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By Kristina Fiore, Staff Writer, MedPage Today
Published: January 09, 2009
Reviewed by Robert Jasmer, MD; Associate Clinical Professor of Medicine, University of California, San Francisco . Earn CME/CE credit
for reading medical news
CORDOBA, Spain, Jan. 9 -- Higher levels of hepatic steatosis in donor livers correspond to poorer outcomes for transplant patients with cirrhosis from hepatitis C virus infection, researchers here said.
Action Points
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■Explain that patient and graft survival significantly decreased among HCV patients given liver grafts with higher levels of steatosis.
■Explain that disease recurrence was earlier, more frequent, and more severe among these patients as well.
Patient and graft survival significantly decreased with degree of steatosis (P=0.008 and P=0.012, respectively), Ruben Ciria, M.D., of Reina Sofia University Hospital here, and colleagues reported in the January issue of Liver Transplantation.
And HCV recurrence was earlier, more frequent, and more severe among these patients (P=0.042), the researchers said.
"Our results show a direct relationship between marginal donors, graft steatosis, and more frequent, more severe, and earlier viral recurrence after orthotopic liver transplantation for HCV-related cirrhosis," the researchers said.
They said that recent studies have shown survival rates among transplanted HCV cirrhosis patients have been falling. A number of factors could be contributing to this decline, they said, but these have not been widely studied.
One possible explanation is that the limited number of available liver donors has led to the increased use of marginal livers that don't meet optimal criteria.
So to assess the influence of donor graft steatosis on outcomes including patient survival, graft survival, and viral recurrence, the researchers analyzed 120 patients who had a liver transplant as a result of HCV cirrhosis from 1995 through 2005 at Reina Sofia University Hospital (62.5% male, mean age 51).
Donor steatosis was categorized as absent (0% to 10%), mild (11% to 30%), moderate (31% to 60%), or severe (>60%).
Patient survival significantly decreased with the degree of hepatic steatosis, the researchers said. Those who received a liver with steatosis levels lower than 30% had a survival rate of 66% at three years, compared with 47% for those who received a liver with steatosis levels of 30% or more (P=0.008).
Graft survival also decreased significantly with the degree of fatty content. For those transplanted with steatosis-free livers, survival at three years was 72%. Survival was 58% for those transplanted with mildly steatotic livers and 43% for those with moderate steatosis. Patients transplanted with severely steatotic livers had a survival rate of 42% at three years (P=0.012).
In a multivariate analysis, the only other statistically significant predictors of graft survival were cold ischemia time greater than 12 hours (P=0.0001) and donor age greater than 55 (P=0.048).
The researchers also found an increased risk of earlier and more frequent viral recurrence associated with donor graft steatosis (P=0.042).
The severity of viral recurrence was greater for patients who received more steatotic livers. For example, at 12 months, 40% of patients given a liver with steatosis of 30% or more had fibrosis scores higher than two, compared with 17% of patients given a liver with steatosis less than 30% (P=0.035).
The researchers compared these findings to 87 patients with end-stage alcoholic liver disease who had a liver transplant during the same time period.
Overall survival at 12 months for these patients given a liver with steatosis of 30% or more was 81%, compared with 63% for the hepatitis C transplant patients.
"These results clearly show the higher impact of donor steatosis on graft survival in [hepatitis C] recipients versus non-[hepatitis C] recipients," the researchers said.
They added that what constitutes a "marginal donor" is not the same for all transplant surgeons, so the definition of a marginal graft will continue to vary between centers "until reliable parameters are available for prospectively predicting early graft function."
They concluded that "further multicenter studies and a global consensus may be necessary to finally assess if the use of expanded criteria grafts is safe for HCV-positive recipients and if the organ allocation system needs to be changed for this cohort of patients."
In an accompanying editorial, Nevin Yilmaz, M.D., and Mitchell L. Shiffman, M.D., of Virginia Commonwealth in Richmond, said they were concerned with the study's conclusions.
They said there was not enough data on the patients with alcohol-induced cirrhosis to be sure the populations were comparable.
They were, however, "intrigued" by the finding that a combination of donor graft steatosis and a prolongation in cold ischemic time was associated with severe reperfusion injury.
"We strongly suggest that cold ischemia time should be limited when the donor graft contains greater than 30% steatosis and that such grafts should be used only with caution for patients with chronic HCV infection."
The researchers and editorialists reported no conflicts of interest.
Primary source: Liver Transplantation
Source reference:
Briceno J, et al "Impact of donor graft steatosis on overall outcome and viral recurrence after liver transplantation for hepatitis C virus cirrhosis" Liver Transpl 2009; 15: 37-48.
Additional source: Liver Transplantation
Source reference:
Yilmaz N, Shiffman ML "Impact of the donor liver with steatosis in patients with hepatitis C virus: Not so fast" Liver Transpl 2009; 15: 4-6.
Monday, June 1, 2009
Donor Factors Affect Success of Liver Transplantation
Donor Factors Affect Success of Liver Transplantation
By Todd Neale, Staff Writer, MedPage Today
Published: May 29, 2009
Reviewed by Dori F. Zaleznik, MD; Associate Clinical Professor of Medicine, Harvard Medical School, Boston and
Dorothy Caputo, MA,RN,BC-ADM,CDE, Nurse Planner Earn CME/CE credit
for reading medical news
LITTLE FALLS, N.J., May 29 -- A donor's age and other identifiable characteristics appear to worsen outcomes for liver transplant recipients, particularly those who are hepatitis C virus (HCV)-positive. But steatosis alone in a donor's liver should not be a disqualifier, according to two new studies.
Action Points
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■Explain to interested patients that these studies identified characteristics that modified outcomes following liver transplantation.
■Point out that donor steatosis was not associated with transplantation outcome, even in patients who were HCV-positive.
In the first study, higher scores on an index predicting the risk of graft failure based on donor factors were associated with poorer outcomes in all liver transplant recipients, although the relationship was amplified in HCV-positive patients, according to Daniel Maluf, M.D., of Virginia Commonwealth University in Richmond, and colleagues.
The second study indicated that steatosis in the donor liver had no effect on the progression of liver disease or three-year survival among transplant recipients, regardless of HCV status, according to Patrizia Burra, M.D., Ph.D., of the University of Padova in Italy, and colleagues.
They did find, however, that recipient age older than 50, diabetes, and impaired liver function shortly after transplantation were independent predictors of mortality.
Both studies were published in the June issue of Liver Transplantation.
Despite increases in the number of liver transplants over the years, about 15% of patients awaiting a new liver will die before receiving one.
To address the organ shortage, researchers have searched for ways to increase the supply of donor organs, including using less-than-perfect livers.
It had been unclear whether using mildly or moderately fatty livers would be associated with worse outcomes, so Dr. Burra and colleagues analyzed the results of 116 consecutive liver transplants at a single center. A total of 56 recipients were HCV-positive.
About half (50.9%) of the donor livers showed no signs of steatosis, while 39.6% had mild steatosis, and 9.5% had moderate or severe steatosis.
As expected, through three years, HCV-positive patients had significantly worse progression of fibrosis (P=0.001), with nearly a quarter having pre-cirrhosis or cirrhosis.
However, there was no association between donor liver steatosis and the progression of liver disease.
In addition, three-year survival was not affected by recipients' HCV status (P=0.4) or by the degree of donor steatosis (P=0.7).
"Judging from the data presented here, albeit with the limits imposed by the relatively small number of patients considered, we would like to support the use of grafts with steatosis, even in HCV," Dr. Burra and colleagues said.
In the second study, Dr. Maluf and colleagues performed a retrospective analysis of 16,678 patients who received liver transplants over a six-and-a-half year period using the Organ Procurement and Transplantation Network Database.
Nearly half of the patients (46%) were HCV-positive.
A donor risk index was calculated using the following variables: age older than 40, black race, shorter height, donation after cardiac death, death by cerebrovascular accident, and death other than trauma, stroke or anoxia, and transplantation using a split or partial graft.
Increasing scores on the index were associated with greater chances of graft failure and death for both HCV-positive and HCV-negative patients.
However, the risk was significantly greater in HCV-positive patients for both graft failure (P=0.004) and death (P=0.0002).
Donor age accounted for 70% of the relationship between the donor risk index and adverse outcomes.
Beyond the donor risk index, the researchers said, "other risk factors such as graft steatosis, high vasopressor drug requirement, and other clinical attributes of the donor must also be carefully considered by the transplant physician and balanced against the risk of recipient mortality while the patient continues on the waiting list in the event that the offer of a graft is declined."
They acknowledged that the study was limited by possible misclassification bias and by the determination of HCV status by serology only.
Sandy Feng, M.D., Ph.D., of the University of California San Francisco, wrote in an accompanying editorial that there is a severe shortage of organs and that transplantation remains the best hope of survival for many.
Considering the organ shortage, she said, "we must vow to take advantage of every available opportunity to realize reasonable survival benefit through transplantation."
Creating "an allocation algorithm that can systematically and objectively account for the variable impact of donor characteristics on liver transplant outcomes within the context of recipient diagnosis and disease severity . . . would be the most equitable and transparent way to distribute the differential risk posed by the donor pool to individual transplant candidates," she said.
The study authors and the editorialist made no financial disclosures.
Primary source: Liver Transplantation
Source reference:
Burra P, et al "Donor livers with steatosis are safe to use in hepatitis C virus-positive recipients" Liver Transpl 2009; 15: 619-28.
Additional source: Liver Transplantation
Source reference:
Maluf D, et al "Impact of the donor risk index on the outcome of hepatitis C virus-positive liver transplant recipients" Liver Transpl 2009; 15: 592-99.
Additional source: Liver Transplantation
Source reference:
Feng S "Increased donor risk: who should bear the burden?" Liver Transpl 2009; 15: 570-73.
By Todd Neale, Staff Writer, MedPage Today
Published: May 29, 2009
Reviewed by Dori F. Zaleznik, MD; Associate Clinical Professor of Medicine, Harvard Medical School, Boston and
Dorothy Caputo, MA,RN,BC-ADM,CDE, Nurse Planner Earn CME/CE credit
for reading medical news
LITTLE FALLS, N.J., May 29 -- A donor's age and other identifiable characteristics appear to worsen outcomes for liver transplant recipients, particularly those who are hepatitis C virus (HCV)-positive. But steatosis alone in a donor's liver should not be a disqualifier, according to two new studies.
Action Points
--------------------------------------------------------------------------------
■Explain to interested patients that these studies identified characteristics that modified outcomes following liver transplantation.
■Point out that donor steatosis was not associated with transplantation outcome, even in patients who were HCV-positive.
In the first study, higher scores on an index predicting the risk of graft failure based on donor factors were associated with poorer outcomes in all liver transplant recipients, although the relationship was amplified in HCV-positive patients, according to Daniel Maluf, M.D., of Virginia Commonwealth University in Richmond, and colleagues.
The second study indicated that steatosis in the donor liver had no effect on the progression of liver disease or three-year survival among transplant recipients, regardless of HCV status, according to Patrizia Burra, M.D., Ph.D., of the University of Padova in Italy, and colleagues.
They did find, however, that recipient age older than 50, diabetes, and impaired liver function shortly after transplantation were independent predictors of mortality.
Both studies were published in the June issue of Liver Transplantation.
Despite increases in the number of liver transplants over the years, about 15% of patients awaiting a new liver will die before receiving one.
To address the organ shortage, researchers have searched for ways to increase the supply of donor organs, including using less-than-perfect livers.
It had been unclear whether using mildly or moderately fatty livers would be associated with worse outcomes, so Dr. Burra and colleagues analyzed the results of 116 consecutive liver transplants at a single center. A total of 56 recipients were HCV-positive.
About half (50.9%) of the donor livers showed no signs of steatosis, while 39.6% had mild steatosis, and 9.5% had moderate or severe steatosis.
As expected, through three years, HCV-positive patients had significantly worse progression of fibrosis (P=0.001), with nearly a quarter having pre-cirrhosis or cirrhosis.
However, there was no association between donor liver steatosis and the progression of liver disease.
In addition, three-year survival was not affected by recipients' HCV status (P=0.4) or by the degree of donor steatosis (P=0.7).
"Judging from the data presented here, albeit with the limits imposed by the relatively small number of patients considered, we would like to support the use of grafts with steatosis, even in HCV," Dr. Burra and colleagues said.
In the second study, Dr. Maluf and colleagues performed a retrospective analysis of 16,678 patients who received liver transplants over a six-and-a-half year period using the Organ Procurement and Transplantation Network Database.
Nearly half of the patients (46%) were HCV-positive.
A donor risk index was calculated using the following variables: age older than 40, black race, shorter height, donation after cardiac death, death by cerebrovascular accident, and death other than trauma, stroke or anoxia, and transplantation using a split or partial graft.
Increasing scores on the index were associated with greater chances of graft failure and death for both HCV-positive and HCV-negative patients.
However, the risk was significantly greater in HCV-positive patients for both graft failure (P=0.004) and death (P=0.0002).
Donor age accounted for 70% of the relationship between the donor risk index and adverse outcomes.
Beyond the donor risk index, the researchers said, "other risk factors such as graft steatosis, high vasopressor drug requirement, and other clinical attributes of the donor must also be carefully considered by the transplant physician and balanced against the risk of recipient mortality while the patient continues on the waiting list in the event that the offer of a graft is declined."
They acknowledged that the study was limited by possible misclassification bias and by the determination of HCV status by serology only.
Sandy Feng, M.D., Ph.D., of the University of California San Francisco, wrote in an accompanying editorial that there is a severe shortage of organs and that transplantation remains the best hope of survival for many.
Considering the organ shortage, she said, "we must vow to take advantage of every available opportunity to realize reasonable survival benefit through transplantation."
Creating "an allocation algorithm that can systematically and objectively account for the variable impact of donor characteristics on liver transplant outcomes within the context of recipient diagnosis and disease severity . . . would be the most equitable and transparent way to distribute the differential risk posed by the donor pool to individual transplant candidates," she said.
The study authors and the editorialist made no financial disclosures.
Primary source: Liver Transplantation
Source reference:
Burra P, et al "Donor livers with steatosis are safe to use in hepatitis C virus-positive recipients" Liver Transpl 2009; 15: 619-28.
Additional source: Liver Transplantation
Source reference:
Maluf D, et al "Impact of the donor risk index on the outcome of hepatitis C virus-positive liver transplant recipients" Liver Transpl 2009; 15: 592-99.
Additional source: Liver Transplantation
Source reference:
Feng S "Increased donor risk: who should bear the burden?" Liver Transpl 2009; 15: 570-73.
Retreating chronic hepatitis C
Hepatology June 6 2009.
Retreating chronic hepatitis C with daily interferon alfacon-1/ribavirin after nonresponse to pegylated interferon/ribavirin:
DIRECT results
Bruce R. Bacon 1 *, Mitchell L. Shiffman 2, Flavia Mendes 3, Reem Ghalib 4, Tarek Hassanein 5, Giuseppe Morelli 6, Shobha Joshi 7, Kenneth Rothstein 8, Paul Kwo 9, Norman Gitlin 10
1Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO
2Hepatology Section, Virginia Commonwealth University Medical Center, Richmond, VA
3Division of Hepatology, University of Miami Miller School of Medicine, Miami, FL
4Liver Institute at Methodist Hospital, Dallas, TX
5Division of Gastroenterology and Hepatology, University of California San Diego, San Diego, CA
6Division of Gastroenterology and Hepatology, University of Florida, Gainesville, FL
7Division of Gastroenterology and Hepatology, Tulane University Hospital and Clinic, New Orleans, LA
8Albert Einstein Center for Liver Disease, Philadelphia, PA
9Division of Gastroenterology and Hepatology, Indiana University School of Medicine, Indianapolis IN
10Division of Gastroenterology and Hepatology, Emory Crawford Long Hospital, Atlanta, GA
email: Bruce R. Bacon (baconbr@slu.edu)
*Correspondence to Bruce R. Bacon, Division of Gastroenterology and Hepatology, Saint Louis University School of Medicine, 3635 Vista Avenue at Grand Boulevard, St. Louis, MO 63110-0250
Potential conflict of interest: Nothing to report.
fax: 314-577-8125
Funded by:
InterMune, Inc.
