August 7, 2010

Issue: Aug 2010 Volume 48, Number 8

Diana Koletzki 1,
Stéphanie Dumont 1,
Hans Vermeiren 1,a,
Bart Fevery 1,
Pieter De Smet 2,b,
Lieven J. Stuyver 1,

1 Virco BVBA, Mechelen, Belgium
2 Tibotec-Virco, Mechelen, Belgium
a Present address: Galápagos, Mechelen, Belgium.
b Present address: MIPS NV, Zwijnaarde, Belgium.

Corresponding author: Dr. Diana Koletzki, Virco BVBA, Generaal De Wittelaan L11 B3, 2800 Mechelen, Belgium Phone: +32 15 461 356, Fax: +32 15 461 955,

Citation Information. Clinical Chemistry and Laboratory Medicine. Volume 48, Issue 8, Pages 1095–1102, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2010.236, Available online: 28/06/2010, August 2010

Publication History: Received: 14/8/2009; accepted: 11/3/2010; published online: 28/06/2010

Abstract

Background: Hepatitis C virus (HCV) genotyping and accurate subtype determination is becoming increasingly important to better understand viral evolution, the development of resistance to STAT-C, and possibly even for the treatment and management of chronic HCV-infected patients.

Methods: A subtyping assay based on a 329-bp sequence of the NS5B region, together with an automated subtype interpretation tool was developed. Clinical samples of the six major genotypes were used to assess assay performance characteristics.

Results: The NS5B BLAST-based subtyping assay showed clinical sensitivity for amplification of 89% (n=603 random samples). Assessment of analytical sensitivity of amplification for genotypes 1, 2, 3 and 4 revealed a suitable performance for high viral load samples and decreased only with low viral loads. The results were 100% and 99% accurate at the genotype and subtype level, respectively. A concordance of 97% on genotype level and 62% on subtype level was observed by comparison with subtype results from 5′ non-coding-based assays with a panel of 276 isolates.

Conclusions: The HCV NS5B subtyping assay has been validated for research use. Based on its performance, it is the method of choice in cases where subtype rather than genotype information is needed.

Clin Chem Lab Med 2010;48:1095–102.

Keywords hepatitis C virus, NS5B, sequencing, subtype

Source
Article Date: 02 Aug 2010 - 1:00 PDT
Recent studies suggest that MSCs possess a greater differentiation potential than once thought and several hepatic differentiation protocols from bone marrow cells have been established. However, the incidence of bone marrow-derived hepatocytes was low. Moreover, a long culture period is needed in most cases.

A research article published in World Journal of Gastroenterology addresses this issue. The research team led by Professor Shao JZ from College of Life Sciences of Zhejiang University used mice to investigate the role of VPA on hepatic differentiation of mBM-MSCs. The article indicates that additional exposure of mBM-MSCs to VPA considerably increased the hepatic differentiation in vitro and improved the liver injury by increased homing efficiency of cells to the damaged site in vivo.

The MSCs were successfully isolated from mouse bone marrow and demonstrated that they could differentiate into hepatocytes induced by VPA and well-defined cytokines in a sequential way. The present study demonstrated that VPA-mediated facilitation of hepatic differentiation was regulated by the selective expression of fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) receptors, the increased amounts of acetylated H3 and H4 as well as the structural de-condensation of chromatin. These results provide new insights into the relationships between the epigenetic modification mediated by VPA and the hepatic differentiation from mBM-MSCs.

These findings may also be applicable to study endoderm differentiation and offers an unlimited source of functional hepatocyte-like cells applicable for pharmaco-toxicological research and testing, also this induced system has numerous potential advantages in clinical application.

Reference:
Dong XJ, Zhang H, Pan RL, Xiang LX, Shao JZ. Identification of cytokines involved in hepatic differentiation of mBM-MSCs under liver-injury conditions. World J Gastroenterol 2010; 16(26): 3267-3278

Source:
Lin Tian
World Journal of Gastroenterology

Source
Hepatology International

DOI: 10.1007/s12072-010-9200-8

Original Article

Chung-Feng Huang, Ming-Yen Hsieh, Jeng-Fu Yang, Wu-Cheng Chen, Ming-Lun Yeh, Ching-I Huang, Chia-Yen Dai, Ming-Lung Yu, Zu-Yau Lin and Shinn-Chern Chen, et al.

Abstract

Background/aims

Serum high sensitivity C-reactive protein (hs-CRP) is a surrogate marker for cardiovascular disease risks and related mortality. However, the features of hs-CRP in chronic HCV infection (CHC) patients have not been fully addressed. This study aimed to elucidate the characteristics of hs-CRP and its correlation with clinical profiles in CHC patients.

Methods

Ninety-five CHC patients and 95 age- and sex-matched healthy controls were enrolled for serum hs-CRP level, biochemical, and metabolic profiles examinations. Sequential changes of hs-CRP levels in CHC patients receiving peginterferon/ribavirin combination therapy were also evaluated.

Results

The mean hs-CRP level of CHC patients was significantly higher than that of healthy controls (0.97 ± 0.11 vs. 0.24 ± 0.07 mg/L, P < 0.001). There was no significant correlation between hs-CRP and both virological and histological factors. CHC patients with a high LDL-C level had significantly higher mean hs-CRP (1.38 ± 0.20 mg/L) than that of patients without (0.59 ± 0.06 mg/L) (P < 0.001). Hs-CRP level was significantly decreased in 83 patients after peginterferon/ribavirin combination therapy (0.24 vs. 0.62 mg/L, P < 0.001), particularly in 68 patients achieving a sustained virological response (0.25 vs. 0.64 mg/L, P < 0.001).

Conclusion

CHC patients had a higher hs-CRP level than healthy controls which could be ameliorated after peginterferon/ribavirin combination therapy.

Keywords High sensitivity C-reactive protein - Hepatitis C virus - Antiviral therapy

References
 
Sources
Journal of Neurology
DOI: 10.1007/s00415-010-5686-1Online First™

Original Communication

Min-Suk Yoon, Mark Obermann, Christina Dockweiler, Roland Assert, Ali Canbay, Sebastian Haag, Guido Gerken, Hans-Christoph Diener and Zaza Katsarava

Abstract

There is growing evidence that hepatitis-C virus (HCV) infection might cause peripheral neuropathy. We aimed to investigate the prevalence, clinical and electrophysiological features of sensory neuropathy in patients with cryoglobulin negative HCV infection. We studied 46 consecutive cryoglobulin negative HCV positive patients (24 of them with and 22 without neuropathic symptoms, NS) and compared to 28 age and gender matched controls. In all patients and controls, clinical neuropathy symptom (NSS) and neuropathy deficit scores (NDS) were assessed and standard nerve conduction velocity (SNCV) and pain related-evoked potentials (PREP) were recorded. Both, SNCV and PREP were abnormal in 13 NS positive patients (13/46, 28%). Abnormal PREP but normal SNCV were found in 5 (5/46, 11%) NS positive and in 2 NS negative patients (2/46, 4%). PREP abnormalities correlated positive with both clinical neuropathy scores (NSS r = 0.62; p < 0.001; NDS r = 0.57; p < 0.001), but not with the duration of the disease, current viral load, or the virus subtype. PREP abnormalities were more frequent (16/33, 48.5%) in HCV patients treated with interferon than in therapy naïve patients (4/13, 30.8%); the difference was, however, not significant. In our present study (1) all virus subtypes are capable of inducing neuropathy, (2) no differences were found between interferon therapy and treatment naive patients, (3) the prevalence of peripheral sensory neuropathy including small sensory fibers (20/46, 43.5%) is higher than previously reported and (4) we found that detection of HCV associated neuropathy depends on the evaluation method.

