Prospective and longitudinal evaluations of telomere length of circulating DNA as a risk predictor of hepatocellular carcinoma in HBV patients.

Prospective and longitudinal epidemiological evidence is needed to assess the association between telomere length and risk of hepatocellular carcinoma (HCC). In 323 cancer-free Korean-American HBV patients with 1-year exclusion window (followed for >1 year and did not develop HCC within 1 year), we measured the relative telomere length (RTL) in baseline serum DNAs and conducted extensive prospective and longitudinal analyses to assess RTL-HCC relationship. We found that long baseline RTL conferred an increased HCC risk compared to short RTL [hazard ratio (HR) = 4.93, P = 0.0005). The association remained prominent when the analysis was restricted to patients with a more stringent 5-year exclusion window (HR = 7.51, P = 0.012), indicating that the association was unlikely due to including undetected HCC patients in the cohort, thus minimizing the reverse-causation limitation in most retrospective studies. Adding baseline RTL to demographic variables increased the discrimination accuracy of the time-dependent receiver operating characteristic analysis from 0.769 to 0.868 (P = 1.0 × 10-5). In a nested longitudinal subcohort of 16 matched cases-control pairs, using a mixed effects model, we observed a trend of increased RTL in cases and decreased RTL in controls along 5 years of follow-up, with a significant interaction of case/control status with time (P for interaction=0.002) and confirmed the association between long RTL and HCC risk [odds ratio [OR] = 3.63, P = 0.016]. In summary, serum DNA RTL may be a novel non-invasive prospective marker of HBV-related HCC. Independent studies are necessary to validate and generalize this finding in diverse populations and assess the clinical applicability of RTL in HCC prediction.

Aspartate aminotransferase to platelet ratio index as a prospective predictor of hepatocellular carcinoma risk in patients with chronic hepatitis B virus infection.

BACKGROUND AND AIM: APRI (aspartate aminotransferase [AST] to platelet ratio index) is widely used to assess fibrosis and cirrhosis risk, especially in hepatitis C virus (HCV)-infected patients. Few studies have evaluated APRI and hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) risk. Prospective evidence is needed to assess whether APRI predicts HCC risk in HBV patients. METHOD: In a prospectively enrolled clinical cohort of 855 HBV patients with a 1-year exclusion window (followed for > 1 year and did not develop HCC within 1 year), the predictive value of APRI in HCC risk was evaluated by Cox proportional hazards model using univariate and multivariate analyses and longitudinal analysis. RESULTS: Higher APRI prospectively conferred a significantly increased risk of HCC in univariate analysis (quartile analysis, P trend = 2.9 × 10(-7) ). This effect remained highly significant after adjusting for common host characteristics but not cirrhosis (P trend = 7.1 × 10(-5) ), and attenuated when cirrhosis is adjusted (P trend = 0.021). The effect remained prominent when the analysis was restricted to patients with a more stringent 2-year exclusion window (P trend = 0.008 in quartile analysis adjusting all characteristics including cirrhosis), indicating that the association was unlikely due to including undetected HCC patients in the cohort, thus minimizing the reverse-causation limitation in most retrospective studies. Longitudinal comparison demonstrated a persistently higher APRI value in HBV patients who developed HCC during follow-up than those remaining cancer free. CONCLUSION: APRI might be a marker of HCC risk in HBV patients in cirrhosis-dependent and -independent manners. Further studies are warranted to validate this finding and test its clinical applicability in HCC prevention.

Circulating mitochondrial DNA content associated with the risk of liver cirrhosis: a nested case-control study.