Valeant Pharmaceuticals International
Three Rivers Pharmaceuticals
ABSTRACT
Up to 50% of patients with chronic hepatitis C fail to respond to initial therapy with pegylated interferon (PEG-IFN) and ribavirin (RBV). With unsuccessful viral eradication, these patients remain at risk for developing progression of their liver disease. Retreatment with PEG-IFN/RBV yields sustained virologic response (SVR) rates that are under 10%. A wholly synthetic interferon, interferon alfacon-1 or consensus interferon (CIFN) given with RBV, was evaluated in patients who failed initial PEG-IFN/RBV therapy. The intent-to-treat analysis included 487 patients; 245 received CIFN 9 g/day and RBV, and 242 received CIFN 15 g/day and RBV. Within this group of patients, 59.3% had documented advanced fibrosis at baseline liver biopsy (stage F3 or F4). SVR rates were 6.9% (17/245 patients) in the 9 g group and 10.7% (26/242) in the 15 g group. In the intent-to-treat analysis, SVR rates were higher among patients with a >2-log10 decrease in hepatitis C virus RNA during prior PEG-IFN/RBV therapy: 11% (4/38) in the 9 g group and 23% (7/31) in the 15 g group. Among patients with lower baseline fibrosis scores (F0-F3), SVR rates were 7.8% (15/192) in the 9g group and 13.1% (23/175) in the 15 g group. In this same group of patients (F0-F3), if a >2-log10 decrease in hepatitis C virus RNA with previous PEG-IFN/RBV treatment was achieved, SVR rates improved to 10.7% and 31.6% in the 9 g and 15 g groups, respectively. CIFN/RBV combination retreatment was safe and well tolerated. Conclusion: Retreatment of PEG-IFN and RBV nonresponders with CIFN and RBV is safe and efficacious and can be considered a retreatment strategy for patients failing previous therapy with PEG-IFN/RBV, especially in interferon-sensitive patients with lower baseline fibrosis scores. (HEPATOLOGY 2009.)
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Received: 21 August 2008; Accepted: 24 January 2009
DIGITAL OBJECT IDENTIFIER (DOI)
10.1002/hep.22871 About DOI
ARTICLE TEXT
Since 2001, the standard of care for patients with chronic hepatitis C has been the combination of pegylated interferon (PEG-IFN) and ribavirin (RBV).[1][2] This combination has produced sustained virologic response (SVR) rates of 50%-60% in patients infected with hepatitis C virus (HCV) genotype 1 who adhere to their therapeutic regimens and 40% in intention-to-treat populations.[1][2] However, because only about 65% of patients become HCV RNA-undetectable when treated with this regimen, more than one-third of all patients are classified as nonresponders. Some of these patients have relatively mild liver disease but may have symptoms of HCV viremia, while other patients have advanced fibrosis and are at risk for developing complications of chronic liver disease, including decompensated cirrhosis and hepatocellular carcinoma, and may require liver transplantation.[3-5]
The optimal approach to PEG-IFN/RBV nonresponders has not been well defined. Some clinicians have used the watchful waitingapproach[6] and are anticipating new antiviral therapies with either protease inhibitors or polymerase inhibitors. However, it remains to be determined just how effective these new agents will be when combined with PEG-IFN and RBV in the retreatment of PEG-IFN/RBV nonresponders.[7]
Alternative therapies have included retreatment with the alternative brand of PEG-IFN not used in the initial therapy, although most results with this approach have been disappointing. Other approaches have included prolonged treatment with PEG-IFN, maintenance therapy, or the use of higher dosages of either PEG-IFN and/or RBV.[8-12] The strategy studied in the current investigation included high doses of daily consensus interferon (CIFN) (Infergen; interferon alfacon-1) 9 or 15 g/day given with RBV.
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Abbreviations
AE, adverse event; bDNA, branched DNA; CIFN, consensus interferon; DIRECT, Daily-Dose Consensus Interferon and Ribavirin: Efficacy of Combined Therapy; Hb, hemoglobin; HCV, hepatitis C virus; ITT, intention to treat; PEG-IFN, pegylated interferon; RBV, ribavirin; REPEAT, REtreatment with PEgasys in PATients Not Responding to Peg-Intron Therapy; SVR, sustained virologic response; TMA, transcription-mediated amplification.
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Patients and Methods
This study, referred to as the DIRECT (Daily-Dose Consensus Interferon and Ribavirin: Efficacy of Combined Therapy) trial, was designed by the sponsor and by several of the academic investigators. The data were managed by the sponsor and the academic investigators. The sponsor performed the statistical analysis. The academic investigators were responsible for the development of the final manuscript and had unrestricted access to the data. An author involved with the design or execution of this study either wrote or edited every section of the manuscript. Both an academic author (B. R. B.) and an industry representative (Michael Beckloff, Three Rivers Pharmaceuticals, Cranberry Township, PA) attest to the completeness and accuracy of the data.
Study Design.
This was a phase 3, randomized, open-label, multicenter, U.S.-based registration trial conducted to investigate the efficacy, tolerability, and safety of daily CIFN at dosages of 9 and 15 g/day (interferon alfacon-1, Infergen; Three Rivers Pharmaceuticals, LLC, Cranberry Township, PA) administered with daily RBV (Ribasphere, Three Rivers Pharmaceuticals, LLC) compared with no treatment in patients who did not respond to prior therapy with either PEG-IFN alfa-2a or alfa-2b and RBV. The trial was divided into 2 sections: DIRECT-001 and DIRECT-002 (Fig. 1).
Figure 1. DIRECT study design: a randomized, open-label study of CIFN and RBV in patients who did not respond to previous combination therapy with PEG-IFN and ribavirin.
[Normal View 33K | Magnified View 66K]
Patients were randomized at a 1:1:1 ratio into three study groups: CIFN 9 g/day (group 1), 15 g/day (group 2) plus oral RBV 1,000-1,200 mg/day (based on body weight), or a control, no-treatment group (group 3). The no-treatment group was mandated by the U.S. Food and Drug Administration in order to provide a comparison of safety for the two treatment groups. It was not anticipated that any of the patients randomized to the control group would have a spontaneous response. After 24 weeks of observation, all patients in the control group of DIRECT-001 were offered randomization into DIRECT-002 to receive CIFN 9 or 15 g/day plus RBV.
At week 24, patients who had undetectable plasma HCV RNA by branched DNA (bDNA) assay, confirmed by transcription-mediated amplification (TMA) assay, or who had a 2-log10 decrease from baseline in HCV RNA were assigned to continue therapy to week 48. Patients with a <2-log10 decrease from baseline in plasma HCV RNA (bDNA assay) were considered nonresponders and were withdrawn from treatment. At week 48, patients with undetectable plasma HCV RNA (by bDNA and TMA assays) were assigned to return for regular visits in the follow-up period (weeks 52, 60, 68, and 72) until 24 weeks after their last dose of study drug (week 72). Patients with detectable plasma HCV RNA (bDNA or TMA assay) at any time between weeks 48 and 72 were classified as relapsers.
All patients who discontinued therapy early at any time were instructed to return for a single follow-up visit 30 days after their last dose of study drug to complete early termination/discontinuation assessments. Patients who had undetectable plasma HCV RNA by bDNA and TMA assays at the time of stopping therapy or at the early termination/discontinuation visit were to return for follow-up plasma HCV RNA assessments through week 72, as long as their plasma HCV RNA levels remained undetectable by both bDNA and TMA assays. Patients who discontinued for nonresponse at weeks 12 or 24 did not undergo a follow-up HCV RNA measurement.
Patients in the no-treatment group in DIRECT-001 were given the option to enroll in DIRECT-002 under the following conditions: if they achieved a <2-log10 decrease in plasma HCV RNA at week 24 compared with baseline or if they had detectable plasma HCV RNA by bDNA or by TMA at week 48. These patients were then treated according to the same protocol followed in DIRECT-001. The results of DIRECT-001 and DIRECT-002 were pooled for the purpose of this analysis.
Patients.
Men and women were eligible for enrollment if they were chronically infected with HCV of any genotype. Chronic infection was identified based on a history of being positive for serum anti-HCV and/or HCV RNA. A liver biopsy performed within 3 years of screening must have demonstrated evidence of chronic HCV infection. Hepatic fibrosis was interpreted by local pathologists based upon the Metavir scoring system. Patients with advanced liver disease, including bridging fibrosis (F3) and cirrhosis (F4), were eligible for the study as long as they had normal liver function as evidenced by serum albumin >3.5 mg/dL, platelet count >75,000/mm3, and no prior episode of hepatic decompensation (variceal hemorrhage, hepatic encephalopathy, ascites, or hepatocellular carcinoma).
Prior nonresponse and adherence after initial therapy with PEG-IFN alfa-2a (180 g/week) or PEG-IFN alfa-2b (1.5 g/kg/week) plus RBV was determined via careful chart review completed by the study site principal investigator and confirmed by an external study monitor. Nonresponders had to have had a <2-log10 decrease in HCV RNA between weeks 12 and 24 or detectable HCV RNA at weeks 24 or 48. Patients were required to have had completed a minimum of 90 days between discontinuation of their prior regimen and the start of the current study medication. All patients had to have received at least 80% of the cumulative standard dosages of PEG-IFN and RBV for at least 38 weeks (80% of the planned treatment duration). Patients were excluded if this previous treatment was prematurely discontinued, dosing was interrupted, or if the dose of PEG-IFN was reduced because of noncompliance, safety, or tolerability issues (including hematologic or psychiatric side effects).
Patients were also excluded if they were pregnant or lactating women or male partners of pregnant women, or if they were not suitable candidates for enrollment or unlikely to comply with the requirements of the study in the opinion of the investigator or sponsor.
Treatment and Assessments.
Screening took place between 8 weeks and 1 day before the first day of treatment. After providing informed consent, patients were screened for inclusion criteria, underwent a physical examination, provided a baseline medical history, and had blood drawn for laboratory testing. After screening, eligible patients were randomized in a 1:1:1 ratio to receive CIFN 9 or 15 g/day plus RBV 1,000 mg/day (body weight <75 kg) or 1,200 mg/day (body weight >75 kg) or no treatment (Fig. 1). RBV was provided as capsules containing 200 mg of active drug. An independent data monitoring committee conducted regular interim safety assessments throughout the study.
Plasma HCV RNA levels were determined first using the bDNA quantitative assay, which has a sensitivity of detection of 615 IU/mL and a reportable range of 615 to 6,920,000 IU/mL. The Bayer TMA assay, with a sensitivity of detection of 5 IU/mL, was used whenever HCV RNA levels were undetectable via bDNA assay.
Patients developing anemia, defined as hemoglobin (Hb) <10 g/dL, were managed by reducing the dose of RBV to 600 mg/day. The use of growth factors was not permitted. If the Hb increased to >10 g/dL, the RBV dose could be increased in 200-mg/day increments as tolerated according to the discretion of the site principal investigator. RBV dose was not increased after being reduced to 600 mg/day for patients with a history of cardiovascular disease whose Hb decreased by 2 g/dL or more during any 4-week period. RBV was permanently discontinued in patients whose Hb dropped below 8.5 g/dL. In those patients with a history of cardiac or cerebrovascular disease, Hb remaining below 12 g/dL after 4 weeks on a reduced dose required permanent discontinuation of RBV. Neutropenia was managed by CIFN dose reduction; in patients whose absolute neutrophil count fell to <0.75 × 109/L, starting doses of 15 g were lowered to 9 g and then to 6 g, and starting doses of 9 g were lowered to 6 g.
Efficacy Variables.
The primary efficacy variable was the proportion of patients with SVR, defined as undetectable plasma HCV RNA by both bDNA and TMA assays at 24 weeks after the last dose of study drug. In addition, SVR was further explored for the effect of race, genotype, sex, age, baseline HCV RNA, presence/absence of cirrhosis, body weight, and previous response to PEG-IFN/RBV.
Safety and Tolerability.
All adverse events (AEs) and serious AEs were recorded for patients who received at least 1 dose of study medication (active-treatment groups) or who completed baseline assessments (no-treatment group). AEs were recorded until either 30 days after the last dose of study medication (active-treatment groups) or until the last study visit (no-treatment group). AEs were graded from 1 to 5 (1, mild; 2, moderate; 3, severe; 4, life-threatening or disabling; 5, death) based on the Common Toxicity Criteria for Adverse Events v3.0. An AE was considered a serious AE if it resulted in death, was life-threatening, required inpatient hospitalization, or resulted in persistent or significant disability or incapacity.
Statistical Methods.
At least 170 patients were needed in each of the three study groups (for a total of 510 patients) to provide an approximately 91% power to detect a difference in SVR between each of the active treatment groups and the no-treatment group. This analysis was performed using a two-sided Fisher's exact test at = 0.05 significance level, with adjustment for multiple comparisons, and assumed an SVR rate of 10% for either of the active treatment groups and 1.2% for the no-treatment group. The study was not powered to detect differences between the 9 g and the 15 g arms.
Data were summarized and analyzed for two patient populations: the intention-to-treat (ITT) population and patients who did not receive any dose modifications. The ITT group consisted of all patients who were randomized to receive CIFN in DIRECT-001, as well as all patients from the no-treatment group in DIRECT-001 who went on to receive at least 1 dose of CIFN in DIRECT-002. Data from the ITT population were used in all efficacy and safety analyses.
Descriptive statistics were determined for continuous variables (patient counts, mean, standard deviation, median, minimum, and maximum) and categorical variables (number and percentage of patients for each category). Percentages were calculated using the number of patients without missing data as the denominator unless otherwise indicated. Calculations of virologic response (both sustained and at specific visits) used the number of ITT patients as the denominator. All statistical testing was conducted at the 0.05 level of significance using SAS software, version 8.2.
Results
Patients and Disposition.
Five hundred fifteen patients were randomized at 44 sites in the United States and Puerto Rico to receive CIFN 9 g/day plus RBV 1,000 or 1,200 mg/day (n = 171), CIFN 15 g/day plus RBV (n = 172), or no treatment (n = 172). Of the 172 patients in the no-treatment group in DIRECT-001, 144 continued on to DIRECT-002. Of these, 74 received CIFN 9 g/day plus RBV 1,000 or 1,200 mg/day, and 70 received CIFN 15 g/day plus RBV 1,000 or 1,200 mg/day. The final ITT population included 487 patients (245 who received CIFN 9 g/day and 242 who received CIFN 15 g/day).
Baseline demographic and clinical characteristics of the two CIFN treatment groups are presented in Table 1. The majority of patients were male (70%) and Caucasian (64%). Of the enrolled patients, 59.3% had advanced liver disease on biopsy, including bridging fibrosis (F3; 35%) or cirrhosis (F4; 25%). In addition, 52% of patients had hepatic steatosis. The average time between biopsy sampling and study day 1 was 1.6 years. Patients included in the DIRECT trial were required to be off PEG-IFN/RBV therapy for at least 3 months prior to starting CIFN therapy. The median washout period between previous treatment and day 1 of CIFN therapy was 448 days (15 months) and 506 days (16.8 months) for the 9 g and 15 g groups, respectively. Sixty-eight percent of the patients had high baseline HCV RNA levels of >850,000 IU/mL. The majority of patients (79%) failed to achieve an early virologic response (at least a 2-log10 drop in HCV RNA from the pretreatment baseline) to previous PEG-IFN therapy.
Table 1. DIRECT-001 and DIRECT-002: Baseline Characteristics
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CIFN 9 g/day + RBV (n = 245) CIFN 15 g/day + RBV (n = 242)
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Mean age ± SD, years 51 ± 6.65 50 ± 6.59
Male 68% 72%
HCV genotype 1 95% 96%
High viral load
850,000 IU/mL 68% 68%
Mean weight ± SD, kg 89.1 ± 18.64 89.4 ± 17.59
Mean body mass index ± SD, kg/m2 29.3 ± 5.20 29.6 ± 5.02
Race
Caucasian 64% 65%
African American 21% 17%
Liver biopsy results
Cirrhosis (F4) 22% 28%
Bridging fibrosis (F3) 36% 34%
(F0-F2) 42% 38%
Steatosis* 52% 51%
Response to prior therapy
<2-log10 drop 78% 80%
>2-log10 drop 15% 12%
Unknown 7% 8%
Washout interval, days
Mean ± SD 453 ± 345 594 ± 372
Median 448 506
--------------------------------------------------------------------------------
Abbreviation: SD, standard deviation.
* Steatosis defined as present or absent.
Antiviral Efficacy.
By ITT analysis, pooled end-of-treatment response via TMA assay in the pooled 9 g arm was 14.7% (36/245), with a subsequent SVR of 6.9% (17/245). In the 15 g arm, pooled end-of-treatment response via TMA assay was 18.5% (45/242), with an SVR rate of 10.7% (26/242). Relapse rates pooled for both arms were 52% (19/36) and 42% (19/45) for the 9 g and 15 g groups, respectively. Post hoc analysis revealed steatosis and time to viral negativity had the most impact on relapse rates.[13] As expected, in DIRECT 001, patients in the no-treatment arm achieved a 0% SVR. In patients who did not have dose modifications, overall SVR rates were 7% in the CIFN 9 g group and 17% in the 15 g group (Fig. 2A,B). The SVR rates were not significantly different between the 001 and 002 arms (P = 0.818). Although the study was not powered to detect differences between the 9 g and 15 g groups, a post hoc analysis revealed no difference in SVR rates between the two (P = 0.141, 95% CI -8.8%-1.2%).