Keywords HCV-infection - Small-fiber neuropathy - Pain-related evoked potentials - Prevalence

References

Source

Non-viral causes of hepatocellular carcinoma

ISSN 1007-9327 CN 14-1219/R World J Gastroenterol 2010 August 7; 16(29): 3603-3615

EDITORIAL

Wojciech Blonski, Kimberly A Forde, Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, United States
Wojciech Blonski, Department of Gastroenterology, Medical University, Wroclaw, 50-556, Poland
David S Kotlyar, Department of Internal Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY 10467, United States

Author contributions: All Authors contributed to this paper.
Correspondence to: Wojciech Blonski, MD, PhD, Research Scholar, Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, United States. wojciech.blonski@uphs.upenn.edu
Telephone: +1-215-5733095 Fax: +1-215-6153756

Received: February 3, 2010 Revised: April 30, 2010
Accepted: May 7, 2010
Published online: August 7, 2010

Abstract

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and represents an international public health concern as one of the most deadly cancers worldwide. The main etiology of HCC is chronic infection with hepatitis B and hepatitis C viruses. However, there are other important factors that contribute to the international burden of HCC. Among these are obesity, diabetes, non-alcoholic steatohepatitis and dietary exposures. Emerging evidence suggests that the etiology of many cases of HCC is in fact multifactorial, encompassing infectious etiologies, comorbid conditions and environmental exposures. Clarification of relevant non-viral causes of HCC will aid in preventative efforts to curb the rising incidence of this disease.

© 2010 Baishideng. All rights reserved.

Key words: Hepatocellular carcinoma; Etiology; Non-alcoholic steatohepatitis; Obesity; Diabetes; Alcohol; Tobacco; Oral contraceptive

Peer reviewer: Marco Vivarelli, MD, Assistant Professor, Department of Surgery and Transplantation, University of Bologna, S.Orsola Hospital, Bologna 40123, Italy

Blonski W, Kotlyar DS, Forde KA. Non-viral causes of hepatocellular carcinoma. World J Gastroenterol 2010; 16(29): 3603-3615 Available from: URL: http://www.wjgnet.com/1007-9327/full/v16/i29/3603.htm DOI: http://dx.doi.org/10.3748/wjg.v16.i29.3603

INTRODUCTION

Hepatocellular carcinoma (HCC) represents an international public health concern as one of the most common and deadly cancers worldwide. Globally, HCC accounts for 85%-90% of primary liver cancers[1] and its lethality is underscored by the fact that it is the third most frequent cause of cancer-related mortality[2]. In those patients who are not transplant candidates, HCC is particularly lethal, with a 5-year survival of less than 5%[3]. In the United States, the incidence of HCC appears to be increasing, with a more than twofold increase observed from 1976 to 2002 (Figure 1)[1,3,4]. A significant proportion of this increase is accounted for by the growing prevalence of hepatitis C virus (HCV) infection[5]. However, other potential causes of HCC are garnering close attention.

Increased body mass index and diabetes with subsequ­ent development of non-alcoholic steatohepatitis (NASH) represent significant risk factors for HCC. This is especially concerning in light of the growing epidemic of obesity in adults and children over the past 25 years[1,5-8]. Other non-viral causes of HCC include iron overload syndromes, alcohol use, tobacco use, oral contraceptive use, aflatoxin exposure and betel quid chewing, a prevalent habit in the developing world. Emerging evidence suggests that the etiology of many cases of HCC is in fact multifactorial, including both viral infections and non-viral environmental and dietary exposures. This review focuses on the non-viral causes of HCC.

HEREDITARY HEMOCHROMATOSIS AND IRON OVERLOAD SYNDROMES

Hereditary hemochromatosis, a condition characterized by excess iron absorption, is caused by mutations in the HFE gene and/or other mutations in the iron metabolism machinery. This condition represents one of the most common autosomal recessive genetic disorders, affecting as many as 1 in 200 people of Northern European descent[9-11]. The HFE gene is required for efficient in vivo iron metabolism and two mutations within the HFE gene product, C282Y and H63D, have been well described in patients with hereditary hemochromatosis[10]. The C282Y mutation, which results in a base pair substitution in which tyrosine is substituted for cysteine at amino acid 282, is found in the homozygous state in up to 83% of patients with hereditary hemochromatosis[10]. The H63D mutation, characterized by substitution of histidine with aspartic acid at codon 63, is present in a minority of cases of hereditary hemochromatosis either in a homozygous state or with one copy of the C282Y mutation, a state referred to as a compound heterozygote[10]. The clinical significance of this latter mutation within the HFE gene, however, continues to be controversial.

The altered iron metabolism seen in hereditary hemochromatosis leads to excess iron storage in the liver and the subsequent development of liver dysfunction. Although other organs systems are also susceptible to iron overload, the liver bears the majority of malignant disease, with those patients with hereditary hemochromatosis being 20 times more likely to develop liver cancer than all other cancers combined[12].

Several population-based and case-control studies have shown that the diagnosis of hereditary hemochromatosis confers a consistent and markedly elevated risk for the development of HCC[12-17]. A sentinel study from the US National Center for Health Statistics found that patients who were diagnosed with hereditary hemochromatosis and who died were 23-fold more likely to have liver cancer compared to those without a diagnosis of hemochromatosis [Proportionate Mortality Ratio (PMR) 22.5, 95% CI: 20.6-24.6)[13]. In addition, the relationship between hereditary hemochromatosis and HCC is modified by diabetes, sex and genetics. Subjects with liver cancer and concomitant diabetes mellitus were 82 times more likely to have a diagnosis of hemochromatosis[13]. Furthermore, a population-based study from Scandinavia found that men with hemochromatosis had a 29-fold increase in risk of liver cancer, whereas women with hemochromatosis had a sevenfold increase in risk[12]. Lastly, highlighting the genetic predisposition of disease and its consequences, an analysis of 5973 first degree relatives of patients with hemochromatosis found that these subjects had a nearly twofold increase in risk of HCC[12].

The presence of a single copy of the C282Y HFE gene mutation, although not diagnostic for hereditary hemochromatosis, has been studied to determine its prevalence and clinical significance in patients with HCC (Figure 2)[18-23].

Researchers comparing 81 patients with cirrhosis and HCC to 128 normal controls observed a significantly higher prevalence of the C282Y mutation in patients with HCC[18]. Another group observed that patients with HCC had a higher frequency of the C282Y mutation when compared to cirrhotic controls without HCC and healthy controls[19]. Additionally, they demonstrated that those subjects with the C282Y mutation had higher levels of serum ferritin, transferrin saturation, and hepatic iron deposition when compared to those without the C282Y mutation[19]. These studies suggest that increased iron load in HCC patients with a C282Y mutation exerts a cause and effect relationship in hepatocarcinogenesis[18,19]. This risk of HCC in patients with the C282Y mutation may not be equally conferred to all however. A recent prospective cohort study from France found that the C282Y mutation and iron overload were associated with a significantly increased risk of HCC among patients with alcoholic cirrhosis but not among those with HCV-related cirrhosis[23].

Contrary to the data presented above, two well-exe­cuted European studies did not find a significant difference in the prevalence of the C282Y mutation between patients with and without HCC[21,22]. Researchers from France observed comparable proportions of the C282Y heterozygous state in 133 cirrhotic patients with HCC and 100 without[21]. Likewise, in another cohort of 162 consecutive patients with HCC, the majority with cirrhosis, the frequency of the C282Y mutation did not differ from historical healthy controls or patients with HCV[22]. Concrete conclusions from these studies might be elusive, however, because of the small sample sizes, differences in the prevalence of the C282Y mutation in the respective populations, and referral bias to tertiary care centers[24].

More studies are therefore needed to determine correctly, in larger populations, the prevalence and effect of a single copy of the C282Y mutation. Additionally, on an individual basis, further study is needed to better characterize the comorbid, demographic and genetic factors that play a role in the risk of HCC in those with a single copy of the C282Y mutation.

It has also been proposed that the H63D mutation is not directly associated with hemochromatosis[10,25]. Certainly, none of the aforementioned studies observed a significant difference in the prevalence of the H63D mutation between patients with and without HCC[18-22]. Future studies are needed to assess further the relationship between this HFE gene mutation and the development of HCC.

Hereditary hemochromatosis is only one of the iron overload syndromes that leads to excessive iron deposition in the liver and other tissues. In fact, those patients with excess total body iron secondary to other etiologies have been shown to have a higher risk of HCC in the absence of genetic hemochromatosis[26-28]. Studies have suggested that conditions such as b thalassemia or iron overload in people of African descent might be associated with an increased risk of HCC[27,29,30]. One such study found that African iron loaded subjects had a 10-fold increase in the risk of developing HCC after adjusting for viral hepatitis, alcohol use and environmental exposures, such as aflatoxin[27]. Regardless of etiology, iron overload is not a benign condition and when recognized, surveillance for HCC should be undertaken.

