Responses are durable for up to 5 years after completion of peginterferon alfa-2a treatment in hepatitis B e antigen-positive patients.

BACKGROUND: In the large randomised NEPTUNE study, peginterferon alfa-2a 180 µg/wk for 48 weeks produced higher hepatitis B e antigen (HBeAg) seroconversion rates 24 weeks post-treatment (36%) than a lower dose (90 µg/wk) and/or shorter duration (24 weeks) (range 14%-26%). AIM: To determine seroconversion rates 5 years after completion of treatment in NEPTUNE. METHODS: HBeAg-positive patients who completed 24 weeks' follow-up in NEPTUNE (with peginterferon alfa-2a 90 µg/wk × 24 weeks [group 1]; 180 µg/wk × 24 weeks [2]; 90 µg/wk × 48 weeks [3] or 180 µg/wk × 48 weeks [4]) were followed up. RESULTS: Three hundred and eighty three of the 544 patients in the original study were enrolled in the long-term follow-up study. Many patients (196 overall; more in groups 1-3 than 4) received nucleos(t)ide analogues or immunomodulators during follow-up, and more patients had missing data at year 5 in groups 2 and 4 (48 weeks, 50/112) than in groups 1 and 3 (24 weeks, 23/103), which confounds the planned per-protocol analysis. HBeAg seroconversion rates in groups 1, 2, 3 and 4 at year 5 were 47.5%, 50.7%, 52.2% and 67.1%, respectively, (odds ratio for group 4 versus 1-3: 2.02; 95% CI 1.21, 3.38), using multiple imputation methods for missing measurements. CONCLUSION: Seroconversion rates are durable for up to 5 years after completion of peginterferon alfa-2a therapy and, consistent with NEPTUNE, the results suggest that the licensed regimen (180 µg × 48 weeks) is more efficacious for HBeAg-positive patients than a lower dose and/or shorter treatment duration.

Great and rapid HBsAg decline in patients with on-treatment hepatitis flare in early phase of potent antiviral therapy.

HBsAg decline during nucleos(t)ide analogue therapy in chronic hepatitis B with lower pretherapy ALT is usually small and slow. This study aimed to investigate why ~10% of such patients showed "rapid HBsAg decline" ≥0.5 log10 IU/mL by month 6 of therapy. Patients with persistent pretherapy ALT <5X ULN who had qHBsAg at baseline, months 6 and 12 of entecavir or tenofovir therapy were studied. "On-treatment ALT elevation" was defined as >10% increase above baseline to >2X ULN during first 6 months of therapy. Of the 256 patients treated, 51 experienced transient "on-treatment ALT elevation" [group A], including 30 (11.7%) with ALT elevation to 2-5X ULN [group A-1] and 21 (8.2%) flared to >5X ULN [group A-2]. The magnitude of qHBsAg decline and rate of "rapid HBsAg decline" by month 6 was significantly greater and more frequent in group A (-0.446 vs -0.042 log10 IU/mL; 45.1 vs 8.8%, respectively, P = 0.000) than in the remaining 205 patients without on-treatment ALT elevation (group B), being greatest in patients with hepatitis flare (group A-2: -0.559 log10 IU/mL and 57.1%, respectively). In patients with therapy ≥2 years, patients with "on-treatment ALT elevation" also showed significantly greater annual HBsAg decline, more frequent to <100 IU/mL and 4 times higher HBsAg seroclearance rate. "On-treatment ALT elevation," especially flare >5X ULN, during entecavir therapy or tenofovir therapy may enhance/accelerate HBsAg decline, suggesting the effect of immune restoration upon potent viral suppression.

Clinical outcomes after spontaneous and nucleos(t)ide analogue-treated HBsAg seroclearance in chronic HBV infection.