Figure 2. Rates of SVR, defined as undetectable viral levels at least 24 weeks after the end of treatment with CIFN and RBV, in (A) the ITT population (n = 487) and (B) patients who did not receive dose modifications (n = 281). Active versus no treatment (n = 172).
[Normal View 22K | Magnified View 42K]
Patients who achieved a complete early virologic response (defined as viral negativity at week 12 via TMA assay) were more likely to demonstrate an SVR than the general study population. In the 9 g group, 81.3% (13/16) of patients with complete early virologic response achieved SVR, whereas in the 15 g group, 63.6% (14/22) of patients with complete early virologic response demonstrated SVR. In patients deemed slow responders (>2-log drop at week 12, viral-negative at week 24), SVR rates were 11.7% (2/17) and 35.4% (11/31) in the 9 g and 15 g groups, respectively. Two patients in the 9 g arm and one patient in the 15 g arm achieved SVR despite being viral-positive at week 24.
Patients achieving the greatest log reduction in terms of viral response to initial PEG-IFN/RBV therapy had the best likelihood of responding to retreatment with CIFN and RBV (Fig. 3A,B). Among F0-F2 patients with >2-log10 decreases in HCV RNA during their prior PEG-IFN/RBV therapy, SVR rates were 13.3% (2/15) and 30.0% (3/10) in the 9 g and 15 g groups, respectively. A similar trend was seen in the 15 g arm in patients with bridging fibrosis (F3). SVR for the F0-F2 group of patients was 8.7% (9/104) in the 9g group and 14.9% (14/94) in the 15 g group. With patients displaying bridging fibrosis only on biopsy, overall SVR was 6.8% (6/88) and 11.1% (9/81) in the 9 and 15 g groups, respectively. Complete SVR for noncirrhotics (F0-F3) was 7.8% (15/192) in the 9 g group versus 13.1% (23/175) in the 15 g group, whereas cirrhotics achieved SVR rates of 3.8% (2/53) and 4.5% (3/67) in the 9 g and 15 g groups, respectively. In the cirrhotic cohort, patients required at least a 1-log drop on prior therapy to benefit from retreatment with CIFN and RBV. African American patients achieved lower SVR rates than Caucasians (4.2% versus 11%, respectively). In this population, pooled analysis between the two dosage arms revealed that 35.4% of the patients had failed at least two or more prior treatment regimens, with 52.7% having obtained a <1-log drop on prior PEG-IFN/RBV therapy. In addition to this, 60.2% had dose reductions while on CIFN/RBV therapy. Finally, non-genotype 1 patients (genotype 2/3) achieved an overall SVR rate of 23.1% (3/13) and 88.8% (8/9) in the 9 g and 15 g groups, respectively. Further univariate predictors of response are discussed in Table 2.
Figure 3. Rates of SVR, defined as undetectable viral levels at 24 weeks after the end of treatment, in the ITT analysis by known previous response to therapy among patients without F0-F2 fibrosis, F3, and with cirrhosis (F4) in the (A) 9 g and (B) 15 g treatment groups. One patient in the 9 g group and two patients in the 15 g group achieving SVR had unknown prior response to PEG-IFN/RBV therapy.
[Normal View 25K | Magnified View 52K]
Table 2. Univariate Predictors of SVR
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n (% SVR) P Value
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Treatment group
9 g/day (n = 245) 17 (6.9) NS
15 g/day (n = 242) 26 (10.7)
Sex
Male (n = 342) 30 (8.8) NS
Female (n = 145) 13 (9.0)
Fibrosis scores
F0-F2 (n = 198) 23 (11.6) NS
F3-F4 (n = 289) 20 (6.9)
Genotype
1 (n = 464) 32 (6.9) <0.001
Non-genotype 1 (n = 23) 11 (47.8)
Viral load
<850,000 IU/mL (n = 155) 25 (16.1) <0.001
>850,000 IU/mL (n = 331) 18 (5.4)
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Abbreviation: NS, not significant.
Safety and Tolerability.
A total of 83.6% of patients in the 9 g group and 71.7% of patients in the 15 g group received at least 80% of their cumulative CIFN dose. The most common reason for early termination was treatment failure. Discontinuation due to not achieving a >2-log drop at week 24 was similar between the pooled dosage arms of 001 and 002 (32.3% versus 28.4%, P value not significant). Other reasons for treatment discontinuation are listed in Table 3.
Table 3. Early Treatment Discontinuation: ITT Population
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Patients, n (%)
--------------------------------------------------------------------------------
CIFN 9 g/day + RBV (n = 245) CIFN 15 g/day + RBV (n = 242) Total (N = 487)
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Treatment failure 127 (51.8) 107 (44.2) 234 (48.0)
<2-log10 reduction in HCV RNA at week 24 96 (39.2) 64 (26.4) 160 (32.9)
Detectable HCV RNA at week 48 31 (12.7) 43 (17.8) 74 (15.2)
Adverse events 35 (14.3) 51 (21.1) 86 (17.7)
Withdrawal of consent 20 (8.2) 18 (7.4) 38 (7.8)
Decision by principal investigator or sponsor 3 (1.2) 9 (3.7) 12 (2.5)
Lost to follow-up 9 (3.7) 9 (3.7) 18 (3.7)
Other 15 (6.1) 7 (2.9) 2 (4.5)
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Table 4 summarizes the most common AEs experienced by patients in the pooled 001 and 002 arms of the DIRECT trial. Most AEs were grade 2 or 3 and were more commonly related to administration of both CIFN and RBV than either drug alone. Most patients experienced at least one AE in the study. Individual AEs resulting from treatment with CIFN were typical of those reported with IFN-based therapy. All AEs were more common in the CIFN 9 and 15 g groups than in the no-treatment group. RBV-induced hemolytic anemia occurred in 6.4% of patients. In general, most AEs were either not drug-related based on the opinion of the study site principal investigator or were thought to be related to the combination of study drugs rather than to either CIFN or RBV alone. The most common AEs leading to dose modifications in both CIFN treatment groups included neutropenia, fatigue, leukopenia, depression, nausea, myalgia, lymphopenia, and anemia. Overall, discontinuations for AEs occurred in 14% of the 9 g group and 21% of the 15 g group in the pooled ITT analysis.
Table 4. Adverse Events Reactions Occurring Regardless of Attribution
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Body System/Preferred Term (MedDRA) Retreatment*
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CIFN 9 g/day for 48 weeks (n = 244) CIFN 15 g/day for 48 weeks (n = 242)
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Blood and lymphatic disorders
Anemia 13 12
Hemolytic anemia 16 19
Leukopenia 24 34
Lymphopenia 7 14
Neutropenia 36 44
Thrombocytopenia 3 5
Eye disorders
Vision blurred 8 7
Gastrointestinal disorders
Abdominal pain 7 7
Abdominal pain upper 8 7
Constipation 9 10
Diarrhea 18 19
Dry mouth (saliva decreased) 3 5
Dyspepsia 6 7
Nausea 45 45
Vomiting 12 19
General disorders and administration site conditions (or body as a whole)
Asthenia 6 9
Fatigue 75 77
Influenza-like illness (or symptoms) 40 42
Injection site erythema 16 16
Injection site pain 3 5
Injection site reaction 15 12
Injection site rash 2 5
Pain (or body pain) 5 6
Pyrexia (or fever) 13 17
Rigors 19 22
Infections
Sinusitis 7 6
Upper respiratory tract infection 5 6
Investigations
Blood ALT increased 4 6
Blood AST increased 6 10
Blood phosphorus decreased 7 5
Blood uric acid increased 3 5
Lymphocyte count decreased 3 5
Neutrophil count decreased 5 4
WBC count decreased 6 5
Weight decrease 16 22
Metabolism and nutrition disorders
Anorexia 15 21
Decreased appetite 17 18
Hyperglycemia 3 7
Hypertriglyceridemia 7 7
Hyperuricemia 8 10
Musculoskeletal and connective tissue disorders
Arthralgia 31 31
Back pain 12 9
Muscle cramp 5 6
Myalgia 24 34
Nervous system disorders
Dizziness 14 19
Dysgeusia 6 95
Headache 46 39
Memory impairment 5 6
Syncope 2 5
Psychiatric disorder
Agitation 6 2
Anxiety 12 11
State of confusion 4 5
Depression 27 25
Insomnia 39 38
Irritability 21 17
Respiratory, thoracic, and mediastinal disorders
Cough 14 17
Dyspnea 15 20
Dyspnea exertional 10 9
Epistaxis 1 5
Pharyngolaryngeal pain 5 6
Skin and subcutaneous tissue disorders
Alopecia 10 10
Dry skin 9 8
Hyperhidrosis (or sweating increased) 2 5
Pruritus 15 11
Rash 17 12
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Only events that occurred at a frequency of 5% in any treatment group of both IRHC-001 and IRHC-002 studies combined are included. Patients can appear more than once in Table 5. All values are percentages.
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; WBC, white blood cell.
* Adverse events reported in patients during treatment or posttreatment observation are listed regardless of attribution to treatment.
Discussion
Retreatment of PEG-IFN/RBV nonresponders with daily CIFN/RBV resulted in an SVR rate of 6.9% with 9 g/day CIFN and 10.7% with 15 g/day CIFN. Patients whose doses were not reduced achieved SVR rates of 7% in the 9 g group and 17% in the 15 g group. These findings are consistent with a previous clinical trial demonstrating encouraging SVR rates with this higher dose of CIFN, 15 g/day.[14] The best response rate, 31.6%, was observed in noncirrhotic patients (F0-F3) who had a partial virologic response with a >2-log10 decline in HCV RNA during their previous course of PEG-IFN treatment.
These results were achieved even though the patients in the DIRECT trial had numerous poor prognostic factors for a successful response. Approximately 95% had HCV genotype 1, about 20% were African American, 68% had a high baseline HCV RNA level of >850,000 IU/mL, and almost 90% had a baseline HCV RNA level of >400,000 IU/mL. Approximately 80% had a <2-log10 decline in HCV RNA during prior treatment. Sixty percent of patients had advanced liver disease, including cirrhosis (25%) and bridging fibrosis (35%), and 52% had steatosis on biopsy. All of these factors have been shown to significantly reduce rates of SVR.
Overall, the alternative strategies for improving SVR in PEG-IFN/RBV nonresponders have not met with success. Two trials of maintenance IFN therapy were evaluated in PEG-IFN/RBV nonresponders with advanced fibrosis or cirrhosis to determine if this strategy can reduce progression to cirrhosis, complications of cirrhosis, hepatocellular carcinoma, the need for liver transplantation, and death.[8][15] The Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis trial demonstrated that maintenance PEG-IFN alfa-2a therapy at a dose of 90 g/week over 3.5 years provided no overall benefit compared with no treatment.[8] Similar results were observed in the Colchicine versus PEG-Intron Long Term study, which compared PEG-IFN alfa-2b 0.5 g/kg/week to colchicine over 3.5 years.[15]
In the recently completed REtreatment with PEgasys in PATients Not Responding to Peg-Intron Therapy (REPEAT) trial, nonresponders and relapsers to previous PEG-IFN alfa-2b and RBV were retreated with either a standard dose of PEG-IFN alfa-2a 180 g/week or a higher dose of 360 g/week for 12 weeks, after which the dose was reduced to the standard dose.[16] Patients who became HCV RNA undetectable by week 24 were treated for either 48 or 72 weeks. In a protocol-defined primary analysis, SVR rates after retreatment with PEG-IFN alfa-2a and RBV were only 7% to 9% with 48 weeks of treatment but increased to 14% to 16% in those patients treated for 72 weeks. This increase in SVR resulted from a decline in relapse with the prolonged course of treatment. The use of the higher induction dose of PEG-IFN alfa-2a, 360 g/week, did not impact SVR rates. The SVR results of this study, using 72 weeks of PEG-IFN alfa-2a are comparable with those achieved with 48 weeks of treatment with CIFN/RBV in the DIRECT trial. Several differences exist between the REPEAT and DIRECT trials that confound direct comparison of the results. It is not known what proportion of patients in the REPEAT trial were treatment-compliant, and the number of relapse patients included in the trial is not clear.[17] Furthermore, the patients enrolled in the DIRECT trial had more advanced liver disease than those in REPEAT (60% versus 27% with stage F3-F4) and contained a higher percentage of African American patients (20% versus 10%).
Recent studies of CIFN and RBV have demonstrated a favorable response in the retreatment of PEG-IFN/RBV nonresponders.[14][18][19] Two open-label trials demonstrated SVR rates ranging from 10% to 37%, with varying CIFN regimens.[17][19] In a third study, 137 consecutive patients who did not become HCV RNA-undetectable during treatment with PEG-IFN alfa-2b with RBV were switched to CIFN 15 g/day for 12 weeks, followed by CIFN three times weekly for an additional 36 weeks with weight-based doses of RBV.[14] SVR rates were noted in 37% of patients who remained on their full doses of therapy. The SVR rate was 27% in African Americans and 41% in Caucasian patients.
Several other new and promising therapies are under development for the treatment of chronic hepatitis C. These include RBV-like molecules, polymerase and protease inhibitors, and novel IFN formulations.[7][20] Two protease inhibitors, Boceprevir and Telaprevir, are the furthest along in development. Phase III studies are in the midst of enrollment, and these agents may gain U.S. Food and Drug Administration approval in 2 to 3 years. In addition, it has already been demonstrated that both protease and polymerase inhibitors will require the use of both IFN and RBV to achieve an SVR in treatment-naïve or treatment-experienced patients.
In conclusion, the current study shows the benefit CIFN holds for difficult-to-treat patients with chronic hepatitis C who have failed to respond to previous treatment with PEG-IFN and RBV. The present study demonstrated that some patients with chronic hepatitis C who have failed to respond to treatment with PEG-IFN and RBV can be successfully retreated with daily CIFN and RBV. The greatest SVR rate during retreatment in the present study was observed in F0-F3 patients who had a partial virologic response during their prior course of treatment. Therefore, once-daily CIFN in combination with RBV can be considered for select patients with chronic HCV who have failed to respond to prior treatment with PEG-IFN and RBV.
Acknowledgements
The authors would like to thank the DIRECT-001 and DIRECT-002 study investigators (Bashar Aqel, Sanjeev Arora, Carl Berg, David Bernstein, Edmund Bini, Scott Cotler, Michael Fallon, Kenneth Flora, Steven-Huy Bui Han, Joanne Imperial, Mark Jonas, Gerond Lake-Bakaar, Anne Larson, Eric Lawitz, Edward Lebovics, Michael Lyons, Gary Matusow, Douglas Meyer, Abdul Nadir, Robert Reindollar, Adrian Reuben, Maribel Rodriguez-Torres, Vinod Rustgi, Michael Ryan, John Santoro, Muhammad Sheikh, Robert Sjogren, Mark Swaim, Helen Te, Tram Tran, Andrzej Triebling, Harlan Wright, and Nizar Zein) for their participation in the studies. Dr. John Kincaid is also acknowledged for his assistance in the preparation of the first draft of the manuscript. All subsequent revisions of this manuscript were made by Bruce R. Bacon and Mitchell L. Shiffman with some input from the authors on the masthead.