NON-ALCOHOLIC FATTY LIVER DISEASE

Several case reports and subsequent observational studies have proposed that non-alcoholic fatty liver disease (NALFD), and more specifically, NASH, confers an elevated risk of developing HCC (Figure 3)[31]. NAFLD is a spectrum of clinical disease that ranges from benign or bland steatosis to NASH. The latter stage of this disease, through a process of chronic inflammation and subsequent hepatic fibrosis, can lead to cirrhosis[32]. The presence of cirrhosis itself is an independent risk factor for the development of HCC[33]. To characterize the natural history of NALFD, 420 patients identified in Olmstead County, MN, USA with the disorder were followed for an average of 7 years to determine overall mortality as well as liver related morbidity and mortality. In this population-based study, NAFLD was associated with a 34% increase in mortality and a significant increase in the risk of HCC, with two cases or 0.5% being diagnosed over the period of follow-up[34]. In subjects with NASH-related cirrhosis, however, the rate of HCC approached 10%[34]. These findings are well aligned with a series of studies from Japan. In one report, among 82 NASH patients treated from 1990 through 2001, six patients with HCC were identified over 11 years of follow-up[35]. All six patients developed HCC in the setting of NASH-related cirrhosis[35]. In an update to this original observation, over a 17-year period, the authors found that among 382 patients with NASH, HCC was diagnosed in 34, 9% of the cohort, with 11 patients diagnosed during a 40-mo mean follow-up[36]. Comparing those NASH patients with and without HCC, multivariate logistic regression analysis identified older age (OR: 1.1, 95% CI: 1.03-1.2) and advanced hepatic fibrosis (OR: 4.2, 95% CI: 1.8-9.7) as independent predictors for the development of HCC[36]. In a prospective study of 118 patients with NASH and advanced liver fibrosis from the same cohort, the observed 5-year cumulative incidence of HCC was 7.6%, with HCC accounting for 46% of all fatalities[36]. In summary, these data highlight an association between NASH cirrhosis and an increase in the incidence of HCC over that of the general population. Therefore, regular HCC surveillance is imperative in patients with NASH cirrhosis.

The impact of NASH on the incidence of HCC may well be underestimated. In advanced fibrosis, an absence of steatosis may be appreciated, a finding which can obscure identification of the underlying etiology of liver injury in these patients. In this case, patients might be classified as having cryptogenic cirrhosis. In a United States study that examined 105 consecutive patients with HCC, after HCV, cryptogenic cirrhosis was the most common etiology of liver injury[37]. Among patients presenting with cryptogenic cirrhosis, 58% had a body mass index (BMI) ≥ 30, 47% had diabetes, and 50% had a prior histological diagnosis of NASH or clinical characteristics consistent with NAFLD. Furthermore, only 23% of subjects with cryptogenic cirrhosis were undergoing surveillance for HCC in comparison to 61% of subjects who had a history of HCV-related liver disease[37]. Clearly, these observations emphasize the importance of HCC surveillance in this group of patients and the failure thus far to appropriately screen for HCC in this disease process.

OBESITY

The prevalence of obesity has increased to epidemic proportions over the last three decades. Excess body mass is classified as overweight if the BMI is > 25 kg/m2 and < 30 kg/m2, or obese if the BMI is ≥ 30 kg/m2. In addition to the increase in an array of disease processes observed with being overweight or obese, both classifications of excess body mass are associated with a higher risk of developing all cancers, including liver cancer[38]. In one population-based study from Sweden, 28 cases of HCC were diagnosed in 28 129 patients from 1965 to 1993, thus conferring an almost threefold higher risk of HCC in obese patients[39]. A recent European case-control study observed a significantly increased risk of HCC among obese (OR: 3.5, 95% CI: 1.3-9.2) or diabetic (OR: 3.5, 95% CI: 1.6-7.7) patients without viral hepatitis. This risk of HCC was even greater if both obesity and diabetes were present comorbid conditions (OR: 11.8, 95% CI: 2.7-51.9)[40]. A Danish study further confirmed these results, finding a twofold increase in liver cancer incidence in obese subjects compared to non-obese subjects[41].

A meta-analysis of 11 cohort studies that evaluated the association between being overweight or obese and liver cancer was published in 2007, and clarified the risk of development of HCC (Table 1)[42]. Of the included studies, seven examined a total of 5037 overweight patients and 10 examined 6042 obese patients[42]. Patients who were overweight had a 17% increase in risk of developing HCC, whereas obese patients had an 89% increase in risk (Figure 4)[42]. Based on the prevalence of HCC, it was estimated that 28% of HCC cases in men and 27% in women were due to being overweight or obese[42].

In addition to an increased risk of developing HCC, overweight or obese patients appear to be at increased risk for HCC-related mortality. In a population-based study of cancer mortality and BMI, men with a BMI of 30-34.9 were found to have a twofold increase in the risk of death from HCC, with a 4.5-fold increase noted in men with BMI > 35[38].

Lastly, via the pathway of the metabolic syndrome with resultant NASH cirrhosis, obese patients have been found to be at an increased risk for HCC occurrence. Many lines of evidence point to the role of cirrhosis as a mediator in these patients. Firstly, patients presenting with cryptogenic cirrhosis were found to have a significantly higher prevalence of obesity than patients with cirrhosis from non-alcoholic hepatitis C or autoimmune liver disease, but a similar prevalence of obesity when compared to patients with documented NASH[43]. These data are supported by a case-control study in which 49 patients with cryptogenic cirrhosis were compared to 98 matched controls with an established cause of cirrhosis. In that study, obesity was significantly more prevalent in the cryptogenic cirrhosis patients[44]. Additionally, a retrospective analysis of 19 271 American patients who had undergone liver transplantation found that there were 653 cases of HCC, and those with a diagnosis of cryptogenic cirrhosis had an 11-fold increase in the risk of having HCC[45]. Therefore, being overweight and obesity, secondary to cryptogenic cirrhosis, or more likely undiagnosed NASH cirrhosis, can increase the risk of developing HCC. Clearly, these data suggest that screening is important for diagnosis of asymptomatic HCC and highlight the need for surveillance in this population.

DIABETES

Diabetes has been found to increase the risk of developing chronic liver disease and HCC[46]. Studies that have compared patients with cryptogenic cirrhosis to patients with a known etiology of their cirrhosis have shown a significantly higher prevalence of diabetes among the latter group[43,44]. Again, as noted with the overweight and obese, a similar prevalence of diabetes has been observed among patients with cryptogenic and NASH cirrhosis[43].

In addition to increasing the prevalence of chronic liver disease, diabetes has also been shown to be an independent risk factor for the development of HCC. In a recent systematic review of 13 case control studies, 11 supported an association between diabetes and the development of HCC[47]. Among the 13 case-control studies, subjects with diabetes were found to have a twofold increase in the risk of HCC; an association that was further strengthened by excluding studies with significant heterogeneity (Figure 5)[47]. This association was also appreciated amongst 12 cohort studies evaluated (Figure 6). The presence of diabetes remained an independent risk factor for HCC after adjustment for alcohol use or viral hepatitis in the studies that evaluated these factors[47]. However, as dictated by the limitations of the studies available in the literature, further well-defined studies are required to account for dietary factors and obesity.

DIET

Several studies have examined whether alterations in diet have an effect on the risk of HCC. A trial from Italy has examined a broad range of dietary habits among 185 patients with HCC and 412 patients without cancer[48,49]. Those with HCC were more likely to consume a large amount of calories, were five times more likely to be former drinkers, and were 30 times more likely to be infected with either HCV or hepatitis B virus (HBV). Among dietary compounds, consumption of iron and thiamine were associated with a significant threefold and twofold increase in risk of HCC, respectively. Conversely, b-carotene and linoleic acid consumption was associated with a reduced risk of HCC[48]. An association between intake of iron was also evaluated according to the presence or absence of viral hepatitis[48]. When compared to appropriate controls, consumption of iron among patients without viral hepatitis was associated with a significantly increased risk of HCC[48]. This increase in risk was not conferred to those with HCV or HBV. In a similar study, those subjects with consumption in the highest quartile for yogurt and milk, white meat and eggs had a significantly lower likelihood of developing HCC[50]. This effect was observed in patients with and without viral hepatitis[50].

