be kept in mind during mass surveys of AFP.     

Increased apoptosis in HepG2.2.15 cells with hepatitis B virus expression by synergistic induction of interferon‐γ and tumour necrosis factor‐α

Background: Interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) were thought to be important immune mediators in host defence against hepatitis B virus (HBV) infection. Aims: To examine the synergistic effect of IFN‐γ and TNF‐α on HBV‐expressing HepG2.2.15 cells and its potential mechanisms. Methods: Cell viability was quantitatively measured by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide assay. Cell morphology was captured using light microscopy. The typical DNA ladder test was performed using agarose gel electrophoresis. HBsAg and HBeAg titre changes were quantified by the enzyme‐linked immunosorbent assay method. Gene expression was analysed using cDNA macroarrays. Results: Interferon‐γ (1000 U/ml) alone or combined with TNF‐α (5 ng/ml) treatment resulted in apoptosis in HepG2.2.15 cells, but no significant apoptosis in the parent non‐virus expressing HepG2 cells. IFN‐γ‐ and TNF‐α‐mediated apoptosis was reduced by lamivudine treatment in HepG2.2.15 cells. IFN‐γ combined with TNF‐α reduced the titre of hepatitis B surface antigen and hepatitis B e antigen in the HepG2.2.15 cell line. For apoptosis‐related gene changes, IFN regulatory factor 1 (IRF‐1) (12.2‐fold), c‐myc (V00568 4.7‐fold, L00058 2.4‐fold) and caspase 7 (2.3‐fold) genes were upregulated in the combination treatment group. Conclusion: Interferon‐γ and TNF‐α play a role in the cell death of HBV‐expressing HepG2.2.15 cells. Expression of HBV leads to IFN‐γ‐ and TNF‐α‐mediated apoptosis in the cells. Increased IRF‐1, c‐myc and caspase 7 gene expression may be responsible for the synergistic induction of apoptosis by IFN‐γ and TNF‐α.     

Long‐term outcome and hepatocellular carcinoma development in chronic hepatitis B or cirrhosis patients after nucleoside analog treatment with entecavir or lamivudine

Aim: We conducted this prospective study to elucidate the long‐term outcome and incidence of hepatocellular carcinoma (HCC) development after nucleos(t)ide analog (NA) treatment in patients with chronic hepatitis B (CHB) or cirrhosis. Methods: CHB or cirrhosis patients without past NA treatment or HCC were started on entecavir (ETV) or lamivudine (LVD), and prospectively followed up with monthly blood tests, and with abdominal imaging every 6 months in CHB and every 3 months in cirrhosis patients. Results: A total of 256 subjects with CHB (n = 194) or cirrhosis (n = 62) received ETV (n = 129) or LVD (n = 127) for 4.25 years (range: 0.41–10.0). After NA treatment, serum HBV DNA, alanine aminotransferase and α‐fetoprotein (AFP) dropped significantly, along with significant increases in serum albumin and prothrombin time. Drug‐resistance developed in 60 cases in the LVD group and in only one case in the ETV group. HCC developed in 35 patients, and the incidence at years 1, 3, 5, 7 and 10 was significantly higher in patients with cirrhosis (8.1%, 17.5%, 43.2%, 46.7% and 53.4%, respectively) than chronic hepatitis (1.6%, 3.5%, 3.5%, 7.1% and 29.6%, respectively), with no difference between ETV and LVD. After NA treatment, the sensitivity/specificity for HCC of AFP and des‐γ‐carboxy prothrombin (DCP) was 45.7%/97.3% and 33.3%/96.2%, respectively, with the specificity of AFP being higher than at baseline (64.4%), at the cut‐off of 10 ng/mL. Conclusion: NA exerted a long‐term efficacy and improved hepatic reservation in CHB and cirrhosis. After NA treatment, AFP dropped to lower than 10 ng/mL with marked elevation of specificity, leading to an earlier detection of HCC.     

New HBV subgenotype D9, a novel D/C recombinant,identified in patients with chronic HBeAg‐negative infection in Eastern India