REFERENCES
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2 Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 2001; 358: 958-965. Links
3 Fartoux L, Degos F, Trepo C, Goria O, Cales P, Tran A, et al. Effect of prolonged interferon therapy on the outcome of hepatitis C virus-related cirrhosis: a randomized trial. Clin Gastroenterol Hepatol 2007; 5: 502-507. Links
4 Helbling B, Jochum W, Stamenic I, Knopfli M, Cerny A, Borovicka J, et al. HCV-related advanced fibrosis/cirrhosis: randomized controlled trial of pegylated interferon alpha-2a and ribavirin. J Viral Hepat 2006; 13: 762-769. Links
5 Kobayashi S, Takeda T, Enomoto M, Tamori A, Kawada N, Habu D, et al. Development of hepatocellular carcinoma in patients with chronic hepatitis C who had a sustained virological response to interferon therapy: a multicenter, retrospective cohort study of 1124 patients. Liver Int 2007;27: 186-191. Links
6 Shiffman ML. Chronic hepatitis C: treatment of pegylated interferon/ribavirin nonresponders. Curr Gastroenterol Rep 2006; 8: 46-52. Links
7 Keeffe EB. Future treatment of chronic hepatitis C. Antivir Ther 2007; 12: 1015-1025. Links
8 DiBisceglie A., Shiffman ML, Everson GT, Lindsay KL, Everhart JE, Wright EC, et al. Prolonged therapy of advanced chronic hepatitis C with low-dose peginterferon. N Engl J Med 2008; 359: 2429-2441. Links
9 Jensen DB, Freilich B, Andreone P, DiBisceglie A, Brandão-Mello CE, Reddy KR, et al. Pegylated interferon alfa-2a (40kD) plus ribavirin (RBV) in prior non-responders to pegylated interferon alfa-2b (12kD)/RBV: final efficacy and safety outcomes of the REPEAT study [abstract LB4].HEPATOLOGY 2007; 46(Suppl): 291A-292A. Links
10 Kaiser S, Hass H, Gregor M. Successful retreatment of peginterferon nonresponder patients with chronic hepatitis C with high dose consensus interferon induction therapy [Abstract 125]. Gastroenterology 2004; 126(Suppl 2): A-668. Links
11 Kaiser S, Hass HG, Bissinger L, Gregor M. Comparison of daily consensus interferon versus peginterferon alfa 2a extended therapy of 72 weeks for peginterferon/ribavirin relapse patients with chronic hepatitis C [Abstract S1060]. Gastroenterology 2006; 130(Suppl 2): A-784. Links
12 Rustgi VK, Esposito S, Hamzeh FM, Shiffman ML. Peginterferon alfa-2a/ribavirin in hepatitis C virus patients nontolerant or nonresponsive to peginterferon alfa-2b/ribavirin. Aliment Pharmacol Ther 2008; 27: 433-440. Links
13 Hassanein T, Ghalib R, Zein N, Rothstein KD, Joshi SN, Kwo PY, et al. Analysis of relapse rates in pegylated interferon and ribavirin non-responders treated with daily consensus interferon and ribavirin [Abstract 1319]. HEPATOLOGY 2007; 46: A1319. Links
14 Leevy CB. Consensus interferon and ribavirin in patients with chronic hepatitis C who were nonresponders to pegylated interferon alfa-2b and ribavirin. Dig Dis Sci 2008; 53: 1961-1966. Links
15 Afdhal NH, Levine R, Brown RJ, Freilich B, O'Brien M, Brass C. Colchicine versus peg-interferon alfa 2b long term therapy: results of the 4 year COPILOT trial [Abstract 3]. J Hepatol 2008; 48(Suppl): S4. Links
16 Jensen D, Di Bisceglie A, Gitlin N, Freilich B, Reddy K, Feinman V, et al. Peginterferon alfa-2a (40KD) plus ribavirin (RBV) in pegylated interferon alfa-2b (12KD)/ribavirin non-responders: week 12 efficacy and safety outcomes of the REPEAT study. Gastroenterology 2006; 130; T1800. Links
17 Jensen D, Di Bisceglie A, Gitlin N, Freilich B, Reddy K, Feinman V, et al. Type of response to prior pegylated interferon alfa-2b (12kd)/rbv predicts subsequent response to retreatment with peginterferon alfa-2a (40KD)/RBV. Gastroenterology 2008; 134: S1937. Links
18 Kaiser S, Boecher W, Lutze B, Sauter B, Bissinger L, Werner C, et al. Treatment of peginterferon/ribavirin nonresponders with daily dosing of consensus interferon and ribavirin: preliminary results of the German Consensus Interferon Multicenter Study [Abstract 1306]. HEPATOLOGY2007; 46 Suppl:817A. Links
19 Ghalib RH, Levine CD, Friedman DA, Rashdan S, Schwartz AG, Weinstein J. Consensus interferon plus ribavirin therapy in patients who are nonresponders or relapsers to prior PEG IFN plus ribavirin. Gastroenterology 2007; 46: M1874. Links
20 Harrison SA. Small molecules and novel treatment for chronic hepatitis C virus infection. Am J Gastroenterol 2007; 102: 2332-2338.
Retreating chronic hepatitis C with daily interferon alfacon-1/ribavirin after nonresponse to pegylated interferon/ribavirin:
DIRECT results
Bruce R. Bacon 1 *, Mitchell L. Shiffman 2, Flavia Mendes 3, Reem Ghalib 4, Tarek Hassanein 5, Giuseppe Morelli 6, Shobha Joshi 7, Kenneth Rothstein 8, Paul Kwo 9, Norman Gitlin 10
1Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO
2Hepatology Section, Virginia Commonwealth University Medical Center, Richmond, VA
3Division of Hepatology, University of Miami Miller School of Medicine, Miami, FL
4Liver Institute at Methodist Hospital, Dallas, TX
5Division of Gastroenterology and Hepatology, University of California San Diego, San Diego, CA
6Division of Gastroenterology and Hepatology, University of Florida, Gainesville, FL
7Division of Gastroenterology and Hepatology, Tulane University Hospital and Clinic, New Orleans, LA
8Albert Einstein Center for Liver Disease, Philadelphia, PA
9Division of Gastroenterology and Hepatology, Indiana University School of Medicine, Indianapolis IN
10Division of Gastroenterology and Hepatology, Emory Crawford Long Hospital, Atlanta, GA
email: Bruce R. Bacon (baconbr@slu.edu)
*Correspondence to Bruce R. Bacon, Division of Gastroenterology and Hepatology, Saint Louis University School of Medicine, 3635 Vista Avenue at Grand Boulevard, St. Louis, MO 63110-0250
Potential conflict of interest: Nothing to report.
fax: 314-577-8125
Funded by:
InterMune, Inc.
Valeant Pharmaceuticals International
Three Rivers Pharmaceuticals
ABSTRACT
Up to 50% of patients with chronic hepatitis C fail to respond to initial therapy with pegylated interferon (PEG-IFN) and ribavirin (RBV). With unsuccessful viral eradication, these patients remain at risk for developing progression of their liver disease. Retreatment with PEG-IFN/RBV yields sustained virologic response (SVR) rates that are under 10%. A wholly synthetic interferon, interferon alfacon-1 or consensus interferon (CIFN) given with RBV, was evaluated in patients who failed initial PEG-IFN/RBV therapy. The intent-to-treat analysis included 487 patients; 245 received CIFN 9 g/day and RBV, and 242 received CIFN 15 g/day and RBV. Within this group of patients, 59.3% had documented advanced fibrosis at baseline liver biopsy (stage F3 or F4). SVR rates were 6.9% (17/245 patients) in the 9 g group and 10.7% (26/242) in the 15 g group. In the intent-to-treat analysis, SVR rates were higher among patients with a >2-log10 decrease in hepatitis C virus RNA during prior PEG-IFN/RBV therapy: 11% (4/38) in the 9 g group and 23% (7/31) in the 15 g group. Among patients with lower baseline fibrosis scores (F0-F3), SVR rates were 7.8% (15/192) in the 9g group and 13.1% (23/175) in the 15 g group. In this same group of patients (F0-F3), if a >2-log10 decrease in hepatitis C virus RNA with previous PEG-IFN/RBV treatment was achieved, SVR rates improved to 10.7% and 31.6% in the 9 g and 15 g groups, respectively. CIFN/RBV combination retreatment was safe and well tolerated. Conclusion: Retreatment of PEG-IFN and RBV nonresponders with CIFN and RBV is safe and efficacious and can be considered a retreatment strategy for patients failing previous therapy with PEG-IFN/RBV, especially in interferon-sensitive patients with lower baseline fibrosis scores. (HEPATOLOGY 2009.)
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Received: 21 August 2008; Accepted: 24 January 2009
DIGITAL OBJECT IDENTIFIER (DOI)
10.1002/hep.22871 About DOI
ARTICLE TEXT
Since 2001, the standard of care for patients with chronic hepatitis C has been the combination of pegylated interferon (PEG-IFN) and ribavirin (RBV).[1][2] This combination has produced sustained virologic response (SVR) rates of 50%-60% in patients infected with hepatitis C virus (HCV) genotype 1 who adhere to their therapeutic regimens and 40% in intention-to-treat populations.[1][2] However, because only about 65% of patients become HCV RNA-undetectable when treated with this regimen, more than one-third of all patients are classified as nonresponders. Some of these patients have relatively mild liver disease but may have symptoms of HCV viremia, while other patients have advanced fibrosis and are at risk for developing complications of chronic liver disease, including decompensated cirrhosis and hepatocellular carcinoma, and may require liver transplantation.[3-5]
The optimal approach to PEG-IFN/RBV nonresponders has not been well defined. Some clinicians have used the watchful waitingapproach[6] and are anticipating new antiviral therapies with either protease inhibitors or polymerase inhibitors. However, it remains to be determined just how effective these new agents will be when combined with PEG-IFN and RBV in the retreatment of PEG-IFN/RBV nonresponders.[7]
Alternative therapies have included retreatment with the alternative brand of PEG-IFN not used in the initial therapy, although most results with this approach have been disappointing. Other approaches have included prolonged treatment with PEG-IFN, maintenance therapy, or the use of higher dosages of either PEG-IFN and/or RBV.[8-12] The strategy studied in the current investigation included high doses of daily consensus interferon (CIFN) (Infergen; interferon alfacon-1) 9 or 15 g/day given with RBV.
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Abbreviations
AE, adverse event; bDNA, branched DNA; CIFN, consensus interferon; DIRECT, Daily-Dose Consensus Interferon and Ribavirin: Efficacy of Combined Therapy; Hb, hemoglobin; HCV, hepatitis C virus; ITT, intention to treat; PEG-IFN, pegylated interferon; RBV, ribavirin; REPEAT, REtreatment with PEgasys in PATients Not Responding to Peg-Intron Therapy; SVR, sustained virologic response; TMA, transcription-mediated amplification.
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Patients and Methods
This study, referred to as the DIRECT (Daily-Dose Consensus Interferon and Ribavirin: Efficacy of Combined Therapy) trial, was designed by the sponsor and by several of the academic investigators. The data were managed by the sponsor and the academic investigators. The sponsor performed the statistical analysis. The academic investigators were responsible for the development of the final manuscript and had unrestricted access to the data. An author involved with the design or execution of this study either wrote or edited every section of the manuscript. Both an academic author (B. R. B.) and an industry representative (Michael Beckloff, Three Rivers Pharmaceuticals, Cranberry Township, PA) attest to the completeness and accuracy of the data.
Study Design.
This was a phase 3, randomized, open-label, multicenter, U.S.-based registration trial conducted to investigate the efficacy, tolerability, and safety of daily CIFN at dosages of 9 and 15 g/day (interferon alfacon-1, Infergen; Three Rivers Pharmaceuticals, LLC, Cranberry Township, PA) administered with daily RBV (Ribasphere, Three Rivers Pharmaceuticals, LLC) compared with no treatment in patients who did not respond to prior therapy with either PEG-IFN alfa-2a or alfa-2b and RBV. The trial was divided into 2 sections: DIRECT-001 and DIRECT-002 (Fig. 1).
Figure 1. DIRECT study design: a randomized, open-label study of CIFN and RBV in patients who did not respond to previous combination therapy with PEG-IFN and ribavirin.
[Normal View 33K | Magnified View 66K]
Patients were randomized at a 1:1:1 ratio into three study groups: CIFN 9 g/day (group 1), 15 g/day (group 2) plus oral RBV 1,000-1,200 mg/day (based on body weight), or a control, no-treatment group (group 3). The no-treatment group was mandated by the U.S. Food and Drug Administration in order to provide a comparison of safety for the two treatment groups. It was not anticipated that any of the patients randomized to the control group would have a spontaneous response. After 24 weeks of observation, all patients in the control group of DIRECT-001 were offered randomization into DIRECT-002 to receive CIFN 9 or 15 g/day plus RBV.
At week 24, patients who had undetectable plasma HCV RNA by branched DNA (bDNA) assay, confirmed by transcription-mediated amplification (TMA) assay, or who had a 2-log10 decrease from baseline in HCV RNA were assigned to continue therapy to week 48. Patients with a <2-log10 decrease from baseline in plasma HCV RNA (bDNA assay) were considered nonresponders and were withdrawn from treatment. At week 48, patients with undetectable plasma HCV RNA (by bDNA and TMA assays) were assigned to return for regular visits in the follow-up period (weeks 52, 60, 68, and 72) until 24 weeks after their last dose of study drug (week 72). Patients with detectable plasma HCV RNA (bDNA or TMA assay) at any time between weeks 48 and 72 were classified as relapsers.
All patients who discontinued therapy early at any time were instructed to return for a single follow-up visit 30 days after their last dose of study drug to complete early termination/discontinuation assessments. Patients who had undetectable plasma HCV RNA by bDNA and TMA assays at the time of stopping therapy or at the early termination/discontinuation visit were to return for follow-up plasma HCV RNA assessments through week 72, as long as their plasma HCV RNA levels remained undetectable by both bDNA and TMA assays. Patients who discontinued for nonresponse at weeks 12 or 24 did not undergo a follow-up HCV RNA measurement.
Patients in the no-treatment group in DIRECT-001 were given the option to enroll in DIRECT-002 under the following conditions: if they achieved a <2-log10 decrease in plasma HCV RNA at week 24 compared with baseline or if they had detectable plasma HCV RNA by bDNA or by TMA at week 48. These patients were then treated according to the same protocol followed in DIRECT-001. The results of DIRECT-001 and DIRECT-002 were pooled for the purpose of this analysis.
Patients.
Men and women were eligible for enrollment if they were chronically infected with HCV of any genotype. Chronic infection was identified based on a history of being positive for serum anti-HCV and/or HCV RNA. A liver biopsy performed within 3 years of screening must have demonstrated evidence of chronic HCV infection. Hepatic fibrosis was interpreted by local pathologists based upon the Metavir scoring system. Patients with advanced liver disease, including bridging fibrosis (F3) and cirrhosis (F4), were eligible for the study as long as they had normal liver function as evidenced by serum albumin >3.5 mg/dL, platelet count >75,000/mm3, and no prior episode of hepatic decompensation (variceal hemorrhage, hepatic encephalopathy, ascites, or hepatocellular carcinoma).
Prior nonresponse and adherence after initial therapy with PEG-IFN alfa-2a (180 g/week) or PEG-IFN alfa-2b (1.5 g/kg/week) plus RBV was determined via careful chart review completed by the study site principal investigator and confirmed by an external study monitor. Nonresponders had to have had a <2-log10 decrease in HCV RNA between weeks 12 and 24 or detectable HCV RNA at weeks 24 or 48. Patients were required to have had completed a minimum of 90 days between discontinuation of their prior regimen and the start of the current study medication. All patients had to have received at least 80% of the cumulative standard dosages of PEG-IFN and RBV for at least 38 weeks (80% of the planned treatment duration). Patients were excluded if this previous treatment was prematurely discontinued, dosing was interrupted, or if the dose of PEG-IFN was reduced because of noncompliance, safety, or tolerability issues (including hematologic or psychiatric side effects).
Patients were also excluded if they were pregnant or lactating women or male partners of pregnant women, or if they were not suitable candidates for enrollment or unlikely to comply with the requirements of the study in the opinion of the investigator or sponsor.
Treatment and Assessments.
Screening took place between 8 weeks and 1 day before the first day of treatment. After providing informed consent, patients were screened for inclusion criteria, underwent a physical examination, provided a baseline medical history, and had blood drawn for laboratory testing. After screening, eligible patients were randomized in a 1:1:1 ratio to receive CIFN 9 or 15 g/day plus RBV 1,000 mg/day (body weight <75 kg) or 1,200 mg/day (body weight >75 kg) or no treatment (Fig. 1). RBV was provided as capsules containing 200 mg of active drug. An independent data monitoring committee conducted regular interim safety assessments throughout the study.
Plasma HCV RNA levels were determined first using the bDNA quantitative assay, which has a sensitivity of detection of 615 IU/mL and a reportable range of 615 to 6,920,000 IU/mL. The Bayer TMA assay, with a sensitivity of detection of 5 IU/mL, was used whenever HCV RNA levels were undetectable via bDNA assay.
Patients developing anemia, defined as hemoglobin (Hb) <10 g/dL, were managed by reducing the dose of RBV to 600 mg/day. The use of growth factors was not permitted. If the Hb increased to >10 g/dL, the RBV dose could be increased in 200-mg/day increments as tolerated according to the discretion of the site principal investigator. RBV dose was not increased after being reduced to 600 mg/day for patients with a history of cardiovascular disease whose Hb decreased by 2 g/dL or more during any 4-week period. RBV was permanently discontinued in patients whose Hb dropped below 8.5 g/dL. In those patients with a history of cardiac or cerebrovascular disease, Hb remaining below 12 g/dL after 4 weeks on a reduced dose required permanent discontinuation of RBV. Neutropenia was managed by CIFN dose reduction; in patients whose absolute neutrophil count fell to <0.75 × 109/L, starting doses of 15 g were lowered to 9 g and then to 6 g, and starting doses of 9 g were lowered to 6 g.
Efficacy Variables.
The primary efficacy variable was the proportion of patients with SVR, defined as undetectable plasma HCV RNA by both bDNA and TMA assays at 24 weeks after the last dose of study drug. In addition, SVR was further explored for the effect of race, genotype, sex, age, baseline HCV RNA, presence/absence of cirrhosis, body weight, and previous response to PEG-IFN/RBV.
Safety and Tolerability.