BACKGROUND AND AIMS: Accumulating evidence has indicated that variations of mitochondrial DNA (mtDNA) content may affect the susceptibility to hepatocellular carcinoma (HCC). However, no study has been conducted to evaluate the association of circulating mtDNA content and the risk of liver cirrhosis, a leading cause of HCC. METHODS: We conducted a nested case-control study including 136 cirrhotic hepatitis B virus (HBV) cases and 136 frequency-matched non-cirrhotic HBV controls. We determined mtDNA content in serum DNA using quantitative real-time PCR and analyzed its association with cirrhosis risk. RESULTS: We found that cirrhotic HBV patients had significantly lower levels of mtDNA content than non-cirrhotic HBV controls (P = 0.0184). Compared to patients with high mtDNA content, those with low mtDNA content had a 2.25-fold increased risk of cirrhosis [odds ratio (OR) 2.25, 95 % confidence interval (CI) 1.26-4.02]. This association exhibited a significant dose relationship as evidenced in both tertile and quartile analyses (P for trend = 0.0018 and 0.0008, respectively). Stratified analyses showed that the association was prominent in younger patients (P = 0.0122), males (P = 0.0069), never smokers (P = 0.0063), never drinkers (P = 0.0078), patients with a family history of HBV infection (P = 0.0062), and patients with low values of aspartate aminotransferase to platelet ratio index (APRI), a commonly used noninvasive marker for cirrhosis (P = 0.0109). Moreover, a joint effect was observed between low mtDNA content and high APRI values on cirrhosis risk (OR 24.07, 95 % CI 6.72-86.24). CONCLUSIONS: Low circulating mtDNA content may confer an increased cirrhosis risk in HBV patients. Further prospective studies are warranted to validate these findings and explore the clinical significance.

Telomere length in circulating serum DNA as a novel non-invasive biomarker for cirrhosis: a nested case-control analysis.

BACKGROUND & AIMS: Previous studies have indicated that telomere length is associated with altered risk of various tumours including hepatitis B virus (HBV)-related hepatocellular carcinoma. However, the association between telomere length and the risk of cirrhosis has not been reported. METHODS: In this nested case-control study, we used real-time quantitative PCR to determine the relative telomere length (RTL) in serum DNA samples from 100 HBV-related cirrhosis cases and 100 frequency-matched HBV controls, and evaluated the associations between RTL and cirrhosis risk by logistic regression analyses. RESULTS: We found that cirrhotic cases had a significantly longer RTL (median, 0.36; range, 0.08-1.87) than non-cirrhotic controls (median, 0.20; range, 0.05-1.11) (P < 0.0001). Compared with subjects with short RTL, those with long RTL had a significantly increased cirrhosis risk [odds ratio, 2.76, 95% confidence interval, 1.50-5.10; P = 0.001]. Quartile analysis further indicated a dose-response effect for this association. Compared with patients with the lowest quartile of RTL, the cirrhosis risk for those with the second, third and highest quartile of RTL was 2.68 (0.91-7.87, P = 0.073), 3.37 (1.32-10.54, P = 0.013) and 6.64 (2.41-18.32, P < 0.0001) respectively (P(trend) <0.0001). Moreover, the area under the receiver operating characteristic curve increased from 0.60 (epidemiological variables) to 0.72 (epidemiological variables plus RTL), with statistically significant difference assessed by bootstrap analysis. CONCLUSIONS: Our study presents the first epidemiological evidence that RTL in serum DNA could potentially be used as a simple, inexpensive and non-invasive marker of cirrhosis risk, a finding that warrants further investigations in independent retrospective and prospective populations.

Cell-free circulating mitochondrial DNA content and risk of hepatocellular carcinoma in patients with chronic HBV infection.

Recent studies have demonstrated a potential link between circulating cell-free mitochondrial DNA (mtDNA) content and cancers. However, there is no study evaluating the association between circulating mtDNA as a non-invasive marker of hepatocellular carcinoma (HCC) risk. We conducted a nested case-control study to determine circulating mtDNA content in serum samples from 116 HBV-related HCC cases and 232 frequency-matched cancer-free HBV controls, and evaluate the retrospective association between mtDNA content and HCC risk using logistic regression and their temporal relationship using a mixed effects model. HCC cases had significantly lower circulating mtDNA content than controls (1.06 versus 2.47, P = 1.7 × 10(-5)). Compared to HBV patients with higher mtDNA content, those with lower mtDNA content had a significantly increased risk of HCC with an odds ratio (OR) of 2.19 (95% confidence interval [CI] 1.28-3.72, P = 0.004). Quartile analyses revealed a significant dose-dependent effect (Ptrend = 0.001) for this association. In a pilot longitudinal sub-cohort of 14 matched cases-control pairs, we observed a trend of dramatically decreased mtDNA content in cases and slightly decreased mtDNA content in controls, with a significant interaction of case-control status with time (Pinteraction = 0.049). Our findings suggest that circulating mtDNA is a potential novel non-invasive biomarker of HCC risk in HBV patients.