BACKGROUND: Both spontaneous and nucleos(t)ide analogue (Nuc)-treated hepatitis B surface antigen (HBsAg) seroclearance are associated with excellent clinical outcomes. AIM: To conduct a case-control study to explore whether there is difference of clinical outcomes between these two groups. METHODS: A total of 312 chronic hepatitis B patients with spontaneous HBsAg seroclearance and 110 patients with Nuc-treated HBsAg seroclearance were recruited retrospectively. Propensity score (PS) matching method produced 98 patients in each group for comparison. The development of hepatocellular carcinoma (HCC), hepatic complications and cumulative incidence of antibody to HBsAg (anti-HBs) was compared. RESULTS: During a mean follow-up period of 107 months after HBsAg seroclearance, five patients developed HCC after a mean period of 75.3 months (four and one patients with spontaneous and Nuc-treated HBsAg seroclearance, respectively) in overall population. One died of pneumonia with sepsis and one experienced variceal bleeding in Nuc-treated patients but none in spontaneous group. The incidence of anti-HBs seroconversion was comparable between spontaneous and Nuc-treated HBsAg seroclearance (69.6% vs. 66.4%, respectively, P = 0.617). There were no significant differences in HCC development (2% vs. 1.1%), overall mortality (0% vs. 1%), variceal bleeding (0% vs. 4.2%) and 6-year cumulative incidence of anti-HBs seroconversion (62.3% vs. 61.5%) among PS-matched patients with spontaneous and Nuc-treated HBsAg seroclearance. CONCLUSIONS: The clinical outcomes between patients with spontaneous and Nuc-treated HBsAg seroclearance are comparable. HCC can develop at a low rate during long-term follow-up and periodic surveillance after HBsAg seroclearance is still mandatory.

Clinical outcomes after interruption of entecavir therapy in HBeAg-negative chronic hepatitis B patients with compensated cirrhosis.

BACKGROUND: Long-term nucleos(t)ide analogues therapy may reduce hepatocellular carcinoma (HCC) in chronic hepatitis B patients with advanced fibrosis or cirrhosis. AIM: To investigate in a retrospective-prospective study whether this beneficial effect would be reduced in cirrhotic patients who discontinued a successful course of entecavir (ETV) therapy. METHODS: The study included 586 hepatitis B e antigen (HBeAg)-negative patients with compensated cirrhosis, mean age of 53.8 ± 10 years and 81% males, treated with ETV for at least 12 months. After ETV therapy for 46.5 ± 22.9 months, 205 patients who achieved hepatitis B virus (HBV) DNA suppression discontinued therapy. The clinical outcomes were assessed and HCC incidence was compared between propensity score (PS)-matched patients who continued and patients who discontinued ETV therapy by Asian Pacific Association for the Study of Liver stopping rule. RESULTS: During a mean duration of 59.3 ± 19 months after start of ETV therapy, nine and six HCC developed in an estimated annual incidence of 2.3% and 1.6% in 154 PS-matched patients who continued and who discontinued ETV therapy, respectively (P = 0.587). Multivariate Cox proportional hazards regression analyses showed that age (HR 1.065, P < 0.001) and HBV DNA (HR 1.216, P = 0.048) were the significant factors for HCC development. The rates of adverse clinical outcomes were comparable. CONCLUSIONS: The clinical outcomes, including HCC, after cessation of a successful course of entecavir therapy in patients with compensated cirrhosis were comparable to those who continued therapy. The results suggest that this strategy of finite therapy is safe and a feasible alternative to indefinite therapy, especially in a low resources setting.

Systematic review: cessation of long-term nucleos(t)ide analogue therapy in patients with hepatitis B e antigen-negative chronic hepatitis B.

BACKGROUND: It has been debated whether finite nucleos(t)ide analogue therapy is feasible in HBeAg-negative chronic hepatitis B. AIM: To review this issue systematically. METHODS: Using text terms HBsAg and various nucleos(t)ide analogues, PubMed was searched between 1995 and 2014 to find studies on therapy >6 months in adult HBeAg-negative chronic hepatitis B patients with off-therapy follow-up >6 months. RESULTS: Twenty-two studies with a total of 1732 patients were identified and included. The median duration of therapy, consolidation therapy and off-therapy follow-up ranged from 6 months to 8 years, 4 to 96 weeks and 6 to 80 months respectively. Patients were monitored with serum ALT and HBV DNA monthly in the first 1-3 months and every 3-6 months afterwards in most studies. The 1-year off-therapy 'virological relapse' (HBV DNA >2000 IU/mL) and 'clinical relapse' (HBV DNA > 2000 IU/mL + ALT elevation) occurred in <70% and <50% of the patients, respectively, and <40% of the patients received re-treatment. These rates were higher in patients with shorter treatment, shorter consolidation therapy and those treated with less potent nucleos(t)ide analogues. Off-therapy severe flares were rare and hepatic decompensation was reported in only one patient with cirrhosis. Biochemical relapse reflecting enhanced immune-mediated hepatocyte killing may lead to a higher chance for off-therapy HBsAg seroclearance and be possibly desirable. CONCLUSION: With an appropriate stopping rule and a proper off-therapy monitoring plan, cessation of long-term nucleos(t)ide analogue therapy prior to HBsAg seroclearance in HBeAg-negative chronic hepatitis B is a feasible alternative to indefinite treatment.