All adverse events (AEs) and serious AEs were recorded for patients who received at least 1 dose of study medication (active-treatment groups) or who completed baseline assessments (no-treatment group). AEs were recorded until either 30 days after the last dose of study medication (active-treatment groups) or until the last study visit (no-treatment group). AEs were graded from 1 to 5 (1, mild; 2, moderate; 3, severe; 4, life-threatening or disabling; 5, death) based on the Common Toxicity Criteria for Adverse Events v3.0. An AE was considered a serious AE if it resulted in death, was life-threatening, required inpatient hospitalization, or resulted in persistent or significant disability or incapacity.
Statistical Methods.
At least 170 patients were needed in each of the three study groups (for a total of 510 patients) to provide an approximately 91% power to detect a difference in SVR between each of the active treatment groups and the no-treatment group. This analysis was performed using a two-sided Fisher's exact test at = 0.05 significance level, with adjustment for multiple comparisons, and assumed an SVR rate of 10% for either of the active treatment groups and 1.2% for the no-treatment group. The study was not powered to detect differences between the 9 g and the 15 g arms.
Data were summarized and analyzed for two patient populations: the intention-to-treat (ITT) population and patients who did not receive any dose modifications. The ITT group consisted of all patients who were randomized to receive CIFN in DIRECT-001, as well as all patients from the no-treatment group in DIRECT-001 who went on to receive at least 1 dose of CIFN in DIRECT-002. Data from the ITT population were used in all efficacy and safety analyses.
Descriptive statistics were determined for continuous variables (patient counts, mean, standard deviation, median, minimum, and maximum) and categorical variables (number and percentage of patients for each category). Percentages were calculated using the number of patients without missing data as the denominator unless otherwise indicated. Calculations of virologic response (both sustained and at specific visits) used the number of ITT patients as the denominator. All statistical testing was conducted at the 0.05 level of significance using SAS software, version 8.2.
Results
Patients and Disposition.
Five hundred fifteen patients were randomized at 44 sites in the United States and Puerto Rico to receive CIFN 9 g/day plus RBV 1,000 or 1,200 mg/day (n = 171), CIFN 15 g/day plus RBV (n = 172), or no treatment (n = 172). Of the 172 patients in the no-treatment group in DIRECT-001, 144 continued on to DIRECT-002. Of these, 74 received CIFN 9 g/day plus RBV 1,000 or 1,200 mg/day, and 70 received CIFN 15 g/day plus RBV 1,000 or 1,200 mg/day. The final ITT population included 487 patients (245 who received CIFN 9 g/day and 242 who received CIFN 15 g/day).
Baseline demographic and clinical characteristics of the two CIFN treatment groups are presented in Table 1. The majority of patients were male (70%) and Caucasian (64%). Of the enrolled patients, 59.3% had advanced liver disease on biopsy, including bridging fibrosis (F3; 35%) or cirrhosis (F4; 25%). In addition, 52% of patients had hepatic steatosis. The average time between biopsy sampling and study day 1 was 1.6 years. Patients included in the DIRECT trial were required to be off PEG-IFN/RBV therapy for at least 3 months prior to starting CIFN therapy. The median washout period between previous treatment and day 1 of CIFN therapy was 448 days (15 months) and 506 days (16.8 months) for the 9 g and 15 g groups, respectively. Sixty-eight percent of the patients had high baseline HCV RNA levels of >850,000 IU/mL. The majority of patients (79%) failed to achieve an early virologic response (at least a 2-log10 drop in HCV RNA from the pretreatment baseline) to previous PEG-IFN therapy.
Table 1. DIRECT-001 and DIRECT-002: Baseline Characteristics
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CIFN 9 g/day + RBV (n = 245) CIFN 15 g/day + RBV (n = 242)
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Mean age ± SD, years 51 ± 6.65 50 ± 6.59
Male 68% 72%
HCV genotype 1 95% 96%
High viral load
850,000 IU/mL 68% 68%
Mean weight ± SD, kg 89.1 ± 18.64 89.4 ± 17.59
Mean body mass index ± SD, kg/m2 29.3 ± 5.20 29.6 ± 5.02
Race
Caucasian 64% 65%
African American 21% 17%
Liver biopsy results
Cirrhosis (F4) 22% 28%
Bridging fibrosis (F3) 36% 34%
(F0-F2) 42% 38%
Steatosis* 52% 51%
Response to prior therapy
<2-log10 drop 78% 80%
>2-log10 drop 15% 12%
Unknown 7% 8%
Washout interval, days
Mean ± SD 453 ± 345 594 ± 372
Median 448 506
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Abbreviation: SD, standard deviation.
* Steatosis defined as present or absent.
Antiviral Efficacy.
By ITT analysis, pooled end-of-treatment response via TMA assay in the pooled 9 g arm was 14.7% (36/245), with a subsequent SVR of 6.9% (17/245). In the 15 g arm, pooled end-of-treatment response via TMA assay was 18.5% (45/242), with an SVR rate of 10.7% (26/242). Relapse rates pooled for both arms were 52% (19/36) and 42% (19/45) for the 9 g and 15 g groups, respectively. Post hoc analysis revealed steatosis and time to viral negativity had the most impact on relapse rates.[13] As expected, in DIRECT 001, patients in the no-treatment arm achieved a 0% SVR. In patients who did not have dose modifications, overall SVR rates were 7% in the CIFN 9 g group and 17% in the 15 g group (Fig. 2A,B). The SVR rates were not significantly different between the 001 and 002 arms (P = 0.818). Although the study was not powered to detect differences between the 9 g and 15 g groups, a post hoc analysis revealed no difference in SVR rates between the two (P = 0.141, 95% CI -8.8%-1.2%).
Figure 2. Rates of SVR, defined as undetectable viral levels at least 24 weeks after the end of treatment with CIFN and RBV, in (A) the ITT population (n = 487) and (B) patients who did not receive dose modifications (n = 281). Active versus no treatment (n = 172).
[Normal View 22K | Magnified View 42K]
Patients who achieved a complete early virologic response (defined as viral negativity at week 12 via TMA assay) were more likely to demonstrate an SVR than the general study population. In the 9 g group, 81.3% (13/16) of patients with complete early virologic response achieved SVR, whereas in the 15 g group, 63.6% (14/22) of patients with complete early virologic response demonstrated SVR. In patients deemed slow responders (>2-log drop at week 12, viral-negative at week 24), SVR rates were 11.7% (2/17) and 35.4% (11/31) in the 9 g and 15 g groups, respectively. Two patients in the 9 g arm and one patient in the 15 g arm achieved SVR despite being viral-positive at week 24.
Patients achieving the greatest log reduction in terms of viral response to initial PEG-IFN/RBV therapy had the best likelihood of responding to retreatment with CIFN and RBV (Fig. 3A,B). Among F0-F2 patients with >2-log10 decreases in HCV RNA during their prior PEG-IFN/RBV therapy, SVR rates were 13.3% (2/15) and 30.0% (3/10) in the 9 g and 15 g groups, respectively. A similar trend was seen in the 15 g arm in patients with bridging fibrosis (F3). SVR for the F0-F2 group of patients was 8.7% (9/104) in the 9g group and 14.9% (14/94) in the 15 g group. With patients displaying bridging fibrosis only on biopsy, overall SVR was 6.8% (6/88) and 11.1% (9/81) in the 9 and 15 g groups, respectively. Complete SVR for noncirrhotics (F0-F3) was 7.8% (15/192) in the 9 g group versus 13.1% (23/175) in the 15 g group, whereas cirrhotics achieved SVR rates of 3.8% (2/53) and 4.5% (3/67) in the 9 g and 15 g groups, respectively. In the cirrhotic cohort, patients required at least a 1-log drop on prior therapy to benefit from retreatment with CIFN and RBV. African American patients achieved lower SVR rates than Caucasians (4.2% versus 11%, respectively). In this population, pooled analysis between the two dosage arms revealed that 35.4% of the patients had failed at least two or more prior treatment regimens, with 52.7% having obtained a <1-log drop on prior PEG-IFN/RBV therapy. In addition to this, 60.2% had dose reductions while on CIFN/RBV therapy. Finally, non-genotype 1 patients (genotype 2/3) achieved an overall SVR rate of 23.1% (3/13) and 88.8% (8/9) in the 9 g and 15 g groups, respectively. Further univariate predictors of response are discussed in Table 2.
Figure 3. Rates of SVR, defined as undetectable viral levels at 24 weeks after the end of treatment, in the ITT analysis by known previous response to therapy among patients without F0-F2 fibrosis, F3, and with cirrhosis (F4) in the (A) 9 g and (B) 15 g treatment groups. One patient in the 9 g group and two patients in the 15 g group achieving SVR had unknown prior response to PEG-IFN/RBV therapy.
[Normal View 25K | Magnified View 52K]
Table 2. Univariate Predictors of SVR
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n (% SVR) P Value
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Treatment group
9 g/day (n = 245) 17 (6.9) NS
15 g/day (n = 242) 26 (10.7)
Sex
Male (n = 342) 30 (8.8) NS
Female (n = 145) 13 (9.0)
Fibrosis scores
F0-F2 (n = 198) 23 (11.6) NS
F3-F4 (n = 289) 20 (6.9)
Genotype
1 (n = 464) 32 (6.9) <0.001
Non-genotype 1 (n = 23) 11 (47.8)
Viral load
<850,000 IU/mL (n = 155) 25 (16.1) <0.001
>850,000 IU/mL (n = 331) 18 (5.4)
--------------------------------------------------------------------------------
Abbreviation: NS, not significant.
Safety and Tolerability.
A total of 83.6% of patients in the 9 g group and 71.7% of patients in the 15 g group received at least 80% of their cumulative CIFN dose. The most common reason for early termination was treatment failure. Discontinuation due to not achieving a >2-log drop at week 24 was similar between the pooled dosage arms of 001 and 002 (32.3% versus 28.4%, P value not significant). Other reasons for treatment discontinuation are listed in Table 3.
Table 3. Early Treatment Discontinuation: ITT Population
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Patients, n (%)
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CIFN 9 g/day + RBV (n = 245) CIFN 15 g/day + RBV (n = 242) Total (N = 487)
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Treatment failure 127 (51.8) 107 (44.2) 234 (48.0)
<2-log10 reduction in HCV RNA at week 24 96 (39.2) 64 (26.4) 160 (32.9)
Detectable HCV RNA at week 48 31 (12.7) 43 (17.8) 74 (15.2)
Adverse events 35 (14.3) 51 (21.1) 86 (17.7)
Withdrawal of consent 20 (8.2) 18 (7.4) 38 (7.8)
Decision by principal investigator or sponsor 3 (1.2) 9 (3.7) 12 (2.5)
Lost to follow-up 9 (3.7) 9 (3.7) 18 (3.7)
Other 15 (6.1) 7 (2.9) 2 (4.5)
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Table 4 summarizes the most common AEs experienced by patients in the pooled 001 and 002 arms of the DIRECT trial. Most AEs were grade 2 or 3 and were more commonly related to administration of both CIFN and RBV than either drug alone. Most patients experienced at least one AE in the study. Individual AEs resulting from treatment with CIFN were typical of those reported with IFN-based therapy. All AEs were more common in the CIFN 9 and 15 g groups than in the no-treatment group. RBV-induced hemolytic anemia occurred in 6.4% of patients. In general, most AEs were either not drug-related based on the opinion of the study site principal investigator or were thought to be related to the combination of study drugs rather than to either CIFN or RBV alone. The most common AEs leading to dose modifications in both CIFN treatment groups included neutropenia, fatigue, leukopenia, depression, nausea, myalgia, lymphopenia, and anemia. Overall, discontinuations for AEs occurred in 14% of the 9 g group and 21% of the 15 g group in the pooled ITT analysis.
Table 4. Adverse Events Reactions Occurring Regardless of Attribution
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Body System/Preferred Term (MedDRA) Retreatment*
--------------------------------------------------------------------------------
CIFN 9 g/day for 48 weeks (n = 244) CIFN 15 g/day for 48 weeks (n = 242)
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Blood and lymphatic disorders
Anemia 13 12
Hemolytic anemia 16 19
Leukopenia 24 34
Lymphopenia 7 14
Neutropenia 36 44
Thrombocytopenia 3 5
Eye disorders
Vision blurred 8 7
Gastrointestinal disorders
Abdominal pain 7 7
Abdominal pain upper 8 7
Constipation 9 10
Diarrhea 18 19
Dry mouth (saliva decreased) 3 5
Dyspepsia 6 7
Nausea 45 45
Vomiting 12 19
General disorders and administration site conditions (or body as a whole)
Asthenia 6 9
Fatigue 75 77
Influenza-like illness (or symptoms) 40 42
Injection site erythema 16 16
Injection site pain 3 5
Injection site reaction 15 12
Injection site rash 2 5
Pain (or body pain) 5 6
Pyrexia (or fever) 13 17
Rigors 19 22
Infections
Sinusitis 7 6
Upper respiratory tract infection 5 6
Investigations
Blood ALT increased 4 6
Blood AST increased 6 10
Blood phosphorus decreased 7 5
Blood uric acid increased 3 5
Lymphocyte count decreased 3 5
Neutrophil count decreased 5 4
WBC count decreased 6 5
Weight decrease 16 22
Metabolism and nutrition disorders
Anorexia 15 21
Decreased appetite 17 18
Hyperglycemia 3 7
Hypertriglyceridemia 7 7
Hyperuricemia 8 10
Musculoskeletal and connective tissue disorders
Arthralgia 31 31
Back pain 12 9
Muscle cramp 5 6
Myalgia 24 34
Nervous system disorders
Dizziness 14 19
Dysgeusia 6 95
Headache 46 39
Memory impairment 5 6
Syncope 2 5
Psychiatric disorder
Agitation 6 2
Anxiety 12 11
State of confusion 4 5
Depression 27 25
Insomnia 39 38
Irritability 21 17
Respiratory, thoracic, and mediastinal disorders
Cough 14 17
Dyspnea 15 20
Dyspnea exertional 10 9
Epistaxis 1 5
Pharyngolaryngeal pain 5 6
Skin and subcutaneous tissue disorders
Alopecia 10 10
Dry skin 9 8
Hyperhidrosis (or sweating increased) 2 5
Pruritus 15 11
Rash 17 12
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Only events that occurred at a frequency of 5% in any treatment group of both IRHC-001 and IRHC-002 studies combined are included. Patients can appear more than once in Table 5. All values are percentages.
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; WBC, white blood cell.
* Adverse events reported in patients during treatment or posttreatment observation are listed regardless of attribution to treatment.
Discussion
Retreatment of PEG-IFN/RBV nonresponders with daily CIFN/RBV resulted in an SVR rate of 6.9% with 9 g/day CIFN and 10.7% with 15 g/day CIFN. Patients whose doses were not reduced achieved SVR rates of 7% in the 9 g group and 17% in the 15 g group. These findings are consistent with a previous clinical trial demonstrating encouraging SVR rates with this higher dose of CIFN, 15 g/day.[14] The best response rate, 31.6%, was observed in noncirrhotic patients (F0-F3) who had a partial virologic response with a >2-log10 decline in HCV RNA during their previous course of PEG-IFN treatment.
These results were achieved even though the patients in the DIRECT trial had numerous poor prognostic factors for a successful response. Approximately 95% had HCV genotype 1, about 20% were African American, 68% had a high baseline HCV RNA level of >850,000 IU/mL, and almost 90% had a baseline HCV RNA level of >400,000 IU/mL. Approximately 80% had a <2-log10 decline in HCV RNA during prior treatment. Sixty percent of patients had advanced liver disease, including cirrhosis (25%) and bridging fibrosis (35%), and 52% had steatosis on biopsy. All of these factors have been shown to significantly reduce rates of SVR.
Overall, the alternative strategies for improving SVR in PEG-IFN/RBV nonresponders have not met with success. Two trials of maintenance IFN therapy were evaluated in PEG-IFN/RBV nonresponders with advanced fibrosis or cirrhosis to determine if this strategy can reduce progression to cirrhosis, complications of cirrhosis, hepatocellular carcinoma, the need for liver transplantation, and death.[8][15] The Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis trial demonstrated that maintenance PEG-IFN alfa-2a therapy at a dose of 90 g/week over 3.5 years provided no overall benefit compared with no treatment.[8] Similar results were observed in the Colchicine versus PEG-Intron Long Term study, which compared PEG-IFN alfa-2b 0.5 g/kg/week to colchicine over 3.5 years.[15]
In the recently completed REtreatment with PEgasys in PATients Not Responding to Peg-Intron Therapy (REPEAT) trial, nonresponders and relapsers to previous PEG-IFN alfa-2b and RBV were retreated with either a standard dose of PEG-IFN alfa-2a 180 g/week or a higher dose of 360 g/week for 12 weeks, after which the dose was reduced to the standard dose.[16] Patients who became HCV RNA undetectable by week 24 were treated for either 48 or 72 weeks. In a protocol-defined primary analysis, SVR rates after retreatment with PEG-IFN alfa-2a and RBV were only 7% to 9% with 48 weeks of treatment but increased to 14% to 16% in those patients treated for 72 weeks. This increase in SVR resulted from a decline in relapse with the prolonged course of treatment. The use of the higher induction dose of PEG-IFN alfa-2a, 360 g/week, did not impact SVR rates. The SVR results of this study, using 72 weeks of PEG-IFN alfa-2a are comparable with those achieved with 48 weeks of treatment with CIFN/RBV in the DIRECT trial. Several differences exist between the REPEAT and DIRECT trials that confound direct comparison of the results. It is not known what proportion of patients in the REPEAT trial were treatment-compliant, and the number of relapse patients included in the trial is not clear.[17] Furthermore, the patients enrolled in the DIRECT trial had more advanced liver disease than those in REPEAT (60% versus 27% with stage F3-F4) and contained a higher percentage of African American patients (20% versus 10%).