Other studies from Japan and Europe have found those who consume a large amount of green vegetables have a significantly lower likelihood of developing HCC[51-53]. One study has shown that eating green vegetables daily, as compared with consumption fewer times per week, had a protective effect against the development of HCC (OR: 0.75, 95% CI: 0.60-0.95)[51]. On the contrary, a Greek study has found no association between vegetable intake and reduction in the risk of developing HCC[54].

In summary, there is evidence to suggest that consumption of yogurt and milk as well as vitamin supplements offers a protective effect against HCC. The enthusiasm for these findings however should be tempered by the fact that the majority of these studies were retrospective in nature.

COFFEE

In addition to its reported association with reductions in bladder cancer and colorectal cancer, coffee consumption has also been extensively studied and appears to have a potentially favorable effect on the prevention of liver diseases, including HCC[55,56]. There are several hypotheses that could explain why consuming coffee attenuates the risk of developing HCC. One hypothesis argues that coffee intake lowers serum levels of g-glutamyl transferase (GGT), which is associated with a lower incidence of HCC[56-59]. Coffee consumption has also been linked to a lower incidence of cirrhosis, which is a major risk factor for the development of HCC[56].

An analysis of two large prospective studies of > 70 000 participants in Japan has shown that those who drank one or more cups coffee daily had a significantly lower risk of developing HCC[60]. A case-control study of 2746 people has found that those who drank three or more cups of coffee were 40% less likely to develop HCC[56]. Similar results have also been found in an array of studies conducted in Europe and Japan[60-65].

Additionally, two meta-analyses that have examined the association between coffee drinking and the risk of developing HCC have recently been published. The first was inclusive of four cohort studies and five case-control studies[66]. In the pooled analysis, a 43% lower risk of developing HCC was found for those who drank more than two cups of coffee per day (RR: 0.57, 95% CI: 0.49-0.67) (Figure 7)[66]. The second meta-analysis examined four cohort studies from Japan and six from Japan and Southern Europe[67]. There were differing definitions of low and high coffee consumption, however, the results of the studies were consistent. In summary, those who drank any coffee compared to non-drinkers had a significantly lower risk of HCC (RR: 0.59, 95% CI: 0.49-0.72). The greater the coffee consumption, the greater the attenuation in HCC risk. Low coffee consumption was associated with a 30% reduction in risk and high consumption with a 55% reduction in HCC risk (Figure 8)[67].

Although these results are impressive and consistent, one must consider that the findings of an inverse relationship between coffee consumption and the risk of HCC might be influenced by bias. Coffee metabolism is impaired in cirrhotic livers as compared to the normal liver. This altered metabolism generates an increase in the untoward side effects of the beverage. Therefore, the presence of liver disease might lead affected patients to consume less coffee. This could result in a falsely negative association. Therefore, the potential bias of this association in the liver disease patient cannot be discounted.

ALCOHOL

The mechanism by which alcohol consumption increases the risk of HCC is primarily through the development of cirrhosis. It has been suggested that heavy alcohol consumption of > 80 g/d ethanol for at least 5 years increases the risk of HCC by nearly fivefold[68]. The risk appears to be proportional to the amount of alcohol consumed. As characterized by a meta-analysis, relative risks of 1.19 (95% CI: 1.12-1.27), 1.40 (95% CI: 1.25-1.56), and 1.81 (95% CI: 1.50-2.19) were associated with the consumption of 25, 50 and 100 g/d alcohol, respectively[69].

In addition to a daily dose response, persistent alcohol consumption appears to have a long-term effect on the risk of HCC occurrence. A prospective case-control study from Japan has observed that heavy alcohol drinkers, defined as > 600 L of alcohol during a lifetime, had a fivefold increase in the risk of HCC in comparison to non-drinkers or those who consumed < 600 L of alcohol (OR: 5.19, 95% CI: 2.53-10.64)[70]. However, the risk of HCC among those who consume low or moderate levels of alcohol remains unknown[1].

An association between genetic polymorphisms of the enzymes participating in the metabolic pathway of ethanol and the increased risk of HCC in heavy alcohol drinkers has been also proposed as a mechanism by which HCC develops. The frequency of aldehyde dehydrogenase 2 (ALDH2) genotype polymorphisms is significantly associated with increased risk of HCC in heavy alcohol drinkers (OR: 2.53, 95% CI: 1.63-58.60)[70]. A study from Italy has observed that, among subjects who consumed > 100 g/d of ethanol and were bearers of the gluthatione S-transferase M1 (GSTM1) null genotype had twice the risk of HCC compared with bearers of the GSTM1 non-null genotype (OR: 8.5, 95% CI: 3.9-18.6 vs OR: 4.5, 95% CI: 2.0-10.0)[71].

SMOKING

Several studies have evaluated the association between smoking and development of primary liver cancer. A prospective cohort study including 4050 men aged ≥ 40 years who were followed-up for an average length of 9 years observed that those who smoked had a threefold increased risk of primary liver cancer when compared to never smokers (RR: 3.3, 95% CI: 1.2-9.5)[72]. Additionally, a study from Korea has found a 50% increase in the risk of primary liver cancer for current male smokers compared to never smokers[73]. In contrast however, a recent population-based case-control study from the United States did not observe a significantly increased risk of primary liver cancer among current male smokers[74]. Male ex-smokers, however, had a significant increase in risk of primary liver cancer, which suggests that there is perhaps a dose or duration response underlying this association[72-74]. Such responses were further explored in the Korean Cancer Prevention study that included 1 283 112 subjects[75]. Although the amount of smoking did not alter the risk of HCC, the duration of smoking significantly increased the risk of HCC for subjects who had smoked for > 20 years when compared to those who had smoked for < 10 years[75].

The association between tobacco and liver cancer and its reliance on host factors such as genetics, sex, and an underlying history of viral hepatitis has also been explored. With respect to the role of genetics, a small study from Japan has evaluated 78 patients with HCC and genetic polymorphisms of tobacco and alcohol-related metabolizing enzymes and 138 hospital controls without cancer. They have demonstrated that cigarette smokers did not have a significantly increased risk of HCC when compared with non-smokers[70]. To analyze the effect of sex, a prospective cohort study that included 83 885 patients followed up for 8 years observed a positive association between smoking and HCC in women who smoked > 10 cigarettes per day (RR: 4.2, 95% CI: 1.3-13.8)[76]. However, no significant increase in the risk of HCC was demonstrated among male smokers[76]. Additionally, to determine the effect of viral hepatitis on the association between HCC and tobacco, a prospective study of 12 008 men observed that smoking significantly increased the risk of HCC only in anti-HCV-positive patients but not in those who were anti-HCV-negative when compared to anti-HCV-negative nonsmoking individuals[77].

In addition to an increase in the risk of developing HCC, it is also suggested in the literature that smoking increases the risk of death in HCC. In the Korean Cancer Prevention cohort study, men who were current smokers had an increased risk of death from HCC[75]. Women who were current smokers did not have the same elevation in risk of HCC-related death as that observed in men[75]. These findings were further replicated in the Japan Collaborative Cohort (JACC) Study that analyzed 65 528 subjects aged 40-79 years[78]. In this cohort, an increased risk of death due to HCC was shown among current and ex-smokers[78]. Further analyses from the JACC cohort demonstrated that cigarette smoking significantly increased the risk of death from HCC in individuals positive for anti-HCV antibody[79].

ORAL CONTRACEPTIVES

Prior to the widespread use of oral contraceptives (OCs), benign liver tumors in young women were rarely observed. In the current case report literature, however, therapy with oral contraceptives appears to be associated with the development of benign liver tumors such as hepatic hemangioma, hepatocellular adenoma or focal nodular hyperplasia[80,81]. Although not well researched, it has been proposed that OCs might also be associated with malignant liver tumors including HCC[82,83]. Rarely, malignant transformation can occur within the context of hepatic adenomas. It is unclear, however, whether the use of OCs influences the likelihood of developing adenoma and that these benign tumors transform.