A long-term study of the effects of antiviral therapy on survival of patients with HBV-associated hepatocellular carcinoma (HCC) following local tumor ablation.

The ultimate goal of antiviral therapy for chronic hepatitis B (CHB) is prevention of hepatocellular carcinoma (HCC). Earlier we reported favorable effects of antiviral therapy on survival of HCC patients following curative tumor ablation (Int J Cancer online 14 April 2010; doi: 10.1002/ijc.25382). It was the first observation made in the United States. We now report 12 year follow-up of this patient group. CHB patients with no prior antiviral therapy with a single HCC (≤ 7 cm) were studied. All patients underwent local tumor ablation as their first option. Patients diagnosed before 1999 received no antiviral treatment while those diagnosed after 1999 received antiviral treatment. Survival between the treated and untreated groups was compared. Among 555 HCC patients seen at our clinic between 1991 and 2013, 25 subjects were eligible. Nine subjects (all male patients, median age 53 years [46-66]) did not receive antiviral therapy while 16 (14 male patients, median age 56 years [20-73]) received treatment. Between the two groups, there was no difference in their median tumor size and levels of alpha-fetoprotein and albumin. However, the survival was significantly different (P = 0.001): the median survival of the untreated was 16 months (3-36 months) while that of the treated was 80 months (15-152 months). Fourteen of 16 treated patients are alive to date with two longest survivors alive for ≥ 151 months. In conclusion, concomitant antiviral therapy for CHB patients with HCC reduces and prevents new/recurrent tumor and improves survival. This novel treatment strategy offers an alternative to liver transplantation in patients with HBV-associated HCC.

Relative telomere length: a novel non-invasive biomarker for the risk of non-cirrhotic hepatocellular carcinoma in patients with chronic hepatitis B infection.

BACKGROUND AND AIMS: Telomere length has emerged as a promising risk predictor of various cancers including hepatocellular carcinoma (HCC). However, the majority of studies in this area measured telomere length in hepatocytes and one in lymphocytes with conflicting results. Moreover, no studies have been reported on using circulating DNA telomere length as a non-invasive HCC biomarker. METHODS: We conducted a nested case-control study to determine the relative telomere length (RTL) in serum DNA from 140 hepatitis B virus (HBV)-related HCC cases and 280 frequency-matched cancer-free HBV controls. RESULTS: Cases had a significantly longer RTL (median, 0.31; range, 0.02-2.31) than controls (median, 0.20; range, 0.01-1.60) (P = 0.003). Consistently, longer RTLs conferred a significantly increased HCC risk compared to short RTLs in a univariate logistic regression analysis (odds ratio [OR] = 1.55, 95% confidence interval [CI] = 1.02-2.33, P = 0.038). This association attenuated after multivariate adjustment (OR = 1.40, 95% CI = 0.90-2.19, P = 0.132). In a quartile analysis, a significant dose-response relationship was noted in univariate analysis (P(trend) = 0.017) which was again attenuated in multivariate analysis (P(trend) = 0.079). Further analyses revealed that the significant association between serum RTL and HCC risk was evident in non-cirrhotic (OR = 3.54, 95% CI 1.58-7.93 P = 0.002), but not cirrhotic (OR = 0.95, 95% CI 0.55-1.64, P = 0.860) HBV patients. Moreover, the significantly increased HCC risk conferred by cirrhosis was modulated by RTL with a significant interaction effect (P(interaction) = 0.013). CONCLUSIONS: RTL in circulating cell-free serum DNA could potentially be used as a novel non-invasive biomarker for non-cirrhotic HCC. Prospective cohort studies are warranted to validate this finding and assess its clinical significance in HCC prevention.

Predictive value of alpha-fetoprotein in the long-term risk of developing hepatocellular carcinoma in patients with hepatitis B virus infection--results from a clinic-based longitudinal cohort.