Efficacy and safety of continuous 4-year telbivudine treatment in patients with chronic hepatitis B.

In the phase-III GLOBE/015 studies, telbivudine demonstrated superior efficacy vs lamivudine during 2-year treatment in HBeAg-positive and HBeAg-negative chronic hepatitis B (CHB). After completion, 847 patients had an option to continue telbivudine treatment for further 2 years. A total of 596 (70%) of telbivudine-treated patients, who were serum HBV DNA positive or negative and without genotypic resistance to telbivudine at the end of the GLOBE/015 trials, were enrolled into a further 2-year extension study. A group of 502 patients completed 4 years of continuous telbivudine treatment and were included in the telbivudine per-protocol population. Amongst 293 HBeAg-positive patients, 76.2% had undetectable serum HBV DNA and 86.0% had normal serum ALT at the end of 4 years. Notably, the cumulative rate of HBeAg seroconversion was 53.2%. Amongst 209 HBeAg-negative patients, 86.4% had undetectable HBV DNA and 89.6% had normal serum ALT. In patients who had discontinued telbivudine treatment due to HBeAg seroconversion, the HBeAg response was durable in 82% of patients (median 111 weeks of off-treatment follow-up). The cumulative 4-year resistance rate was 10.6% for HBeAg-positive and 10.0% for HBeAg-negative patients. Most adverse events were mild or moderate in severity and transient. Renal function measured by estimated glomerular filtration rate (eGFR) increased by 14.9 mL/min/1.73 m(2) (16.6%) from baseline to 4 years (P < 0.0001). In conclusion, in HBeAg-positive and HBeAg-negative CHB patients without resistance after 2 years, two additional years of telbivudine treatment continued to provide effective viral suppression with a favourable safety profile. Moreover, telbivudine achieved 53% of HBeAg seroconversion in HBeAg-positive patients.

Basal core promoter mutation is associated with progression to cirrhosis rather than hepatocellular carcinoma in chronic hepatitis B virus infection.

BACKGROUND: As most cases of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) have concurrent cirrhosis, viral factors identified to be associated with HCC might be related to cirrhosis rather than HCC. METHODS: Hepatitis B virus DNA levels, genotypes and precore/basal core promoter (BCP) mutants were compared between cirrhotic HCC and non-cirrhotic HCC patients. Age- and sex-matched case-control studies were performed to identify the risk factors. RESULTS: Hepatitis B virus DNA levels showed no significant difference between non-cirrhotic HCC patients (n=20) and cirrhotic HCC patients (n=140) or 1 : 3 age- and sex-matched cirrhotic HCC patients (n=60), but genotype C and BCP mutant were significantly more prevalent in the latter than in the former. In multiple logistic regression, BCP mutant but not genotype C correlated significantly with the presence of cirrhosis in HCC patients. Compared with inactive carriers (n=60), non-cirrhotic HCC patients (n=20) had significantly higher HBV DNA levels but no difference in HBV genotypes and precore/BCP mutants. Furthermore, HBV DNA levels, the distribution of HBV genotypes and the prevalence of precore/BCP mutants all failed to show any significant difference between cirrhotic HCC patients (n=60) and cirrhotic patients without HCC (n=60). CONCLUSION: Basal core promoter mutant is associated with progression to cirrhosis rather than HCC in chronic HBV infection.

Serial HBV DNA levels in patients with persistently normal transaminase over 10 years following spontaneous HBeAg seroconversion.

Earlier studies addressing the hepatitis B virus (HBV) DNA cut-off level for inactive chronic HBV infection largely involved patients with normal alanine aminotransferase (ALT) for only 1-2 years and based on a single time HBV DNA assay. This study was conducted to address this issue using serial HBV DNA assays in patients with persistently normal ALT (PNALT) over 10 years following spontaneous hepatitis B e antigen (HBeAg) seroconversion. Serial serum specimens (mean 9 samples per patient) of 62 patients with PNALT and no disease progression over 10 years (median 18.1 years) after spontaneous HBeAg seroconversion were assayed for HBV DNA. Excluding assays within 1 year after HBeAg seroconversion, 21% and 82.3% of the patients with PNALT had HBV DNA levels persistently lower than 4 log(10) and 5 log(10) copies/mL, respectively, and only 8% had a level ≥ 5 log(10) copies/mL in at least two assays. Of the 27 patients with PNALT defined by ALT <30 U/L for male and <19 U/L for female, only 33% had serum HBV DNA level persistently <4 log(10) copies/mL. There was no significant difference in the serial HBV DNA changes among patients with different gender, HBV genotype or age at HBeAg seroconversion. Liver biopsy in nine patients invariably showed minimal necroinflammation and one showed Ishak fibrosis score 4. These results suggest that 5 log(10) copies/mL (20,000 IU/mL) is a more appropriate cut-off HBV DNA level for inactive chronic HBV infection in the setting of PNALT.