Within the literature, there have been 14 cases of hepatic adenoma with focal malignant transformation to HCC in women taking OCs[83-93]. The mean age of these patients at the time of diagnosis of malignant transformation was 36 years (range: 23-57 years) and the mean duration of OC use was 11 years (range: 1 mo-20 years)[83-93]. Although difficult to obtain from the literature, the frequency of HCC among hepatic adenomas appears to vary from 5% to 18%[89,92-94].

To evaluate further the risk of HCC in the setting of OC use, several observational studies have been conducted. A recent meta-analysis of 12 case-control studies, including 739 cases and 5223 controls, which evaluated the risk of HCC among women using OCs indicated that there was no increase in risk of HCC with short-term use; defined as < 5 years of exposure[95]. An analysis of all studies in the aforementioned meta-analysis yielded a pooled unadjusted OR of 1.57 (95% CI: 0.96-2.54)[95]. An adjusted analysis, which accounted for variables such as age, race and parity, did not yield significant findings (Figure 9)[95]. On the contrary, six studies have observed a significantly increased risk of HCC among women taking OCs for > 5 years; an increase in risk of 2-20-fold[95]. However, given the variable periods of duration used in each of the studies, a pooled estimate of risk could not be generated[95]. Based on these results, further studies are required to evaluate the association between OCs and the risk of HCC and how such risk is modified by duration of OC use. Additionally, it should be noted that an association between new-generation OCs with lower doses of hormones and the risk of HCC has not yet been explored.

BETEL QUID

The chewing of betel quid is woven into the cultural fabric of up to 20% of the world population. Betel quid consists of the nut of the Areca catechu palm (areca nut), betel leaf or fruit from Piper betle and red slaked paste[96]. These ingredients have been shown to have genotoxic, mutagenic and tumorigenic properties[97-102]. A case-control study from Taiwan has shown that betel quid chewing was an independent risk factor for liver cirrhosis (OR: 3.56, 95% CI: 1.41-8.96)[103].

Recently, two prospective case-control studies from Asia also have observed a significant association between betel quid chewing and the incidence of HCC. One such study included 263 pairs of age- and sex-matched patients with HCC and healthy controls and observed that betel quid chewing was an independent risk factor for HCC, with a threefold risk noted (OR: 3.49, 95% CI: 1.74-6.96). The aggregate risk increased with increasing duration and/or quantity of consumption[96]. These data were further supported by a study from Taiwan, including 420 age- and sex-matched patients with HCC and liver cirrhosis, liver cirrhosis only and healthy controls. In this study, a nearly sixfold and nearly twofold increased risk of HCC was observed in patients with HCC compared with healthy controls and patients with liver cirrhosis, respectively[104]. Additionally, they found an additive interaction between betel quid chewing and chronic HBV and/or HCV infection.

AFLATOXIN

Aflatoxin B1 (AFB1) is the major metabolite of the molds Aspergillus fumigatus and Aspergillus parasiticus. These molds grow on a variety of food products that are stored in warm and damp conditions or are cultivated in countries with hot and humid climates[1,105]. AFB1 induces a single nucleotide substitution in codon 249 in the p53 tumor suppressor gene, which results in the change of the amino acid arginine to serine[106,107]. This mutation is present in up to 50% of patients with HCC who are indigenous to geographic regions with high exposure to AFB1[108-111]. On the other hand, this mutation is absent in patients with HCC from regions with low exposure to AFB1[112,113]. Moreover, it has been recently demonstrated that AFB1-albumin adducts in patients with HCC correlate significantly with the presence of plasma DNA hypermethylation and mutations in the p16 and p53 tumor suppressor genes[114].

Several studies have evaluated an association between the risk of HCC and exposure to AFB1. A prospective case-control study from China which included 18 244 middle-aged men showed that individuals with the presence of urinary aflatoxin biomarkers had a significantly increased risk of HCC after adjusting for HBV surface antigen seropositivity and cigarette smoking[115]. These data were further supported by a community-based cohort study from Taiwan which found that elevated AFB1 exposure measured by detectable AFB1-albumin adducts was an independent risk factor for HCC after adjustment for important confounders (OR: 5.5, 95% CI: 1.2-24.5)[116].

It should be stressed that areas with high exposure to AFB1 are also characterized by a high prevalence of HBV infection. AFB1 is independent of the risk conferred by HBV, however concomitant exposure to both HBV and AFB1 markedly increases the risk of HCC. When compared to those without HBV infection and absence of urinary AFB1 markers, the risk of HCC was 60 times higher in patients with HBV infection and a concomitant elevation of urinary AFB1 markers (RR: 59.4, 95% CI: 16.6-212.0)[115]. Patients with HBV infection and normal urinary AFB1 markers had sevenfold increase in risk of HCC when compared to appropriate controls[115].

CONCLUSION

Multiple non-viral factors have been implicated in the development of HCC. Hemochromatosis and iron overload syndromes have consistently been shown to dramatically increase the rate of HCC. Additionally, factors such as obesity and diabetes, which operate via NASH cirrhosis or perhaps independently, have also been demonstrated to increase the risk of HCC. This phenomenon has closely mirrored the epidemic of obesity over the last 15-25 years.

With respect to other exposures, although alcohol and tobacco clearly increase the risk of HCC development and mortality, other exposures such as coffee and high levels of vegetable consumption may be protective against this condition. Further studies are urgently needed to determine the pathogenesis that underlies the occurrence of HCC in the setting of these exposures, as well as the way in which such risk is modified by environmental and host characteristics such as genetics.

Clarification of relevant non-viral causes of HCC will help to focus clinicians on those risk factors that are modifiable. With more information, future surveillance efforts will be more appropriately targeted toward populations at greatest risk. This multilevel preventative approach will hopefully lead to a reduction in incidence of non-viral HCC, and a decrease in the patient morbidity and mortality as well as the societal economic burden associated with HCC.

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S- Editor Wang YR L- Editor Kerr C E- Editor Ma WH

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Friday, 06 August 2010

Legislation introduced today by Senator John Kerry, "The Viral Hepatitis and Liver Cancer Control and Prevention Act," would transform our nation's approach to identifying and treating chronic viral hepatitis B and C and is urgently needed to prevent a looming tidal wave of fatal cases costing our health care system tens of billions of dollars annually by 2024, the National Viral Hepatitis Roundtable (NVHR) said today. NVHR is the national coalition of public, private, and voluntary organizations dedicated to reducing the incidence of infection, morbidity, and mortality from viral hepatitis.

"On behalf of over 5 million Americans infected with chronic viral hepatitis, we very much appreciate Senator Kerry's leadership and are gratified to have his support," said Ms. Lorren Sandt, NVHR Chair and Executive Director of Caring Ambassadors Program, based in Portland, OR. "Senator Kerry's legislation is urgently needed to modernize our nation's public health response to chronic viral hepatitis. Screening and early intervention are critical to achieving better outcomes for infected patients and must be a national priority. Otherwise, our system will incur - each and every year -- thousands of avoidable deaths and billions of dollars in unnecessary costs."

"Viral hepatitis is a silent killer," Senator John Kerry said in introducing the legislation. "Most people don't even know they have hepatitis until it causes liver damage or even cancer years after the initial infection. We can easily avoid these needless tragedies with prevention, surveillance programs, and by educating Americans about this deadly disease. The bill I'm introducing today will help create a national strategy to combat and prevent hepatitis, hopefully ending this silent affliction's often deadly consequences."

The legislation authorizes funding of nearly $600 million over the next five years to direct the HHS Secretary to develop a national strategy to prevent and control viral hepatitis. The Kerry legislation is supported by at least 102 community-based organizations from coast to coast that provide viral hepatitis counseling, screening, and treatment programs. A copy of the letter the groups sent to Senator Kerry is attached. The Kerry bill is the companion to bipartisan legislation pending in the US House of Representatives, HR 3974, which currently has 61 cosponsors.