BACKGROUND: Although serum level of alpha-fetoprotein (AFP) has long been used to complement imaging tests in the screening and diagnosis of hepatocellular carcinoma (HCC), whether it can be used as a predictive marker of long-term risk for developing HCC in patients with hepatitis B virus (HBV) has not been extensively evaluated and thus remains controversial. METHODS: We retrospectively conducted a clinic-based longitudinal cohort study including 617 Korean American patients with HBV who had been followed for up to 22 years (median follow-up time, 6.2 years) to evaluate the association between baseline serum AFP level and the long-term risk of HCC. RESULTS: The median baseline AFP value of these patients was 3.8 ng/ml. Compared to patients with lower-than-median AFP value, those with higher-than-median baseline serum AFP had a significantly increased risk of developing HCC with a hazard ratio (HR) of 2.73 (95% confidence interval [CI] 1.25-5.99), independent of other major HCC risk factors. In addition, we calculated the cumulative incidence of HCC during different years of follow-up time by baseline serum AFP, and found that the cumulative incidence of HCC was significantly higher in HBV patients with high baseline serum AFP compared to those with low baseline serum AFP in each of the five follow-up time periods examined. CONCLUSIONS: Our results indicated that AFP was a strong independent prospective predictor of long-term HCC risk in high-risk HBV patients. More targeted prevention and early detection of HCC may be considered for these patients.

Comprehensive analysis of common serum liver enzymes as prospective predictors of hepatocellular carcinoma in HBV patients.

BACKGROUND: Serum liver enzymes are frequently tested in clinics to aid disease diagnosis. Large observational studies indicated that these enzymes might predict cancer risk and mortality. However, no prospective study has reported on their relationships with the risk of HBV-related hepatocellular carcinoma (HCC). METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the predictive values of four routinely tested liver enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase [ALP], and gamma-glutamyltransferase [GGT]) in HCC risk in a prospectively enrolled clinical cohort of 588 Korean American HBV patients. For all four enzymes, the baseline level as well as the average and maximum levels during the first 1 or 2 years of follow-up were analyzed using multivariate Cox proportional hazards model. Patients were categorized into a normal or an elevated group based on the clinical cut-off of each enzyme. During a median follow-up of 7.5 years, 52 patients (incidence rate, 8.8%) developed HCC. The incidence rates were higher in the elevated groups for all four enzymes. The most significant finding was for GGT, with the highest incidence rate of 16.4% in the elevated group compared to 4.6% in the normal group (P<0.001). Compared to patients with normal baseline GGT, those with elevated GGT exhibited a significantly increased HCC risk with a hazards ratio (HR) of 2.60 (95% confidence interval [CI], 1.41-4.77, P = 0.002). Further analyses revealed a cumulative effect between baseline GGT and ALP (HR = 3.41, 95% CI 1.54-7.56, P = 0.003). CONCLUSIONS SIGNIFICANCE: Serum GGT might predict HCC risk in HBV patients individually or jointly with other enzymes.

Hepatitis B virus infection in 6,130 unvaccinated Korean-Americans surveyed between 1988 and 1990.

OBJECTIVES: During the past decades, the influx of immigrants from hepatitis B virus (HBV) endemic regions has brought significant changes in the prevalence of HBV-associated liver diseases and hepatocellular carcinoma (HCC) in the United States. Our program, which was intended to identify those in need of hepatitis B vaccination, helped us to learn of the natural history of HBV infection in Korean Americans. METHODS: Between November of 1988 and May 1990, we screened 6,130 Korean Americans in the eastern United States for HBV infection. RESULTS: The overall hepatitis B surface antigen (HBsAg) (+) rate was 6.1%, with 8.0% for males and 4.4% for females. The carrier rate peaked in subjects between the ages of 21 and 40 yr. The HBsAg (+) rate for 452 U.S.-born children was lower (2.7%) than that of 623 Korean-born (5.5%). None received hepatitis B immune-globulin or HBV vaccination. The vertical transmission rate was 30.3% in children born to HBsAg (+) mothers and 100% in those born to hepatitis B e antigen (HBeAg) positive mothers. In contrast, the paternal transmission rate was low; 10.3% in children with HBsAg (+) fathers and 19.2% in those with HBeAg (+) fathers. Another significant observation was the unexpected finding of ongoing liver diseases in incidentally identified carriers. Evaluation of 139 asymptomatic adult carriers revealed that 42% had elevated liver enzymes and 11% had already developed liver cirrhosis. CONCLUSION: These findings strongly suggest the need for active HBV screening of immigrants from endemic regions and, most importantly, the need for careful monitoring of the carriers.