Shorter durations and lower doses of peginterferon alfa-2a are associated with inferior hepatitis B e antigen seroconversion rates in hepatitis B virus genotypes B or C.

UNLABELLED: As there is currently a lack of consensus on the most appropriate dose and duration of peginterferon alfa-2a (PEG-IFNα-2a) therapy in hepatitis B e antigen (HBeAg)-positive patients, the efficacy and safety of either 24 or 48 weeks' duration and 90 µg/week or 180 µg/week doses were compared. HBeAg-positive patients (n = 544; 34% genotype B, 51% genotype C) were randomized to receive PEG-IFNα-2a (2 × 2 factorial design) for 24 or 48 weeks and at 90 µg/week or 180 µg/week and included in the per-protocol population. The primary efficacy endpoint of the noninferiority study was HBeAg seroconversion 6 months posttreatment. The prespecified odds ratio (OR) noninferiority margin was 1.88 with a one-sided significance level of 0.025. The highest rates of HBeAg seroconversion 6 months posttreatment were in the 180/48 arm (36.2% versus 14.1%-25.8% in the other arms). When the dose and duration arms were pooled, the OR for noninferiority of 24 weeks versus 48 weeks was 2.17 (95% confidence interval [CI] 1.43, 3.31; P = 0.749) and for 90 µg versus 180 µg was 1.79 (95% CI 1.18, 2.72; P = 0.410). As the upper limit of the 95% CI of the ORs were >1.88, 24 weeks were inferior to 48 weeks and 90 µg/week was inferior to 180 µg/week. The highest rates of response in the 180/48 arm were achieved by patients with HBsAg <1,500 IU/mL at Week 12 (58%) or Week 24 (57%), whereas patients with HBsAg >20,000 IU/mL did not respond. Adverse events were typical of those associated with PEG-IFNα-2a. CONCLUSION: Compared with lower doses and shorter durations, the licensed PEG-IFNα-2a treatment regimen (180 µg/48 weeks) was the most efficacious and beneficial for HBeAg-positive patients predominantly infected with hepatitis B virus genotypes B or C.

Temporal relationship between hepatitis B virus enhancer II/basal core promoter sequence variation and risk of hepatocellular carcinoma.

BACKGROUND AND AIMS: To investigate the temporal relationship between sequence variation in the enhancer II (EnhII), basal core promoter (BCP), and precore regions of hepatitis B virus (HBV) and the risk of hepatocellular carcinoma (HCC), we conducted a nested case-control study within a cohort of 4841 male HBV carriers who were recruited during the period 1988-1992. METHODS: The HBV DNA sequence was determined in baseline blood samples taken from 132 incident cases and 204 controls. Base exchanges during follow-up in 71 cases were compared with 81 controls with samples taken during a similar length of follow-up. RESULTS: Nine single nucleotide polymorphisms in the EnhII/BCP regions (six of which were genotype C HBV related) were associated with subsequent risk of HCC. The strength of these associations decreased as the lag time between baseline measurement and diagnosis increased over 3 years. However, an increased disease risk in subjects with BCP double variants (mostly T1762/A1764) or genotype C HBV-related variants was evident 9 years or more before diagnosis. The BCP double variants (odds ratio, 1.92 (95% confidence interval, 1.14 to 3.25)) were statistically significantly associated with HCC risk even after adjusting for alanine aminotransferase levels, antibodies against HBV e antigen, HBV genotype, HBV viral load, and other sequence variants. Longitudinal analysis indicated that the increased HCC risks for at-risk sequence variants were attributable to the persistence of these variants. CONCLUSIONS: HCC risk is associated with sequence variation in the EnhII/BCP regions of HBV, and persistence of at-risk sequence variants is critical for HCC development.

Sustained virological response to interferon reduces cirrhosis in chronic hepatitis C: a 1,386-patient study from Taiwan.