Source: National Viral Hepatitis Roundtable

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Also See: Kerry Introduces Bill to Fight Viral Hepatitis
"SVR was as high as 70% among patients with no fibrosis, declining in a step-wise fashion to rates of 31% for those with bridging fibrosis (n=97), and only 10% for those with cirrhosis (n=30).....patients with advanced fibrosis appear unlikely to achieve a SVR without a RVR, and very unlikely if HCV RNA is detectable at week 8 or 12......The low SVR rates among patients with advanced fibrosis in the CHARIOT study were characterized by two clearly outlined virological patterns: (1) slower early virological responses and (2) considerably higher virological relapse...... A marked step-wise decline in SVR was evident by fibrosis stage: F0 (70%); F1 (60%); F2 (51%); F3 (31%); F4 (10%) (p<0.0001)......Virological relapse rates were similar in early disease stages (F0, 16%; F1, 23%; F2, 26%), but increased markedly in advanced fibrosis (F3, 50%; F4, 80%) (p<0.0001). Cumulative PEG-IFNα-2a and ribavirin doses were similar among patients with F3/4 and F0-2 within treatment arms through week 4, 8, 12, and week 24.......Patients with advanced fibrosis with initial undetectable HCV RNA at week 4, 8, 12, were less likely to achieve an SVR compared to those in the corresponding virological response group without advanced fibrosis. Among patients without advanced fibrosis and a RVR, the proportion with a SVR (PPV) was 80%, within the 77-90% RVR PPV range from other studies [26], [27], [28]. However, only 63% of patients with advanced fibrosis and RVR achieved a SVR.....Pivotal studies among genotype 1 patients for the protease inhibitors telaprevir and boceprevir have included individuals with advanced fibrosis, therefore data on efficacy and safety in this sub-group is eagerly awaited."

"The slower initial HCV RNA decline, in patients with advanced fibrosis, was despite receiving similar cumulative dosages of PEG-IFNα-2a and ribavirin to patients without advanced fibrosis, indicating that differences in treatment tolerability in relation to dose modifications and/or discontinuations are not the explanation for the lower initial HCV RNA decline or higher relapse rates seen in advanced fibrosis. A potential explanation for our findings is the relationships between fibrosis stage, intrahepatic HCV RNA replication, and HCV clearance. Although patients with advanced fibrosis do not have higher serum HCV RNA levels, they have a higher percentage of hepatocytes with evidence of HCV RNA replication [19]. Viral kinetic studies have shown that patients with HCV genotype 1 and advanced fibrosis have slower second phase HCV RNA decline than those with less fibrosis [20], consistent with our week 4 and 8 response findings. Further, recent HCV modeling based on HCV genotype 1 patients indicates that patients with cirrhosis have lower SVR rates due to a higher percentage of HCV-infected hepatocytes [21]. An additional explanation would relate to potential immunological differences by extent of liver disease. Immunological characteristics including up-regulation of intrahepatic Th1-like cytokines and interferon-stimulated genes (ISGs) are known to influence both fibrosis progression and IFN-based treatment response [22], [23]. Finally, the recently reported association, between IL28B genetic polymorphisms and PEG-IFN/RBV treatment outcome in treatment naïve patients with chronic HCV genotype 1, indicates that host genetic characteristics are important for treatment responsiveness [24], [25]. If IL28B genetic polymorphisms associated with poor HCV treatment response are also found to be linked to liver disease progression, host genetic characteristics could explain the differential response by fibrosis stage."

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Articles in Press

Low virological response and high relapse rates in hepatitis C genotype 1 patients with advanced fibrosis despite adequate therapeutic dosing

Wendy S.C. Cheng1, Stuart K. Roberts2, Geoffrey McCaughan3, William Sievert4, Martin Weltman5, Darrell Crawford6, William Rawlinson7, Philippa S. Marks8, James Thommes9, Bishoy Rizkalla10, Motoko Yoshihara10, Gregory J Dore811Corresponding Author Informationemail address, on behalf of the CHARIOT Study Group

Corrected Proof

ABSTRACT

Background & Aims

The impact of fibrosis stage on chronic hepatitis C virus (HCV) treatment response was explored in CHARIOT, a study of high dose peginterferon alfa-2a (PEG-IFNα-2a) induction therapy in treatment naïve genotype 1 infection.

Methods

Eight hundred and ninety-six patients were randomised 1:1 to 360µg (n=448) or 180µg (n=448) PEG-IFNα-2a weekly with RBV 1000-1200mg/day for 12weeks followed by 36weeks of 180µg PEG-IFNα-2a weekly plus RBV 1000-1200mg/day. Virological responses were assessed at week 4, 8, 12, 24, 48 (end of therapy), and 24weeks following therapy (sustained virological response, SVR). As previously reported, there was no significant difference in SVR in the induction (53%) and standard (50%) arms, therefore the pooled study population was used for analysis of SVR and relapse.

Results

A marked step-wise decline in SVR was evident by fibrosis stage: F0 (70%); F1 (60%); F2 (51%); F3 (31%); F4 (10%) (p<0.0001). Early virological responses were lower among F3/4 patients, including rapid virological response (RVR) (21% vs. 34% for F3/4 and F0-2, respectively) (p=0.0072), and the RVR positive predictive value was also lower (63% vs. 80%). Virological relapse rates were similar in early disease stages (F0, 16%; F1, 23%; F2, 26%), but increased markedly in advanced fibrosis (F3, 50%; F4, 80%) (p<0.0001). Cumulative PEG-IFNα-2a and ribavirin doses were similar among patients with F3/4 and F0-2 within treatment arms through week 4, 8, 12, and week 24.

Conclusions

Low virological response in hepatitis C genotype 1 patients with advanced fibrosis is not explained by inadequate cumulative PEG-IFN and ribavirin doses.

Introduction

Pegylated interferon and ribavirin therapy for chronic hepatitis C virus (HCV) infection achieves a sustained virological response (SVR) in 70-80% in genotype 2/3, but only 40-55% in genotype 1 infected patients [1], [2], [3]. Treatment response rates are particularly poor among patients with genotype 1 infection and advanced fibrosis (bridging fibrosis and compensated cirrhosis), with SVR rates of 15-40% [2], [3], [4], [5], [6], [7] reported from generally small study populations.

HCV-related cirrhosis is associated with a high risk of progression to hepatic decompensation, hepatocellular carcinoma (HCC), and liver-related mortality [8], [9], [10]. Although interferon-based HCV viral clearance is more difficult to achieve in patients with advanced fibrosis, there are clear benefits for those who do, with lower risk of progression to advanced liver disease complications [11], [12], [13], and regression of fibrosis in many [14], [15].

The CHARIOT trial was a large international randomized controlled trial of peginterferon (PEG-IFN) alfa-2a induction dosing in combination with ribavirin in treatment naïve patients with chronic HCV genotype 1 infection [16]. Despite enhanced early virological responses, induction therapy provided no improvement in SVR (53% and 50% in induction and standard therapy arms, respectively). Preliminary analyses of the influence of fibrosis stage on treatment outcomes, following induction and standard therapy, demonstrated poor SVR rates among patients with advanced fibrosis (28% and 24%) compared to those with moderate (51% and 50%) and no or minimal fibrosis (64% and 60%) [16]. The aim of this analysis was to further evaluate the impact of fibrosis stage on treatment response within CHARIOT, including the predictive value of week 4, 8, and 12 responses, and virological relapse. In particular, the contribution of cumulative PEG-IFNα-2a and ribavirin dose to the lower virological responses observed in patients with advanced fibrosis was explored.

Discussion

This comprehensive evaluation of the impact of fibrosis stage on PEG-IFNα-2a and ribavirin treatment response, within a large randomized trial population of treatment naïve patients with chronic HCV genotype 1 infection, has demonstrated several important findings. A marked difference in response by fibrosis stage was found, with very encouraging responses for patients with early liver disease, but poor responses for those with advanced fibrosis. High virological relapse was also seen in patients with advanced fibrosis. The predictive values of early virological responses for SVR differed by fibrosis stage grouping, with the major finding that patients with advanced fibrosis appear unlikely to achieve a SVR without a RVR, and very unlikely if HCV RNA is detectable at week 8 or 12. Only small differences in cumulative PEG-IFNα-2a and ribavirin dose by fibrosis stage were apparent, including through to week 48 among patients with an end-of-treatment response. Thus, low HCV treatment response and high virological relapse rates among patients with advanced fibrosis are not explained by inadequate cumulative therapeutic dosing.