Genome diversity is a hallmark of hepatitis B virus (HBV), which allowed its classification into 10 genotypes (A–J) and numerous subgenotypes. Among them, Genotype D is currently segregated into eight subgenotypes (D1–D8). Here, we report the identification and characterization of a novel subgenotype within genotype D of HBV from chronic hepatitis B e antigen (HBeAg)‐negative patients of Eastern India. Phylogenetic tree analysis based on complete genome sequences revealed that six of 39 HBV/D isolates formed a distinct cluster supported by high bootstrap value and had nucleotide divergence >4% relative to the known D subgenotypes (D1–D8), justifying their assignment into a new subgenotype (D9). By comparing the amino acid sequences of the four ORFs of HBV/D9 with D1–D8, 36 specific residues, including a unique one (E₁₁₂ in the core region), were identified that could be considered as a signature of D9. Further analysis by Simplot, BootScan and jpHMM demonstrated that D9 resulted from a discrete recombination with genotype C over the precore–core region. This type of recombination has not been described previously as all C/D recombinants reported so far possessed genotype C backbones with mosaic fragments derived from HBV/D. Interestingly, compared to other subgenotypes of HBV/D, D9 isolates had a higher frequency of mutations (A1762T and G1764A) in the basal core promoter region that had been implicated in the development of hepatocellular carcinoma. Further investigations are needed to determine the overall prevalence and clinical significance of these newly characterized D9 strains and to assess the impact of inter‐genotypic recombination on viral properties.     

Long‐term efficacy and safety of peginterferon in the treatment of children with HBeAg‐positive chronic hepatitis B

Achieving ‘clinical cure’ in children with chronic hepatitis B (CHB) with safe and effective antiviral treatment is an unmet medical need. Peginterferon (PegIFN) has higher hepatitis B s antigen (HBsAg) clearance than nucleoside analogs (NUC). Currently, studies on interferon (IFN) in the treatment of Chinese children with CHB are relatively rare. This study aimed to further explore the efficacy of PegIFNα‐2a as an antiviral treatment in Chinese children and analyse the long‐term follow‐up after drug discontinuation. We enrolled 118 patients with CHB (2‐16 years old, 79 cases are males) treated with PegIFNα‐2a by the author in the Third People's Hospital of Kunming City from February 2009 to February 2015. The course of treatment was 52 weeks, with a follow‐up period of 104 weeks. All the patients completed at least 1 dose, of which 104 completed at least 36 weeks of treatment and 104 weeks of follow‐up. During treatment and follow‐up, indicators such as alanine aminotransferase (ALT), hepatitis B virus (HBV) DNA and HBV serological markers were monitored, and the efficacy and safety of PegIFNα‐2a in the treatment of CHB patients were observed. Hepatitis B e antigen (HBeAg) clearance and seroconversion rates were 53.8% and 49%, respectively, when the drug was discontinued; 72.1% and 72.1%, respectively, at the end of the follow‐up; and 98.2% and 98%, respectively, for sustained response. HBsAg clearance and seroconversion rates were 48.1% and 47.1%, respectively, when the drug was discontinued; 53.8% and 52.9%, respectively, at the end of the follow‐up; and 94% and 95.9%, respectively, for sustained response. The HBV DNA suppression rate was 89.4% when the drug was discontinued, 90.4% at the end of the follow‐up and 97.8% for sustained response. Two patients had virological relapse (2.3%) during follow‐up; however, no clinical relapse occurred. Multivariate regression analysis showed that genotype B, weight < 25 kg or between 25 and 45 kg, and reduction of HBsAg by more than 1 log following 24 weeks of treatment were independent predictors of HBsAg clearance at the end of follow‐up. Adverse events that occurred during treatment were similar to those reported in previous clinical studies on PegIFN. The results of this study showed that PegIFN was safe and effective in the treatment of children with CHB, and sustained response could be achieved after treatment. PegIFN treatment of children with CHB helps more achieve ‘clinical cure’.     

Increased peripheral RORα and RORγt mRNA expression is associated with acute‐on‐chronic hepatitis B liver failure

Summary. T helper cells17 (Th17) have accurate but inconclusive roles in the pathogenesis of acute‐on‐chronic hepatitis B liver failure (ACHBLF). Retinoic acid‐related orphan receptor γ t(RORγt) and RORα are two lineage‐specific nuclear receptors directly mediating Th17 differentiation. This study was aimed to evaluate the gene expression of RORα and RORγt and their potential role in ACHBLF. Forty patients with liver failure, 30 with chronic hepatitis B (CHB) and 20 healthy controls were studied. The mRNA levels of RORα and RORγt in peripheral mononuclear cells were determined by quantitative real‐time polymerase chain reaction. The frequency of peripheral Th17 cells was determined using flow cytometry. The serum levels of interleukin‐6(IL‐6), transforming growth factor –β (TGF‐β), interleukin‐17(IL‐17), interleukin‐23(IL‐23) and interferon‐γ (IFN‐γ) were measured by enzyme‐linked immunosorbent assay. The frequency of peripheral Th17 cells in patients with liver failure was significantly increased compared to patients with CHB and controls. The peripheral mRNA levels of RORα and RORγt in hepatitis B‐associated acute‐on‐chronic liver failure were significantly higher than in patients with CHB and controls as were the serum levels of IL‐6 and TGF‐β. The serum level of IFN‐γ in patients with acute‐on‐chronic liver failure from HBV was significantly higher than patients with CHB but lower than controls. In patients with acute‐on‐chronic liver failure associated with HBV, RORγt, IL‐6 and IL‐23 were positively correlated with the frequency of Th17 cells, while RORα, TGF‐β and IFN‐γ had no correlation with the latter. The mRNA level of RORγt was positively correlated with model of end‐stage liver disease (MELD) score, but there was no correlation of RORα and MELD score. RORγt plays an important role in the pathogenesis of acute‐on‐chronic HBV‐associated liver failure and might be considered to be a candidate factor consistent with the severity of disease.     