BACKGROUND: The long-term benefits of interferon-based therapy on preventing cirrhosis at non-cirrhotic stage in chronic hepatitis C patients are not fully clarified. AIM: To evaluate the effectiveness of interferon-based therapy regarding to cirrhosis prevention in non-cirrhotic chronic hepatitis C patients. METHODS: A total of 1386 biopsy-proven, non-cirrhotic chronic hepatitis C patients (892 received interferon-based therapy and 494 untreated) were enrolled. RESULTS: Fifty-six untreated and 51 treated (24 sustained virologic responders and 27 non-responders) patients developed cirrhosis during a mean follow-up period of 5.0 (1-16) and 5.1 (1-15.3) years, respectively. The annual incidences of cirrhosis in untreated and treated groups were 2.26 and 1.11% (non-responders: 1.99%, sustained responders: 0.74%), respectively. The 15-year cumulative incidence of cirrhosis was significantly lower in treated (9.9%) than untreated patients (39.8%, P = 0.0008, log-rank test). The 14.5-year cumulative incidence of cirrhosis was significantly lower in sustained responders (4.8%) compared with non-responders (21.6%, P = 0.0007) and untreated patients (36.6%, P < 0.0001). The difference was not significant between non-responders and untreated controls. Cox proportional hazards regression showed sustained virologic responders and younger age were independent negative factors for cirrhosis development. CONCLUSION: A sustained virologic response secondary to IFN-based therapy could reduce cirrhosis development in chronic hepatitis C patients.

Chronic hepatitis B virus infection acquired in childhood: special emphasis on prognostic and therapeutic implication of delayed HBeAg seroconversion.

In high endemic areas of hepatitis B virus (HBV) infection, the vast majority of infection is acquired perinatally or during early childhood. The age of the patient is, therefore, almost equivalent to the duration of HBV infection. The natural history of chronic HBV infection consists of three chronological phases: immune tolerance, immune clearance and low replicative phases. The prevalence of hepatitis B e antigen (HBeAg) in asymptomatic HBV carriers is around 90% before 15 years of age, and decreases remarkably to less than 10% after 40 years of age. The immune clearance phase is characterized by a series of hepatitis flares and remissions. These will be followed eventually by HBeAg seroconversion, which is usually accompanied by remission of liver disease and confers favourable outcome. However, patients with persistent HBeAg seropositivity over 40 years of age are associated with a significantly higher risk for progression to cirrhosis than those with HBeAg seroconversion before 40 years of age, and thus should be considered as patients with 'delayed' HBeAg seroconversion. Antiviral or immunomodulatory therapy should be considered seriously for these patients.

Does increased body mass index with hepatic steatosis contribute to seroclearance of hepatitis B virus (HBV) surface antigen in chronic HBV infection?

BACKGROUND: Overweight and hepatic steatosis have been increasingly recognized recently. This study aimed to test whether substantial amount of fatty infiltration in liver, which may interfere with cytoplasmic distribution of hepatitis B surface antigen (HBsAg), can contribute to HBsAg seroclearance in HBsAg carriers. METHODS: Clinical and laboratory data including ultrasound grading of hepatic steatosis were studied in 54 HBsAg carriers with HBsAg seroclearance, and the results were compared with 108 age- and sex-matched carriers with HBsAg persistence. RESULTS: Body mass index and ultrasound grading of hepatic steatosis were significantly higher in HBsAg carriers with HBsAg seroclearance than in those with HBsAg persistence. The degrees of hepatic steatosis correlated significantly with body mass index (P<0.001). The prevalence of mild hepatic steatosis showed no significant difference (33% (18/54) vs 31% (33/108), P=0.72), but moderate-severe hepatic steatosis was significantly more prevalent in patients with HBsAg seroclearance (33% (18/54) vs 13% (17/108), P=0.01). HBsAg carriers with moderate and severe hepatic steatosis were associated with a 3.2-fold (95% confidence interval: 1.2-8.4, P=0.02) and 3.9-fold (95% confidence interval: 1.1-14.2, P=0.04), respectively, increased odds of HBsAg seroclearance compared to those without hepatic steatosis. CONCLUSION: Moderate-severe hepatic steatosis may contribute to HBsAg seroclearance in HBsAg carriers.

Hepatitis B virus genotype B is associated with better response to thymosin alpha1 therapy than genotype C.