These further analyses within the CHARIOT study provide robust evidence for the influence of fibrosis stage on treatment response. SVR was as high as 70% among patients with no fibrosis, declining in a step-wise fashion to rates of 31% for those with bridging fibrosis (n=97), and only 10% for those with cirrhosis (n=30). We have combined patients with bridging fibrosis and cirrhosis because of the relatively smaller number of patients with cirrhosis. An Italian randomized controlled trial, in a treatment naive genotype 1 population, demonstrated similar trends among patients receiving PEG-IFNα-2b and ribavirin, with SVR rates declining from 60% for Ishak stage 1 to 7% for stage 6 [7]. However, the influence of fibrosis stage on PEG-IFN and ribavirin therapy outcomes in other studies has been based on two disease stage groupings only (generally advanced fibrosis and earlier disease) [2], [3], [18]. Further, key studies have not provided genotype-specific SVR by disease stage, including the pivotal studies of PEG-IFN and ribavirin therapy by Fried et al. [18] and Manns et al. [3].

Low treatment response rates have been described in other studies of PEG-IFN and ribavirin involving treatment naïve patients with genotype 1 and advanced fibrosis, with SVR rates generally 15-40% [2], [3], [4], [5], [6], [7]. In the Hadziyannis et al. study of PEG-IFNα-2a and ribavirin treatment duration and ribavirin dosing, SVR for patients with genotype 1 receiving 48weeks therapy and higher ribavirin dose (1000-1200mg/day) was 41% and 57% among those with and without advanced fibrosis, respectively [2]. A French randomized controlled trial within a METAVIR F3/4 population (51% with cirrhosis) demonstrated SVR rates among the sub-group of treatment naïve genotype 1 patients (n=104) of 24% and 17%, respectively, for standard (1.5µg/kg/week) and low dose (0.75µg/kg/week) PEG-IFNα-2b in combination with ribavirin (800mg/day) for 48weeks [5]. Another randomized controlled trial of PEG-IFNα-2b and ribavirin (800-1200mg/day), in patients with established cirrhosis, showed an SVR of 24% in the genotype 1 sub-group [6].

The low SVR rates among patients with advanced fibrosis in the CHARIOT study were characterized by two clearly outlined virological patterns: (1) slower early virological responses and (2) considerably higher virological relapse. Although SVR and relapse rates were considerably lower and higher, respectively, among patients with advanced fibrosis, when non-SVR patients were analyzed separately within the two fibrosis groupings, the proportions failing therapy due to non-response at week 12, virological breakthrough, and virological relapse were similar.

The slower initial HCV RNA decline, in patients with advanced fibrosis, was despite receiving similar cumulative dosages of PEG-IFNα-2a and ribavirin to patients without advanced fibrosis, indicating that differences in treatment tolerability in relation to dose modifications and/or discontinuations are not the explanation for the lower initial HCV RNA decline or higher relapse rates seen in advanced fibrosis. A potential explanation for our findings is the relationships between fibrosis stage, intrahepatic HCV RNA replication, and HCV clearance. Although patients with advanced fibrosis do not have higher serum HCV RNA levels, they have a higher percentage of hepatocytes with evidence of HCV RNA replication [19]. Viral kinetic studies have shown that patients with HCV genotype 1 and advanced fibrosis have slower second phase HCV RNA decline than those with less fibrosis [20], consistent with our week 4 and 8 response findings. Further, recent HCV modeling based on HCV genotype 1 patients indicates that patients with cirrhosis have lower SVR rates due to a higher percentage of HCV-infected hepatocytes [21]. An additional explanation would relate to potential immunological differences by extent of liver disease. Immunological characteristics including up-regulation of intrahepatic Th1-like cytokines and interferon-stimulated genes (ISGs) are known to influence both fibrosis progression and IFN-based treatment response [22], [23]. Finally, the recently reported association, between IL28B genetic polymorphisms and PEG-IFN/RBV treatment outcome in treatment naïve patients with chronic HCV genotype 1, indicates that host genetic characteristics are important for treatment responsiveness [24], [25]. If IL28B genetic polymorphisms associated with poor HCV treatment response are also found to be linked to liver disease progression, host genetic characteristics could explain the differential response by fibrosis stage.

A further feature of the virological response patterns, between patients with and without advanced fibrosis, was the differential outcomes within similar virological response groupings. Patients with advanced fibrosis with initial undetectable HCV RNA at week 4, 8, 12, were less likely to achieve an SVR compared to those in the corresponding virological response group without advanced fibrosis. Among patients without advanced fibrosis and a RVR, the proportion with a SVR (PPV) was 80%, within the 77-90% RVR PPV range from other studies [26], [27], [28]. However, only 63% of patients with advanced fibrosis and RVR achieved a SVR. The high NPV of RVR for SVR among patients with advanced fibrosis (84%) makes extension of therapy a potential strategy for evaluation in this group. Extension of PEG-IFN and ribavirin therapy, from 48 to 72weeks among patients with HCV genotype 1 and non-RVR, has reduced relapse rates and increased SVR [29], [30], but data on this specific strategy among patients with advanced fibrosis is not available.

Although combination therapy with PEG-IFN, ribavirin, and direct acting antivirals (DAA) such as protease or polymerase inhibitors has shown great promise in phase I and II evaluation, most of these trials have enrolled relatively few patients with advanced fibrosis (patients with established cirrhosis have generally been excluded) and disease stage-specific response data is not available [31], [32]. Pivotal studies among genotype 1 patients for the protease inhibitors telaprevir and boceprevir have included individuals with advanced fibrosis, therefore data on efficacy and safety in this sub-group is eagerly awaited.

Results

Patient characteristics

A total of 871 patients were enrolled, randomized (1:1) to induction and standard therapy, and received study drug (intention to treat analysis population). Baseline liver histology was obtained in 625 of 871 patients (71.8%). Fibrosis stage was F3/4 in 127 patients (14.6%), F0-2 in 498 patients (57.2%), and missing in 246 patients (28.2%). F3/4 patients were older with higher body weight and baseline ALT levels compared to F0-2 patients (Table 1). All F3/4 patients had compensated liver disease (Child-Pugh Score 5 in 124 and 6 in 3 patients). The distribution of patient characteristics for those with biopsy staging were similar to those with missing staging, apart from a lower proportion of Caucasians (80% vs. 90%), and a higher baseline ALT level (median: 54 vs. 44U/L) in those with biopsy (Table 1). Laboratory markers of advanced fibrosis were similar among those with and without biopsy staging including AST/ALT ratio, serum albumin, serum total bilirubin, INR, and platelet count (Table 1). Only six (2.4%) patients with missing biopsy had a Child-Pugh Score of >5.

Virological responses

Induction therapy provided approximately 10% higher early virological responses including undetectable HCV RNA proportions at week 4, 8, and 12 for both F3/4 and F0-2 groups (Fig. 1). However, early virological responses were lower among F3/4 patients in both induction and standard groups compared to corresponding F0-2 patient groups; this difference was apparent by week 4. A rapid virological response (RVR) was achieved in 25% and 18% of F3/4 patients on induction and standard therapy, respectively, compared to 39% and 28% in F0-2 patients.

Fig. 1. Rates of undetectable HCV RNA (<15IU/ml) during treatment (week 4, 8, 12, 24, 48) and at 24weeks post-treatment follow up in both the induction and standard dose treatment groups according to fibrosis group (F0-2, F3/4).


A similar disparity in virological response between F3/4 and F0-2 groups was seen at week 24 and week 48 (ETR), however, this disparity widened considerably by week 72, with SVR rates of 24-28% in F3/4 patients compared with 55-58% among F0-2 patients (p<0.0001) (Fig. 1).

The randomized groups were combined (given similar SVR rates) to explore the impact of individual fibrosis stage on treatment responses. A marked trend in SVR by fibrosis stage was evident with progressive declines in response with increasing fibrosis: F0 (70%); F1 (60%); F2 (51%); F3 (31%); F4 (10%) (p<0.0001) (Fig. 2A). Virological relapse rates were similar in early disease stages (F0, 16%; F1, 23%; F2, 26%), but increased markedly in advanced fibrosis (F3, 50%; F4, 80%) (p<0.0001) (Fig. 2B). Among patients without SVR, the percentage with non-response at week 12, virological breakthrough, and virological relapse was similar for patients with F0-2 (n=216; 27%, 36%, 37%, respectively) and patients with F3/4 (n=94; 30%, 28%, 42%, respectively).