Recent studies of CIFN and RBV have demonstrated a favorable response in the retreatment of PEG-IFN/RBV nonresponders.[14][18][19] Two open-label trials demonstrated SVR rates ranging from 10% to 37%, with varying CIFN regimens.[17][19] In a third study, 137 consecutive patients who did not become HCV RNA-undetectable during treatment with PEG-IFN alfa-2b with RBV were switched to CIFN 15 g/day for 12 weeks, followed by CIFN three times weekly for an additional 36 weeks with weight-based doses of RBV.[14] SVR rates were noted in 37% of patients who remained on their full doses of therapy. The SVR rate was 27% in African Americans and 41% in Caucasian patients.
Several other new and promising therapies are under development for the treatment of chronic hepatitis C. These include RBV-like molecules, polymerase and protease inhibitors, and novel IFN formulations.[7][20] Two protease inhibitors, Boceprevir and Telaprevir, are the furthest along in development. Phase III studies are in the midst of enrollment, and these agents may gain U.S. Food and Drug Administration approval in 2 to 3 years. In addition, it has already been demonstrated that both protease and polymerase inhibitors will require the use of both IFN and RBV to achieve an SVR in treatment-naïve or treatment-experienced patients.
In conclusion, the current study shows the benefit CIFN holds for difficult-to-treat patients with chronic hepatitis C who have failed to respond to previous treatment with PEG-IFN and RBV. The present study demonstrated that some patients with chronic hepatitis C who have failed to respond to treatment with PEG-IFN and RBV can be successfully retreated with daily CIFN and RBV. The greatest SVR rate during retreatment in the present study was observed in F0-F3 patients who had a partial virologic response during their prior course of treatment. Therefore, once-daily CIFN in combination with RBV can be considered for select patients with chronic HCV who have failed to respond to prior treatment with PEG-IFN and RBV.
Acknowledgements
The authors would like to thank the DIRECT-001 and DIRECT-002 study investigators (Bashar Aqel, Sanjeev Arora, Carl Berg, David Bernstein, Edmund Bini, Scott Cotler, Michael Fallon, Kenneth Flora, Steven-Huy Bui Han, Joanne Imperial, Mark Jonas, Gerond Lake-Bakaar, Anne Larson, Eric Lawitz, Edward Lebovics, Michael Lyons, Gary Matusow, Douglas Meyer, Abdul Nadir, Robert Reindollar, Adrian Reuben, Maribel Rodriguez-Torres, Vinod Rustgi, Michael Ryan, John Santoro, Muhammad Sheikh, Robert Sjogren, Mark Swaim, Helen Te, Tram Tran, Andrzej Triebling, Harlan Wright, and Nizar Zein) for their participation in the studies. Dr. John Kincaid is also acknowledged for his assistance in the preparation of the first draft of the manuscript. All subsequent revisions of this manuscript were made by Bruce R. Bacon and Mitchell L. Shiffman with some input from the authors on the masthead.
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14 Leevy CB. Consensus interferon and ribavirin in patients with chronic hepatitis C who were nonresponders to pegylated interferon alfa-2b and ribavirin. Dig Dis Sci 2008; 53: 1961-1966. Links
15 Afdhal NH, Levine R, Brown RJ, Freilich B, O'Brien M, Brass C. Colchicine versus peg-interferon alfa 2b long term therapy: results of the 4 year COPILOT trial [Abstract 3]. J Hepatol 2008; 48(Suppl): S4. Links
16 Jensen D, Di Bisceglie A, Gitlin N, Freilich B, Reddy K, Feinman V, et al. Peginterferon alfa-2a (40KD) plus ribavirin (RBV) in pegylated interferon alfa-2b (12KD)/ribavirin non-responders: week 12 efficacy and safety outcomes of the REPEAT study. Gastroenterology 2006; 130; T1800. Links
17 Jensen D, Di Bisceglie A, Gitlin N, Freilich B, Reddy K, Feinman V, et al. Type of response to prior pegylated interferon alfa-2b (12kd)/rbv predicts subsequent response to retreatment with peginterferon alfa-2a (40KD)/RBV. Gastroenterology 2008; 134: S1937. Links
18 Kaiser S, Boecher W, Lutze B, Sauter B, Bissinger L, Werner C, et al. Treatment of peginterferon/ribavirin nonresponders with daily dosing of consensus interferon and ribavirin: preliminary results of the German Consensus Interferon Multicenter Study [Abstract 1306]. HEPATOLOGY2007; 46 Suppl:817A. Links
19 Ghalib RH, Levine CD, Friedman DA, Rashdan S, Schwartz AG, Weinstein J. Consensus interferon plus ribavirin therapy in patients who are nonresponders or relapsers to prior PEG IFN plus ribavirin. Gastroenterology 2007; 46: M1874. Links
20 Harrison SA. Small molecules and novel treatment for chronic hepatitis C virus infection. Am J Gastroenterol 2007; 102: 2332-2338.
Juicing and the LIVER
Liver
Liver is one of the strongest organs of the human body. There is a saying about liver; “liver doesn’t break easily and when it does it does not heal easily”. With the world moving at rocket pace, we are forced to live unhealthy lives leading to toxic build-up in our body. The heart, kidney, and liver are at greatest risks of being damaged. They take a lot of functional load and literally run the entire machine between themselves. Liver secretes many enzymes which fights any toxic build-up. To stimulate the enzymes we take resort to various natural foods in fruits, broccoli, lemons, and so on. There are many juices which help the liver detoxify effortlessly. They help in activating those enzymes of defense that hibernate in liver passages otherwise. There are juice fasting and smoothie cleansing recipes which guard the liver against the toxin concentration. For juice fasting liver detox, it becomes important to invest in a capable juicer, one which has the ability to sift the pulp from the juice through centrifugal force. This is a health investment and must be done without two-thoughts. Few juices contain fruit essences comprising of rich traces of vitamins and polyphenols. It is thus important for the juicer to retain the entire paramount nutrient. Juices like apple-celery, cucumber, tomato, beet juice, papaya, and cherry grape help a great deal in enhancing liver’s defense system.
There is a liver clean recipe which impulse and practice both turn towards. It’s the alkaline recipe which has been successfully used for liver detoxification. Its active ingredients are 2 big grapefruits, 300ml of distilled water, 2 tablespoons of Udo’s Choice, 1-2 cloves of fresh garlic, 2 inches of fresh root ginger, 4 lemons. Experimental ingredients which can be used are acidophilus at 1 teaspoon and optional ingredient is cayenne in small quantity. The juices of grapefruit and lemon should be squeezed in a blender and kept aside. Garlic should be grated along with ginger and squeezed into the already blended juice (preferably through garlic press). Water must then be added along with Udo’s and acidophilus powder, and blended for another 30 seconds.
This is a potent remedy for detoxifying the entire system; liver gets a fillip, it has no side effects, and a little breath pile produced due to garlic is effectively taken care of by grape juice. This also is a great method to remove hangovers. The juice is recommended on the weekend mornings and should be prefixed with light exercises. It is also advisable to do some breathing after exercises to let the lymph flow properly into the system.
All these liver recipes make great forces of liberation for the body and let it dish out the necessary enzymes for detox. They keep liver in shape and help liver keep us going. Without the effective incorporation of these natural remedies, into our lifestyles we continually run the risk of being defeated by our metabolism. Times are such that external forces like pollution have already deflated our natural detoxifying mechanism. Let’s dutifully indulge in these liver detoxifying juices and guard ourselves from big cardiovascular and liver diseases.
Juicing For Nutrition Information
Fruits and vegetables are excellent choices of a wide range of vitamins, minerals, and other important nutrients, including phytochemicals. Phytochemicals are compounds that have been shown to combat cancer. Healthful substances are being discovered all the time in fruits and vegetables and so there is no one supplement pill that can contain all of the healthy compounds that occur naturally in our foods. Each plant seems to produce particular phytochemicals that work agains cancer in particular ways, it is suggested that a rich assortment of fruits and vegetables be included in your diet. It is also recommended that you should consume two glasses of live juices a day for health maintenance. Four glasses a day is recommended if you want speed healing and recovery from illness.
Juicing is an excellent means of adding fruits and vegetables to your diet. Since juice contains the whole fruit or vegetable - except for the fiber, which is the indigestible part of the plant - it contains virtually all of the plants' health promoting properties. Because fresh juices are made from raw fruits and vegetables, all of the components remain intact. Vitamin C and other water-soluable vitamins can be damaged by overprocessing or over cooking. Enzymes, which are proteins needed for digestion and other important functions, can also be damaged by cooking. Fresh juice, however, provides all of the plants' healthful ingredients in a form that is easy to digest and absorb. In fact, it has been estimated that fruit and vegetable juices can be assimilated in 20 to 30 minutes.
The juices listed should be made fresh in your kitchen and consumed immediately. Many commercial juices are heat-treated to lengthen shelf life. This process can destroy important nutrients. In addition, preservatives may have been added. This includes sugars, salt and other compounds. Freshly made juices can lose some of their nutrients by being allowed to sit for long periods of time. By buying the best produce available, properly preparing it for juicing, and processing it in your own juicer, you will produce the most healthful, nutrient-rich drinks possible.
There are generally three catagories of juices.
GREEN JUICES or "GREEN DRINKS"
Green juices cleanse the body of pollutants and have a rejuvenating effect. Made from a variety of green vegetables, green juices are rich in chlorophyll, which helps to purify the blood, build red blood cells, detoxify and heal the body, and provide the body with fast energy. Green juices can be made with alfalfa sprouts, cabbage, kale, dandelion greens, spinach, and other green vegetables, including wheatgrass. Wheatgrass juice is particularly important in any cancer treatment, especially when radiation therapy is involved. To sweeten and dilute your green juices, try adding fresh carrot and apple juice. No other fruit juices should be added. Steam=distilled water is another good addition. Although green juices have great health benefits, they should be consumed in moderation. Try drinking about 8 to 10 ounces a day. The following is an excellent "green drink":
VEGETABLE JUICES
Fresh vegetable juices are restorers and builders. They boost the immune system, remove acid wastes, and balance the metabolism. They also aid in the control of obesity by removing excess body fat. Among the most healthful and delicious of the vegetable juices are beet, cabbage, carrot, celery, cucumber, kale, parsley, turnip, spinach, watercress, and wheatgrass juice. Carrot juice is probably the most popular of the juices, and is packed with beta-carotene, the vitamin A precursor that helps fight cancer. Because carrots are the sweetest of the vegetables, their juice is not just delicious on its own, but is great for mixing with other vegetables to increase their appeal. On the other hand, strong-flavored vegetables - broccoli, celery, onions, parsley, rutabaga, and turnips, for instance - should be used in small amounts only. Garlic is a great addition to vegetable drinks. Before juicing, drop the garlic into vinegar for 1 minute to destroy any bacteria or mold on the surface. To avoid irritating the lining of the intestinal tract, use only 1 clove of fresh garlic in 2 glasses of juice. For the greatest health benefits, use many different vegetables when making your juices. That way, you will provide your body with a variety of important nutrients.
FRUIT JUICES
Fruit juices help to cleanse the body and nourish it with important nutrients, including cancer-fighting antioxidants. Although any fruit can be used, certain juices are particularly healthful and delicious. One favorite cleansing juice is watermelon. To make this refreshing drink, place a whole piece of watermelon - with rind intact - in the juicer. Other delicious juices can be made with apples, apricots, bananas, berries, citrus fruits, kiwi, melons, pears - with just about any fruit that you want to use. You can enjoy fruit juices at any time of the day. About 10 to 12 ounces per day is recommended. The following is just one of the delicious fruit juice drinks you can make at home.
Preparing Produce for Juicing
Juicing is an easy way to make delicious drinks that can boost your health and help you treat a number of disorders. By following these guidelines, you will ensure that your juices are as pure, nutrient-rich, and appetizing as possible. Whenever possible, buy and use organically grown produce. This is produce that is grown without the use of pesticides and other harmful chemicals. This prevents chemical residues from ending up in your juice. If you are unable to obtain organically grown fruits and vegetables, peel or thoroughly wash the produce, using a vegetable brush to remove chemical residues and waxes. Most health food stores carry vegetable washes that will help remove any residues. When purchasing potatoes for juicing, avoid those with a green tint, and be sure to remove any sprouts or eyes. The chemical solanine, which gives the potatoe the green cast, can cause diarrhea, vomiting and abdominal pain. When using organically grown produce, feel free to leave the skin on in most cases. Do remove the skin, though, before juicing apricots, grapefruits, kiwis, oranges, papayas, peaches, and pineapple. The skins of oranges and grapefruits are quite bitter, and also contain a toxic substance that should not be consumed in large amounts. Because kiwis and papayas are tropical fruits, their skins are likely to contain residues of the harmful sprays often used in foreign countries, where some chemicals outlawed in the United States may still be legal. The skins of pineapples are too thick to be processed by most juicers. When juicing fruits, leave in small seeds, except when using apples, apricots, and peaches. These fruit seeds (pits) actually contain cyanide, a toxic substance. Because of their size and hardness, all pits must be removed. Juice most produce with stems and leaves intact. However, remove carrot and rhubarb greens, as they contain toxic substances. When using soft fruits that contain very little water = avocados, bananas, and papayas, for instance - prepare these fruits in a blender rather than using a juicer. Then stir these fruits into other juices.
Liver is one of the strongest organs of the human body. There is a saying about liver; “liver doesn’t break easily and when it does it does not heal easily”. With the world moving at rocket pace, we are forced to live unhealthy lives leading to toxic build-up in our body. The heart, kidney, and liver are at greatest risks of being damaged. They take a lot of functional load and literally run the entire machine between themselves. Liver secretes many enzymes which fights any toxic build-up. To stimulate the enzymes we take resort to various natural foods in fruits, broccoli, lemons, and so on. There are many juices which help the liver detoxify effortlessly. They help in activating those enzymes of defense that hibernate in liver passages otherwise. There are juice fasting and smoothie cleansing recipes which guard the liver against the toxin concentration. For juice fasting liver detox, it becomes important to invest in a capable juicer, one which has the ability to sift the pulp from the juice through centrifugal force. This is a health investment and must be done without two-thoughts. Few juices contain fruit essences comprising of rich traces of vitamins and polyphenols. It is thus important for the juicer to retain the entire paramount nutrient. Juices like apple-celery, cucumber, tomato, beet juice, papaya, and cherry grape help a great deal in enhancing liver’s defense system.
There is a liver clean recipe which impulse and practice both turn towards. It’s the alkaline recipe which has been successfully used for liver detoxification. Its active ingredients are 2 big grapefruits, 300ml of distilled water, 2 tablespoons of Udo’s Choice, 1-2 cloves of fresh garlic, 2 inches of fresh root ginger, 4 lemons. Experimental ingredients which can be used are acidophilus at 1 teaspoon and optional ingredient is cayenne in small quantity. The juices of grapefruit and lemon should be squeezed in a blender and kept aside. Garlic should be grated along with ginger and squeezed into the already blended juice (preferably through garlic press). Water must then be added along with Udo’s and acidophilus powder, and blended for another 30 seconds.
This is a potent remedy for detoxifying the entire system; liver gets a fillip, it has no side effects, and a little breath pile produced due to garlic is effectively taken care of by grape juice. This also is a great method to remove hangovers. The juice is recommended on the weekend mornings and should be prefixed with light exercises. It is also advisable to do some breathing after exercises to let the lymph flow properly into the system.
All these liver recipes make great forces of liberation for the body and let it dish out the necessary enzymes for detox. They keep liver in shape and help liver keep us going. Without the effective incorporation of these natural remedies, into our lifestyles we continually run the risk of being defeated by our metabolism. Times are such that external forces like pollution have already deflated our natural detoxifying mechanism. Let’s dutifully indulge in these liver detoxifying juices and guard ourselves from big cardiovascular and liver diseases.