Early α‐fetoprotein response as a predictor for clinical outcome after localized concurrent chemoradiotherapy for advanced hepatocellular carcinoma

Backgrounds: There are limitations in using only radiological criteria to evaluate treatment outcomes in hepatocellular carcinoma (HCC). α‐fetoprotein (AFP) is regarded as an indicator of tumour activity in HCC. Aims: We present a novel correlation between AFP response and survival outcome in patients treated with localized concurrent chemoradiotherapy (CCRT). Materials: From 2005 to 2008, 187 locally advanced HCC patients underwent localized CCRT (external beam radiotherapy at 45 Gy over 5 weeks plus a concurrent hepatic arterial infusion of 5‐fluorouracil during the first/fifth week), followed by repetitive hepatic arterial infusional chemotherapy (HAIC) with 5‐fluorouracil and cisplatin. Among them, 149 with an elevated baseline AFP level (>20 ng/ml) were finally studied. AFP response was defined as >50% decrease from baseline, 1 month after the completion of localized CCRT. Results: Patients' characteristics were as follows: median age (52 years); Child–Pugh class A/B (n=137/12 respectively); and portal vein thrombosis (n=118). AFP responders (101 patients) had better objective responses than AFP non‐responders (48 patients) after CCRT (44.5 vs. 12.5%; P<0.001) and subsequent HAIC (51.5 vs. 16.7%; P<0.001). Both median progression‐free survival (PFS, 8.1 vs. 3.9 months; P<0.001) and overall survival (OS, 13.3 vs. 5.9 months; P<0.001) were longer in AFP responders than AFP non‐responders. In multivariate analysis, AFP response and objective response were independent factors affecting PFS and OS. Furthermore, AFP non‐responders were more likely to have extrahepatic metastasis within 6 months of treatments initiation than AFP responders (59.5 vs. 25.9%; P<0.001). Conclusions: Early AFP response may be useful not only in predicting prognosis and treatment response but also in establishing optimized treatment plans for HCC.     

Early prediction of sustained virological response at day 3 of treatment with albinterferon‐α‐2b in patients with genotype 2/3 chronic hepatitis C

Background: Albinterferon‐α‐2b (albIFN) is a long‐acting fusion polypeptide composed of albumin and IFN‐α‐2b. In a phase 2 study of albIFN 1500 μg q2wk or q4wk in patients with genotype 2/3 chronic hepatitis C, albIFN demonstrated sustained virological response (SVR) rates of 62–77% (intent‐to‐treat population). Aims: To assess the association of initial viral kinetics during albIFN therapy with baseline factors and SVR prediction. Methods: In all, 43 patients were treated with albIFN 1500 μg (q2wk/q4wk) plus ribavirin (RBV) 800 mg/day for 24 weeks. Hepatitis C virus (HCV)‐RNA levels were measured by real‐time polymerase chain reaction, insulin resistance by homeostasis model assessment of insulin resistance (HOMA‐IR) and serum albIFN levels by enyzme‐linked immunosorbent assay. Prediction analysis was performed in a per protocol 28‐patient subset who were ≥80% adherent to albIFN/RBV and had HCV‐RNA levels measured at treatment day 3. Results: Day‐3 HCV‐RNA level and first‐phase viral decline as well as second‐phase slope of viral decline were significantly associated with SVR. In adherent patients, 82.1% had a day‐3 viral load <4.2 log₁₀ IU/ml or first‐phase decline >1.25 log₁₀ IU/ml, which was predictive of SVR, both positively (95.7%; sensitivity: 100%) and negatively (100%; specificity: 83.3%). As low first‐phase decline was associated with a high pretreatment HOMA‐IR index (P=0.004) and a low day‐3 serum albIFN level (P=0.01). Conclusions: First‐phase viral decline with albIFN/RBV was predictive of SVR in this study and may be modulated in part by IR.