Fig. 2. Sustained virological response and virological relapse by fibrosis stage. (A) Rates and their 95% confidence intervals of sustained virological response by individual fibrosis stage in the combined induction and standard treatment arm population. The total number of patients in each fibrosis sub-group is given at the bottom of each column. (B) Rates and their 95% confidence intervals of virological relapse by individual fibrosis stage in the combined induction and standard treatment arm population. The total number of patients with an end-of-treatment response in each fibrosis sub-group is given at the bottom of each column.


The impact of early treatment response was further characterized in the overall population by determining SVR among fibrosis groups (F0-2, F3/4) within four mutually exclusive response groups based on the week of initial undetectable HCV RNA (week 4, 8, 12, 24). A lower proportion of F3/4 patients reached undetectable HCV RNA by week 4 (21% vs. 34%) and week 8 (19% vs. 26%) (p<0.0001 for comparison of time to undetectable HCV RNA) (Fig. 3A). Furthermore, the SVR rates for each treatment response group were lower for F3/4 patients compared to F0-2 patients, including the rapid virological response group (week 4, 63% vs. 80%) (Fig. 3B). Thus, patients with advanced fibrosis had both slower initial viral decline and lower likelihood of SVR within each early treatment response group.

In a multivariate analysis among patients with all baseline parameters available for inclusion (n=507), METAVIR fibrosis score was associated with SVR (p=0.0194). Adjusted odds ratios for SVR in comparisons with F4 patients were 8.9 (1.8-43.8), 6.3 (1.5-26.6), 5.7 (1.4-23.6), and 2.7 (0.6-11.5) for patients with F0, F1, F2, and F3, respectively.

Predictors of response

The predictive value of early virological response in determination of SVR was also examined, among fibrosis groups and randomized arms (Table 2). The positive predictive value (PPV) of RVR for SVR was lower among F3/4 patients in induction (60%) and standard (67%) arms compared to F0-2 patients (79% and 82%, respectively). Of note, the negative predictive value (NPV) of RVR for SVR was considerably higher among F3/4 patients (83-84%) compared to F0-2 patients (55-57%) (Table 2). Among F3/4 patients the PPV for SVR was lower and NPV higher at week 8. Also, the PPV of week 12 response for SVR, both EVR and cEVR, were considerably lower among F3/4 patients compared to F0-2 patients (Table 2), while NPV of EVR was 100% for all groups (note that patients in both arms without an EVR, continued therapy to week 24 then to week 48 if HCV RNA was undetectable at week 24).

Cumulative therapeutic dosing

The proportions of patients receiving >80% and >95% of planned PEG-IFNα-2a dose by week 12 and 24 were lower among patients on induction therapy, but similar among patients with F3/4 and F0-2 (Table 3). There was a marginally lower proportion with >95% cumulative dosage of PEG-IFNα-2a among F3/4 patients compared to F0-2 patients in the standard arm by week 12 (83.6% vs. 89.3%) and 24 (76.1% vs. 81.0%). The proportions with receipt of >80% and >95% of planned ribavirin dose were similar across randomized and fibrosis groupings, including >95% receipt by week 12 (F0-2: induction 80.1%, standard 83.9%; F3/4: induction 83.3%, standard 83.6%) and week 24 (F0-2: induction 74.6%, standard 74.8%; F3/4: induction 75.0%, standard 73.1%) (Table 3).

Further analyses were undertaken among patients who received greater than or equal to 80% of cumulative PEG-IFN and ribavirin dosage. A marked trend in SVR by fibrosis stage remained with progressive declines in response with increasing fibrosis: F0 (78%); F1 (75%); F2 (70%); F3 (46%); F4 (13%). Virological relapse rates were similar in early disease stages (F0, 19%; F1, 23%; F2, 26%), but again increased markedly in advanced fibrosis (F3, 53%; F4, 85%).

In a logistic regression analysis the odds ratio for SVR was 0.55 (95% CI 0.46-0.66, p<0.0001) per one stage increase in fibrosis stage.

Safety and tolerability

The serious adverse event rate was 12-15% for patients with F3/4 compared to 9-10% for patients with F0-2 (Table 4). Treatment discontinuation due to adverse events or laboratory abnormalities was similar among patients with F3/4 (8-9%) and F0-2 (7-9%) (p=0.86). Although dose modification of PEG-IFNα-2a was more common among patients in the induction than standard treatment group (26% vs. 19%) (p=0.040), there was no significant difference by fibrosis group (p=0.44). In contrast, no significant difference in ribavirin dose modification was seen for both treatment (p=0.21) or fibrosis group stratification (p=0.47). Hemotological toxicity was greater among patients with F3/4, with significantly higher rates of thrombocytopenia (platelets<50x109/L) (p<0.0001) and severe anemia (hemoglobin <8.5g/dl) (p=0.009).

Methods

CHARIOT study population

The CHARIOT trial methods and patient population have been described in detail [16]. Eligible subjects included treatment naïve adults aged 18-75years with chronic HCV genotype 1 infection and compensated liver disease (Child-Pugh Score <7). Initial recruitment (in Australia from August 2004) excluded patients with cirrhosis. The protocol was revised in mid-2006 to allow inclusion of patients with normal serum ALT level and absence of liver biopsy staging of disease consistent with changes to Australian subsidized treatment requirements for serum ALT elevation (December 2005) and pre-treatment liver biopsy (April 2006). In addition, patients with compensated cirrhosis were able to enroll. Also, from mid-2006, recruitment commenced through several other countries (New Zealand, Thailand, Canada, Mexico, Argentina), with pre-treatment liver biopsy staging required in all these countries apart from New Zealand.

Patients meeting screening eligibility criteria were randomly assigned 1:1 to receive PEG-IFNα-2a either in an induction dose or standard dose regimen. The induction regimen consisted of 360µg of PEG-IFNα-2a weekly, for the first 12weeks, followed by 180µg of PEG-IFNα-2a weekly, for 36weeks and 1000/1200mg of ribavirin daily for 48weeks. The standard dose regimen involved 180µg of PEG-IFNα-2a weekly and 1000/1200mg of ribavirin daily for 48weeks. Patients in both arms without an early virological response (EVR) at week 12 continued therapy to week 24; patients with detectable HCV RNA at week 24 ceased therapy.

Cumulative exposure to PEG-IFNα-2a and ribavirin was determined by calculation of the percentage of planned dose received through week 4, 8, 12, 24, and 48. Reductions from maximum dose occurred through both clinician-directed dose modification and patient non-adherence, with adherence assessed via recording the injections and doses of PEG-IFNα-2a and ribavirin at each visit according to the patient's detailed statements, and via documentation of drugs dispensed through pharmacy records.

Assessments and efficacy endpoints

Quantitative serum HCV RNA levels were measured at baseline and at weeks 4, 8, 12, 24, 48, and 72 (Roche Ampliprep/Cobas TaqMan HCV Test with a detection limit of 15IU/ml). Treatment response endpoints were defined as undetectable HCV RNA, including the primary endpoint at week 72, and were based on Taqman results below the level of detection (both undetectable and detectable <15IU/ml).

Liver biopsies were taken within 36months of treatment and scored by local pathologists. Fibrosis stage was classified according to the METAVIR system [17] as F0 (no), F1 (mild), F2 (moderate), F3 (severe), and F4 (cirrhosis). Advanced fibrosis included patients with either F3 or F4 fibrosis stage.

Statistical analysis

The intent-to-treat analysis population was defined as all patients randomized who received at least one dose of study medication. Percentages are calculated for categorical parameters. Means are calculated for the continuous variables. Area under the curve (AUC) of HCV RNA through week 24 is calculated and has been analyzed with F3/4 vs. F0-2 and treatment groups as factors in ANCOVA. A logistic regression analysis was conducted for SVR with METAVIR score as a prognosis factor. A multivariate logistic regression analysis was also conducted to evaluate the independent impact of fibrosis stage on SVR, with the following baseline variables included: treatment group, age, gender, race, body weight, METAVIR fibrosis score, screening HCV RNA level, subtype (1b or non-1b), ALT, AST/ALT ratio, hemoglobin, white blood cell count, platelet count, serum creatinine, serum albumin, total bilirubin, alkaline phosphatase, gamma-glutamyl transferase (GGT), and international normalized ratio (INR). Due to the exploratory nature of the analyses, no alpha-adjustments were applied to account for multiple significance testing.

Source