Juicing For Nutrition Information
Fruits and vegetables are excellent choices of a wide range of vitamins, minerals, and other important nutrients, including phytochemicals. Phytochemicals are compounds that have been shown to combat cancer. Healthful substances are being discovered all the time in fruits and vegetables and so there is no one supplement pill that can contain all of the healthy compounds that occur naturally in our foods. Each plant seems to produce particular phytochemicals that work agains cancer in particular ways, it is suggested that a rich assortment of fruits and vegetables be included in your diet. It is also recommended that you should consume two glasses of live juices a day for health maintenance. Four glasses a day is recommended if you want speed healing and recovery from illness.
Juicing is an excellent means of adding fruits and vegetables to your diet. Since juice contains the whole fruit or vegetable - except for the fiber, which is the indigestible part of the plant - it contains virtually all of the plants' health promoting properties. Because fresh juices are made from raw fruits and vegetables, all of the components remain intact. Vitamin C and other water-soluable vitamins can be damaged by overprocessing or over cooking. Enzymes, which are proteins needed for digestion and other important functions, can also be damaged by cooking. Fresh juice, however, provides all of the plants' healthful ingredients in a form that is easy to digest and absorb. In fact, it has been estimated that fruit and vegetable juices can be assimilated in 20 to 30 minutes.
The juices listed should be made fresh in your kitchen and consumed immediately. Many commercial juices are heat-treated to lengthen shelf life. This process can destroy important nutrients. In addition, preservatives may have been added. This includes sugars, salt and other compounds. Freshly made juices can lose some of their nutrients by being allowed to sit for long periods of time. By buying the best produce available, properly preparing it for juicing, and processing it in your own juicer, you will produce the most healthful, nutrient-rich drinks possible.
There are generally three catagories of juices.
GREEN JUICES or "GREEN DRINKS"
Green juices cleanse the body of pollutants and have a rejuvenating effect. Made from a variety of green vegetables, green juices are rich in chlorophyll, which helps to purify the blood, build red blood cells, detoxify and heal the body, and provide the body with fast energy. Green juices can be made with alfalfa sprouts, cabbage, kale, dandelion greens, spinach, and other green vegetables, including wheatgrass. Wheatgrass juice is particularly important in any cancer treatment, especially when radiation therapy is involved. To sweeten and dilute your green juices, try adding fresh carrot and apple juice. No other fruit juices should be added. Steam=distilled water is another good addition. Although green juices have great health benefits, they should be consumed in moderation. Try drinking about 8 to 10 ounces a day. The following is an excellent "green drink":
VEGETABLE JUICES
Fresh vegetable juices are restorers and builders. They boost the immune system, remove acid wastes, and balance the metabolism. They also aid in the control of obesity by removing excess body fat. Among the most healthful and delicious of the vegetable juices are beet, cabbage, carrot, celery, cucumber, kale, parsley, turnip, spinach, watercress, and wheatgrass juice. Carrot juice is probably the most popular of the juices, and is packed with beta-carotene, the vitamin A precursor that helps fight cancer. Because carrots are the sweetest of the vegetables, their juice is not just delicious on its own, but is great for mixing with other vegetables to increase their appeal. On the other hand, strong-flavored vegetables - broccoli, celery, onions, parsley, rutabaga, and turnips, for instance - should be used in small amounts only. Garlic is a great addition to vegetable drinks. Before juicing, drop the garlic into vinegar for 1 minute to destroy any bacteria or mold on the surface. To avoid irritating the lining of the intestinal tract, use only 1 clove of fresh garlic in 2 glasses of juice. For the greatest health benefits, use many different vegetables when making your juices. That way, you will provide your body with a variety of important nutrients.
FRUIT JUICES
Fruit juices help to cleanse the body and nourish it with important nutrients, including cancer-fighting antioxidants. Although any fruit can be used, certain juices are particularly healthful and delicious. One favorite cleansing juice is watermelon. To make this refreshing drink, place a whole piece of watermelon - with rind intact - in the juicer. Other delicious juices can be made with apples, apricots, bananas, berries, citrus fruits, kiwi, melons, pears - with just about any fruit that you want to use. You can enjoy fruit juices at any time of the day. About 10 to 12 ounces per day is recommended. The following is just one of the delicious fruit juice drinks you can make at home.
Preparing Produce for Juicing
Juicing is an easy way to make delicious drinks that can boost your health and help you treat a number of disorders. By following these guidelines, you will ensure that your juices are as pure, nutrient-rich, and appetizing as possible. Whenever possible, buy and use organically grown produce. This is produce that is grown without the use of pesticides and other harmful chemicals. This prevents chemical residues from ending up in your juice. If you are unable to obtain organically grown fruits and vegetables, peel or thoroughly wash the produce, using a vegetable brush to remove chemical residues and waxes. Most health food stores carry vegetable washes that will help remove any residues. When purchasing potatoes for juicing, avoid those with a green tint, and be sure to remove any sprouts or eyes. The chemical solanine, which gives the potatoe the green cast, can cause diarrhea, vomiting and abdominal pain. When using organically grown produce, feel free to leave the skin on in most cases. Do remove the skin, though, before juicing apricots, grapefruits, kiwis, oranges, papayas, peaches, and pineapple. The skins of oranges and grapefruits are quite bitter, and also contain a toxic substance that should not be consumed in large amounts. Because kiwis and papayas are tropical fruits, their skins are likely to contain residues of the harmful sprays often used in foreign countries, where some chemicals outlawed in the United States may still be legal. The skins of pineapples are too thick to be processed by most juicers. When juicing fruits, leave in small seeds, except when using apples, apricots, and peaches. These fruit seeds (pits) actually contain cyanide, a toxic substance. Because of their size and hardness, all pits must be removed. Juice most produce with stems and leaves intact. However, remove carrot and rhubarb greens, as they contain toxic substances. When using soft fruits that contain very little water = avocados, bananas, and papayas, for instance - prepare these fruits in a blender rather than using a juicer. Then stir these fruits into other juices.
Hepatitis C virus-induced hepatocarcinogenesis
Hepatitis C virus-induced hepatocarcinogenesis
Birke Bartosch123, Robert Thimme4, Hubert Blum4, Fabien Zoulim123Corresponding Author Informationemail address
Articles in Press
Jnl of Hepatology
published online 25 May 2009.
Although there is strong evidence that hepatitis C virus (HCV) is one of the leading causes of hepatocellular carcinoma (HCC), there is still much to understand regarding the mechanism of HCV-induced transformation. While liver fibrosis resulting from long-lasting chronic inflammation and liver regeneration resulting from immune-mediated cell death are likely factors that contribute to the development of HCC, the direct role of HCV proteins remains to be determined. In vitro studies have shown that HCV expression may interfere with cellular functions that are important for cell differentiation and cell growth. However, most studies were performed in artificial models which can only give clues for potential mechanisms that need to be confirmed in more relevant models. Furthermore, the difficulty to identify HCV proteins and infected liver cells in infected patients, contributes to the complexity of our current understanding. For these reasons, there is currently very little experimental evidence for a direct oncogenic role of HCV. Further studies are warranted to clarify these issues.
"HCV proteins interact with a number of host factors and signaling pathways and thus contribute to the progression from chronic hepatitis C to liver cirrhosis and HCC"
"Apart from chronic HCV infection other risk factors for HCC development are among others HBV infection, obesity in men, diabetes mellitus, heavy alcohol use and hereditary hemochromatosis. Successful clearance of chronic HCV infection has been shown to reduce the overall liver-related mortality and HCC incidence, providing further evidence for a causal role of HCV in this cancer"...."clinical data show a regression of lymphoma after successful treatment of HCV infection supporting the concept of HCV infection as a cause of lymphoma development in humans"
"In HCV-infected patients, host and environmental factors appear to be more important than viral factors in determining progression of the liver disease to cirrhosis and HCC. These factors include: older age at diagnosis (>55 years: 2- to 4-fold increased risk) [44], [45], duration of infection [30], male sex (2- to 3-fold increased risk) [46], severity of liver disease at presentation, co-morbidities such as porphyria cutanea tarda [47], heavy alcohol intake [3], [48], [49], [50], diabetes mellitus [51], [52], steatosis [53], [54], obesity [52], [55] and coinfections, especially with HBV [26], [56]. Slightly elevated serum bilirubin levels, decreased platelet counts and skin manifestations of liver disease, such as vascular spiders and/or palmar erythema correlate with the HCC risk"
"all HCV genotypes have been shown to interfere with glucose homeostasis, often at early stages in HCV infection"....."the initial or early stages of HCV infection are strongly associated with IR. In contrast, at late stages of disease and in particular in tumorigenesis, transformed cells have been shown to require more glucose and to upregulate insulin sensitivity and glucose uptake"
"in the context of chronic inflammation, the interplay between ER/oxidative stress, steatosis and IR induces a pro-oncogenic microenvironment that results in fibrogenesis and genomic instability. Even though HCV has been reported to have direct transforming properties, the liver microenvironment is thought to significantly modulate the transformation process because HCC develops in chronic HCV infection only over long periods of time."
"In chronic HCV infection, pro-carcinogenic cofactors are steatosis, oxidative stress and insulin resistance (IR). Thus chronic hepatitis C shares many similarities with non-alcoholic fatty liver disease (NAFLD), which may lead to non-alcoholic steatohepatitis (NASH) and HCC"
"a complex interplay between steatosis, ER/oxidative stress and IR (insulin resistance), whose underlying molecular mechanisms remain largely undefined, can lead to chronic liver inflammation, apoptosis and fibrogenesis that are central to the development of liver cirrhosis and HCC in patients with chronic hepatitis C."
"Similar to NAFLD, ER/oxidative stress, steatosis and IR are involved in the pathogenesis of chronic HCV infection, either as metabolic predisposition or directly induced by HCV (Fig. 1 and Table 2). An increased prevalence of steatosis and IR has been observed in patients with HCV infection and has prognostic implications, as it is associated with faster progression to cirrhosis and HCC as well as with a poorer response to treatment. In patients infected with HCV genotypes 1 and 2, steatosis often develops in the context of a pre-existing diabetes, IR or increased body mass index."..."HCV is thought to induce steatosis by interfering with lipid secretion and degradation and by increasing lipid synthesis"
9. Conclusions
Our current view is that the mechanism of HCV-induced HCC is multifactorial. However, because of the lack of adequate models, it has been difficult to demonstrate the specific roles of HCV proteins and the liver environment in the malignant transformation of hepatocytes. To identify and characterize these mechanisms, primary human hepatocyte cultures supporting chronic HCV infection would be most useful to examine the accumulation of transforming events, leading to the selection of transformed cells after several cell passages. An immunocompetent animal model, susceptible to chronic HCV infection, would be important to analyze not only the different viral proteins, but also the liver microenvironment involved in HCC development (including IR, steatosis, oxidative stress, cytokine expression in response to HCV expression, liver regeneration, fibrosis, etc.). The recent discovery of cellular co-receptors required for virus-entry and the better understanding to the molecular biology of HCV replication should open new avenues to address these important questions.
Associate Editor: K. Koike
1. Introduction
Chronic hepatitis C virus (HCV) infection is characterized by inflammatory lesions in the liver, often accompanied by intrahepatic lipid accumulation (steatosis) and progressive fibrosis of variable degrees, and long-term progression to cirrhosis and hepatocellular carcinoma (HCC) [1], [2]. HCC incidence has increased sharply over recent decades and has been attributed to chronic HCV infection. Chronic HCV infection, therefore, is a major risk factor for HCC development. Indeed, each year, 4-5% of patients with chronic hepatitis C develop HCC. Serological markers of HCV infection in patients with HCC range from 27% up to 80%, and HCV infection increases the risk for HCC development by an estimated 17-fold compared to healthy individuals [3], [4], [5], [6] (Table 1). Host, environmental and viral factors appear to play an important role in determining progression of chronic hepatitis C to liver cirrhosis and HCC, a process that frequently takes several decades (Fig. 1). The molecular mechanisms underlying HCC development remain ill-defined. So far, it has not been possible to correlate specific changes in gene expression patterns with HCC development. HCV does not integrate into its host genome and has a predominantly cytoplasmic life cycle [7]. Hepatocarcinogenesis, therefore, must involve several indirect mechanisms including the interplay between chronic inflammation, steatosis, fibrosis and oxidative stress and their pathological consequences. In addition, several HCV proteins have been shown to have direct oncogenic effects and to upregulate mitogenic processes. Increased cell proliferation in a setting of oxidative stress leads to accumulation of DNA damage and is thought to compromise gene and chromosome stability and to form the genomic basis for the malignant transformation of the hepatocyte. Here, we review the epidemiology of HCV-induced HCC and the potential underlying molecular mechanisms.
2. HCV infection: the virus and the disease
In the 1970s and 1980s, serological analyses developed for the detection of hepatitis A virus (HAV) and hepatitis B virus (HBV) infection, respectively, indicated that the majority of transfusion-transmitted hepatitis was not caused by either HAV or HBV and was therefore termed non-A, non-B hepatitis (NANBH). The etiological agent of NANBH was discovered in 1989 and was termed HCV. Based on its structural and functional organization HCV was classified into the family of the Flaviviridae, where it forms its own genus H epacivirus’ [8]. The HCV genome is a single-stranded, positive sense RNA of approx. 9600 nt in length [9] with genetic heterogeneity, resulting in its classification into six different genotypes. The HCV genome contains short non-coding regions (NCR) at each end. The coding sequence is translated into a polyprotein that is processed by viral and cellular proteases. The 5′-region of the genome encodes the structural proteins, including the nucleocapsid protein (core) and two envelope glycoproteins (E1 and E2) that form the viral particle, followed by a number of non-structural proteins (NSI), designated NS2 to NS5B in the 3′-region.
HCV is considered hepatotropic, and only man and chimpanzees are susceptible to HCV infection and disease [10], [11]. While HCV RNA has been unequivocally detected in hepatocytes in liver biopsies from chronically infected patients and chimpanzees, the HCV genome seems to replicate also in cells of lymphoid origin and dendritic cells [12], [13], [14]. Circulating HCV particles have a diameter of 35–50nm and are frequently associated with either immune globulins or very low density lipoproteins (vLDL) [15]. Indeed, the vLDL biosynthesis machinery plays a pivotal role in the life cycle of HCV [15], [16], [17].
Risk factors for HCV transmission include transfusion of blood and blood products, transplantation of solid organs from infected donors, injecting drug use, unsafe therapeutic injections and occupational exposure to blood [18]. The rate of transmission after a needle-stick injury from HCV positive blood ranges from 0 to 10% in most studies. The rate of perinatal HCV transmission is 4–7% and occurs only when HCV RNA is detectable in maternal serum at delivery. Importantly, coinfection with HIV increases the rate of perinatal transmission 4- to 5-fold [18].
Persistence of HCV infection occurs in the majority of HCV-infected individuals. Indeed, acute hepatitis C resolves spontaneously only in about 10–40% of cases [19], [20]. Chronic hepatitis C is characterized by the persistence of elevated aminotransferase levels and HCV RNA in serum, but is otherwise generally asymptomatic. The rate of progression to severe liver disease is highly variable. Factors that promote clinical progression include alcohol intake, coinfection with HIV and/or HBV, male sex and older age at infection [2].
The estimated prevalence of HCV infection worldwide is 2.2% and active or passive vaccination is not available to date. Antiviral combination therapy with a pegylated interferon and ribavirin is usually administered only to patients with more advanced and progressive disease [19], [20], [21] due to cost, side effects and limited efficacy, especially in individuals infected with HCV genotype 1. Therefore, several novel antiviral agents are currently being evaluated including NS3-4A protease inhibitors, RNA dependent RNA polymerase inhibitors and different immune therapies [22].
3. Association between HCV infection and development of HCC or other malignancies
Chronic HCV infection is a major risk factor for HCC development and serological markers of HCV infection are found in up to 80% of patients with HCC in some areas of the world [23], [24] (Table 1). HCV infection is estimated to increase the risk for HCC development up to 17-fold [3], [4]. Host, environmental and viral factors appear to play an important role in determining progression of chronic hepatitis C to liver cirrhosis and HCC [2]. Some [25], [26], [27], [28], but not all clinical studies [29], [30], [31], [32], suggest that the risk of HCC development is associated with certain HCV genotypes, particularly genotype 1b. Apart from chronic HCV infection other risk factors for HCC development are among others HBV infection, obesity in men, diabetes mellitus, heavy alcohol use and hereditary hemochromatosis. Successful clearance of chronic HCV infection has been shown to reduce the overall liver-related mortality and HCC incidence, providing further evidence for a causal role of HCV in this cancer [33].
Apart from HCC, HCV is also a well-established risk factor of lymphoproliferative syndromes such as type II mixed cryoglobulinemia [34] and malignant lymphoma. Indeed, HCV infection increases the risk of B-cell non-Hodgkin lymphoma (B-NHL) 2- to 10-fold [35], [36] (Table 1). This association is particularly striking in southern Europe but much less in northern Europe and north America [35], [36], suggesting that differences in HCV prevalence in these geographic regions, in control populations and in methods of HCV detection may account for these findings. The mechanisms underlying HCV-related lymphoma development, including the contributing host and viral factors [37] remain to be identified. Interestingly, clinical data show a regression of lymphoma after successful treatment of HCV infection supporting the concept of HCV infection as a cause of lymphoma development in humans [38]. HCV infection has also been linked to the development of intrahepatic cholangiocarcinoma (ICC) [39], [40], [41]. It remains unclear, however, whether this association is independent from the underlying liver disease/cirrhosis.