Hepatitis B virus (HBV) genotype has been reported to correlate with response to interferon treatment in several studies. The relationship between HBV genotype and thymosin alpha1 (T-alpha1) treatment is unknown. We retrospectively examine HBV genotypes, precore and core promoter mutations in patients treated with Talpha1 and analyse the correlation between complete response [alanine aminotransferase (ALT) normalization plus seroclearance of HBeAg and HBV-DNA] and HBV genotype. It consisted 98 patients with chronic hepatitis B randomly allocating to three groups: (i) T6 group (n = 32) received a 26-week course of Talpha1 1.6 mg two times a week; (ii) T12 group (n = 34) received the same regimen as T6 group, but Talpha1 therapy extended for 52 weeks; (iii) T0 group (n = 32) served as a control and was followed up for 18 months without specific treatment. Stepwise logistic regression analysis showed that genotype (OR, 3.747; 95% CI, 1.066-13.170; P = 0.039), precore mutation (OR, 6.285; 95% CI, 1.874-21.086; P = 0.003) and Talpha-1 treatment (OR, 12.045; 95% CI, 2.220-65.354; P = 0.004) as independent factors associated with complete response. The complete response of Talpha-1 therapy was higher in patients with genotype B compared to patients with genotype C (52%vs 24%; P = 0.036) and in patients with precore mutation (64%vs 19%; P = 0.002). In conclusion, genotype, presence of precore mutation and Talpha-1 therapy were independent predictors to complete response. Genotype B, compared to genotype C, is associated with a higher response rate to T-alpha1 therapy.

Switching to adefovir monotherapy after emergence of lamivudine-resistant mutations in patients with liver cirrhosis.

Switching to adefovir (ADV) monotherapy is effective in patients with lamivudine (LAM)-resistant hepatitis B virus (HBV) mutations (rtM204 I/V). However, it was recommended to continue LAM therapy for months after starting ADV therapy for safety concern. The safety and efficacy of switching to ADV monotherapy was examined in compensated and decompensated patients with liver cirrhosis. The clinical, biochemical and virological responses were compared between ADV monotherapy in 18 cirrhotic patients and ADV add-on LAM therapy in 10 comparable cirrhotic patients with LAM-resistant rtM204 I/V. After switching to ADV monotherapy, Child-Pugh's score, serum alanine aminotransferase (ALT), bilirubin, albumin and HBV DNA levels improved significantly (P < 0.01). Serum HBV DNA response, defined as HBV DNA decreased to below 10(5) copies/mL or > or =2 log(10) reduction form baseline, was achieved in all patients. A transient ALT flare without concurrent changes in serum bilirubin or prothrombin time was observed in only two patients (11%). The efficacy and safety profile was similar to those with ADV add-on LAM therapy. In conclusion, switching to ADV monotherapy after emergence of LAM-resistant rtM204 I/V is effective and safe in cirrhotic patients, even in those with hepatic decompensation. To stop LAM and switch to ADV in patients with breakthrough is a reasonably safe and cost-effective approach.

Localization of a susceptibility locus for hepatocellular carcinoma to chromosome 4q in a hepatitis B hyperendemic area.

Chromosome 4q is one of the most common regions with a high frequency of allelic loss in hepatocellular carcinoma (HCC). To identify the HCC-susceptibility locus on chromosome 4q, we have performed linkage and family-based association analyses on Chinese families with HCC from Taiwan, where hepatitis B is hyperendemic. Using 77 microsatellite markers spanning chromosome 4q on 52 multiplex families, we found suggestive evidence of linkage to 4q22.3-28.1 with a maximum two-point heterogeneity LOD (HLOD) score of 2.55 at marker D4S3240 on chromosome 4q25. Multipoint analyses with microsatellite markers in the region 4q22.3-28.1 resulted in a maximum HLOD score of 3.12 and a maximum nonparametric linkage (NPL) Z score of 1.98 (pointwise P=0.0080; region-wide empirical P=0.021) for D4S3240. The evidence for linkage to D4S3240 was seen mostly in a subset of 28 families lacking affected parents, which showed multipoint HLOD and NPL scores of 3.25 and 2.79 (pointwise P=0.0028; region-wide empirical P=0.008), respectively. Family-based association analyses of the 77 microsatellite markers in 191 families (53 multiplex plus 138 singleton families) using the pedigree disequilibrium test provide further support for observed linkage. Additional genotyping in the 52 multiplex families informative for linkage analyses was performed for 29 single-nucleotide polymorphisms around D4S3240. A common haplotype (at markers rs7442180 and rs221330) positioned approximately 873 kb away from D4S3240 was associated with HCC, with P=0.0074.