Prospective and retrospective cohort studies of patients with HCV infection have shown the role of the duration of chronic hepatitis in HCC development and the link between HCC development and liver cirrhosis. These studies demonstrated the sequential occurrence of advanced liver fibrosis and the development of HCC. The incidence of HCC development was estimated to be between 3 and 5%/year in patients with liver cirrhosis [42], [43]. In HCV-infected patients, host and environmental factors appear to be more important than viral factors in determining progression of the liver disease to cirrhosis and HCC. These factors include: older age at diagnosis (>55 years: 2- to 4-fold increased risk) [44], [45], duration of infection [30], male sex (2- to 3-fold increased risk) [46], severity of liver disease at presentation, co-morbidities such as porphyria cutanea tarda [47], heavy alcohol intake [3], [48], [49], [50], diabetes mellitus [51], [52], steatosis [53], [54], obesity [52], [55] and coinfections, especially with HBV [26], [56]. Slightly elevated serum bilirubin levels, decreased platelet counts and skin manifestations of liver disease, such as vascular spiders and/or palmar erythema correlate with the HCC risk [26], [56]. Specific HLA class II alleles have also been associated with progression of chronic hepatitis C to decompensated cirrhosis or HCC. In this context, studies documented an association between DRB1∗1301/2 alleles and an asymptomatic HCV infection [44]. Further, an association between the HLA DQ02 allele and HCC development has been reported [44].
4. HCV-induced hepatocarcinogenesis
The mechanisms underlying the progression of HCV infection to HCC, which usually takes many years or decades, remain ill-defined [57], [58]. Transcriptomics and proteomics have helped to identify many genetic and epigenetic alterations associated with HCC clusters. However, the changes of gene expression identified in tumor cells are very heterogeneous, raising the question whether yet unidentified, specific changes at early, preneoplastic stages trigger the transformation process and whether differentiated hepatocytes or stem cells are at the origin of HCC [59], [60].
HCV is the only RNA virus with a predominantly cytoplasmic life cycle [7], [8]. All potentially pro-oncogenic events are therefore likely to be restricted to the cytoplasm, suggesting indirect mechanisms of hepatocarcinogenesis. While HCV infection leads to chronic inflammation, steatosis, fibrosis and oxidative DNA damage, several HCV proteins have been shown to have direct oncogenic effects and to upregulate mitogenesis [57], [61] (Table 2). The accumulation of oxidative stress and DNA damage in a setting of restricted cell cycle checkpoint control and/or accelerated cell division, is thought to compromise gene and chromosome stability and to form the genomic basis for the malignant transformation (Fig. 1). Indeed, in chronic HCV infection, changes in mitogen-activated protein kinase (MAPK) signaling, that regulates both cell metabolism and growth, are frequently detected [62], [63]. Markers of intracellular oxidative stress have also been found to be increased in patients with chronic HCV infection [64], [65] as well as HCV core transgenic mice [66], [67]. However, direct interactions of the various HCV proteins with host cell factors have also been shown to lead to changes in cellular signaling cascades involved in regulation of cell metabolism and division and seem to be sufficient to induce hepatocarcinogenesis [66], [68]. Overall, it is thought that the synergism between chronic inflammation and direct virus–host cell interactions triggers the malignant transformation of hepatocytes. The requirement for such a synergism would also explain the slow ‘multi-step’ transformation process that underlies human HCC development. Indeed, a considerable time lag between HCV infection and the development of cirrhosis and HCC is common and also explains the heterogeneity of genetic and epigenetic alterations observed in different HCCs [57], [58], [69].
5. HCV-induced changes in the hepatic glucose and lipid metabolism
Similar to NAFLD, ER/oxidative stress, steatosis and IR are involved in the pathogenesis of chronic HCV infection, either as metabolic predisposition or directly induced by HCV (Fig. 1 and Table 2). An increased prevalence of steatosis and IR has been observed in patients with HCV infection and has prognostic implications, as it is associated with faster progression to cirrhosis and HCC as well as with a poorer response to treatment. In patients infected with HCV genotypes 1 and 2, steatosis often develops in the context of a pre-existing diabetes, IR or increased body mass index. By comparison, in patients infected with HCV genotype 3, steatosis is directly induced by HCV, because it correlates with the viral load and reverses with response to antiviral treatment [71]. HCV is thought to induce steatosis by interfering with lipid secretion and degradation and by increasing lipid synthesis. The HCV core protein, which localizes to the surface of lipid droplets and mediates viral assembly in close association with the cellular fatty acid metabolism [16], as well as some HCV non-structural proteins, have been shown to interfere with very low density lipoprotein (vLDL) secretion [72], [73]. HCV infection also upregulates lipid synthesis [63], inhibits fatty acid oxidation [74], [75] and increases release of fatty acids from adipocytes [71]. Overall the effects of HCV proteins on lipid synthesis, secretion and oxidation seem to be most pronounced in HCV genotype 3 infection, but also occur in patients infected with other genotypes. Besides changes in the lipid metabolism, HCV core and several non-structural proteins, induce systemic oxidative stress and related signaling by various mechanisms [76]. With respect to IR, all HCV genotypes have been shown to interfere with glucose homeostasis, often at early stages in HCV infection [71]. The mechanism underlying IR and its severity seem again to be genotype dependent. HCV has been shown to interfere with insulin signaling by proteasomal degradation of IRS-1 and -2 either via SOCS proteins or the PI3K/Akt/mTOR pathway, as well as by IRS-1 inactivation via transforming growth factor (TGF)-α and PI3K/Akt [77]. Thus, the initial or early stages of HCV infection are strongly associated with IR. In contrast, at late stages of disease and in particular in tumorigenesis, transformed cells have been shown to require more glucose and to upregulate insulin sensitivity and glucose uptake [78].
6. From liver inflammation to liver cancer
The interdependence between steatosis, IR and oxidative stress is important for disease progression in NAFLD as well as in hepatitis C and induces tissue damage and inflammation with activation of hepatic stellate cells (HSCs). Activated HSCs become responsive to both proliferative and fibrogenic cytokines and undergo epithelial to mesenchymal trans-differentiation (EMT) into contractile myo-fibroblast-like cells, that synthesize extracellular matrix (ECM) components, which accumulate over time to form fibrous scars or fibrosis. Ultimately, regenerating hepatocytes become enclosed by scar tissue and form nodules that define cirrhosis. HSCs are activated by products and effectors of oxidative stress and growth factors, cytokines, adipokines and chemokines, secreted by hepatocytes, Kupffer and inflammatory cells that infiltrate the liver in response to infection. The cytokine TGF-β, a potent inhibitor of epithelial cell growth and tumor suppressor, is a key regulator of EMT and also has pro-oncogenic functions. Importantly, recent findings indicate that TGF-β induces EMT not only in HSCs but possibly also in hepatocytes [79]. TGF-β signaling is upregulated in fibrosis in HCV-infected patients and stimulates ECM deposition and accumulation. IR may link fibrosis and steatosis, since it stimulates HSCs to deposit ECM. Several signaling cascades are involved in fibrogenesis, including SMADs, PI3K-Akt and various MAPK pathways, such as p38 and JNK. While SMADs are indispensable for EMT, TGF-β signaling via SMAD interacts with other signaling pathways to mediate pro-oncogenic EMT. JNK activation by the pro-inflammatory cytokine interleukin (IL)-1β can shift TGF-β signaling from tumor suppression to oncogenesis with increased fibrogenesis, cell motility and transactivation of cell cycle regulatory genes [79], [80]. Thus, in the context of chronic inflammation, the interplay between ER/oxidative stress, steatosis and IR induces a pro-oncogenic microenvironment that results in fibrogenesis and genomic instability. Even though HCV has been reported to have direct transforming properties, the liver microenvironment is thought to significantly modulate the transformation process because HCC develops in chronic HCV infection only over long periods of time.
7. Direct oncogenic effects of HCV
Apart from complex interactions among themselves, HCV proteins interact with a number of host factors and signaling pathways and thus contribute to the progression from chronic hepatitis C to liver cirrhosis and HCC (Table 2). By modulating gene transcription and translation as well as post-translational events, the HCV proteins interfere with innate immunity to favor viral persistence and liver inflammation; they alter cell signaling, apoptosis, membrane physiology and protein trafficking, induce oxidative stress, genomic instability as well as malignant transformation. Among the HCV proteins core, NS3, NS4B and NS5A have all been shown to have transforming potential when transiently or stably expressed in cell culture, or in transgenic mice expressing the different viral proteins or the HCV polyprotein [81], [82], [83], [84].
The HCV core protein is a highly conserved, basic protein that multimerizes, probably in conjunction with microtubules [85] to form the viral nucleocapsid and to package the viral RNA genome. It is localized at the cytoplasmic surface of the ER and on lipid droplets, and the latter observation is likely related to the induction of liver steatosis observed in HCV-infected patients as well as in transgenic mice overexpressing HCV core [86], [87], [88]. Core has also been shown to localize to the outer membranes of mitochondria [89] and is involved in changes of apoptosis and lipid metabolism as well as in malignant transformation. Among many interactions with cellular factors, core has been shown to induce ROS production via interaction with heat shock protein Hsp60 [90], to bind the tumor suppressor proteins p53 [91], [92], p73 [93] and pRb [94]. Core also inhibits the expression of the cyclin-dependent kinase (CDK) inhibitor p21/Waf [95]. p21 is a transcriptional target of p53 and blocks the cyclin/CDK complexes involved in cell-cycle control and tumor formation. Core induces activation of the Raf1/MAPK pathway [96], [97], protects cells from serum starvation and growth arrest and drives cells into proliferation. NF-κB transcription has been shown to be activated [98], [99], [100] and repressed [101] by HCV core. HCV core has also been shown to activate the Wnt/b-catenin pathway, which controls cell proliferation, DNA synthesis and cell-cycle progression [102]. Furthermore, HCV core variants have been shown to interact with SMAD3 and to inhibit the TGF-β pathway [103]. TGF-β signaling not only controls cell proliferation, differentiation and apoptosis but also stimulates liver regeneration and fibrogenesis through its actions on the extracellular matrix (see above). TGF-β levels are frequently increased in patients with chronic HCV infection and correlate with the degree of fibrosis [104], [105]. Finally, HCV core protein associates with cellular membranes [88], [106] and lipid vesicles [106], binds to apolipoprotein II [107], [108] and reduces microsomal triglyceride transfer protein activity [108], resulting in impaired assembly and secretion of vLDL, steatosis and oxidative stress. These in vitro findings are likely to be relevant for HCV pathogenesis because transgenic mice expressing HCV core protein also develop steatosis [108], [109] and HCC [67], [68].
Overexpression of E2 inhibits eIF2α phosphorylation by the dsRNA-activated protein kinase (PKR) or the ER-stress signaling kinase PERK [110], [111]. Similarly, overexpression of NS4A, NS4B, or NS4A-4B has been reported to induce an ER stress-mediated unfolded protein response, to reduce ER-to-Golgi trafficking, to inhibit protein synthesis, and to cause cytopathic effects [112], [113], [114], [115], [116]. NS2 has been shown to inhibit the cellular proapoptotic molecule CIDE-B [117] and to downregulate transcription [118].
NS3-4A serine protease has been reported to interact with p53 to repress p21 function, to block activation of the transcription factors IRF-3 and NF-κB and to antagonize the innate antiviral defenses by interfering with RIG-1, MDA5 and TLR3 mediated signal transduction [119], [120], [121]. Indeed, RIG-I inactivation has been shown to render Huh-7 cells permissive to HCV replication [122], [123].
NS5A has been shown to interact with the geranylgeranylated cellular protein FBL2 [124], an F-box motif containing protein that is probably involved in targeting cellular proteins of yet unknown identity for ubiquitylation and degradation. A number of studies suggest that NS5A is also involved in IFN resistance [8] and one possible mechanism may be its ability to induce expression of the type I interferon anatagonist IL-8 [125]. In addition, NS5A has been described to contain an ‘interferon sensitivity determining region’ (ISDR), that has been described to mediate inhibition of PKR, an activator of the innate immunity [83], [126], [127]. The accumulation of mutations in this region is thought to correlate with treatment efficacy [128], [129]. Importantly, overexpression of NS5A has been reported to induce a number of effects in cells, including oxidative stress, activation of signaling pathways such as STAT-3, PI3K, and NF-κB and altered transcriptional regulation including p21 [130], [131], [132] and of pRB [133]. Other NS5A interaction partners include apoliporotein A1, the major protein found on HDLs, the tumor suppressor p53, Grb-2, an adaptor protein involved in mitogen signaling, SRCAP, an adenosine triphosphatase (ATPase) that activates cellular transcription, karyopherin β3, a protein involved in nuclear trafficking, Cdk1/2, cyclin-dependent and Fyn, Hck, Lck, and Lyn, Src-family kinases [8], [134], [135], [136], [137]. It has also been reported that NS5A expression in the context of the HCV polyprotein results in the inhibition of the transcription factor Forkhead as well as in the phosphorylation and inactivation of the GSK-3, leading to accumulation of β-catenin and stimulation of β-catenin-dependent transcription [138]. Finally, NS5A dependent activation of upstream binding factor, a Pol I DNA binding transcription factor, which occurs as a result of up-regulation of both cyclin D1 and CDK4, leads to enhancement of rRNA transcription activation [139].
8. Genetic and epigenetic changes in HCV-induced hepato-carcinogenesis
HCCs are genetically very heterogeneous tumors. This is not unexpected, given the number of etiological factors implicated in its development, the complexity of hepatocyte physiology and the advanced stage at which HCCs are usually diagnosed. Genome-wide analysis of genetic alterations occurring in HCC revealed two major mechanisms of hepatocarcinogenesis. In the first, genetic alterations are acquired in the context of elevated oxidative stress caused by the vicious circle between chronic inflammation, IR and steatosis; in the second, transformation is induced by β-catenin mutations that dysregulate the Wnt pathway [140]. So far, it has not been possible to correlate chromosome instability with a consistent pattern of proto-oncogene activation in HCC, but several growth factor signaling pathways are frequently affected, including insulin-growth factor (IGF)-, hepatocyte growth factor-, Wnt-, TGF-α/EGF- and TGF-β-signaling [58], [141]. The interplay between these pathways and their respective roles and contributions to HCC development remain to be elucidated, however. One of the most commonly affected pathways involved in cell cycle check control is the p53 pathway, that limits cell survival and proliferation in response to telomere shortening and oncogene activation in order to ensure genome integrity. Loss of p53 by e.g. deletion, mutation, degradation or direct inhibition during progression of chronic hepatitis C to cirrhosis likely results in proliferation of hepatocytes with shortened telomeres or chromosomal damage and to predispose to hepatocarcinogenesis. Indeed, p21 expression, a downstream target of p53 that blocks cell entry into the S phase, is increased in cirrhosis when presumably significant numbers of hepatocytes with genome damage have accumulated, but is lost in premalignant liver lesions and HCC [142]. Besides p53, the retinoblastoma (Rb) pathway is another major checkpoint that limits cell proliferation in response to DNA damage, telomere shortening and oncogene activation. In human HCC, the Rb pathway is defective in more than 80% of cases [143]. Moreover, gankyrin, an inhibitor of p53 and Rb check point function is overexpressed in the vast majority of HCCs [144]. Expression of insulin-like growth factor IGF-2 is frequent and thought to be an early event in hepatocarcinogenesis, present in more than 60% of dysplastic nodules and HCC [145]. IGF-2 receptor impairs cell proliferation by promoting degradation of the IGF-2 mitogen and by activation of TGF-β signaling [146].
β-Catenin pathway activation is very common in hepatocarcinogenesis and detectable in more than 50% of HCCs. It can directly induce hepatocyte transformation without the need for multiple genetic/epigenetic alterations [147]. β-Catenin activation is mainly induced by β-catenin gene mutations and/or Wnt signaling pathway alterations [143]. Wnt/frizzled/β-catenin signaling is mediated by a complex interaction between a Wnt ligand (Wnt) and a Frizzled receptor (Fzd), mostly in cooperation with the low density lipoprotein receptor (LDLR)-related proteins LRP-5 or -6. Normally, the Wnt/β-catenin pathway is involved in cell growth and proliferation as well as developmental control and cell adhesion. Cellular levels of β-catenin are tightly regulated by proteasome-dependent degradation, which is in turn controlled by the activity of the APC and Axin1 proteins, and the glycogen synthase kinase-3b (GSK-3b). A recent report indicates that Fzd-7, which stabilizes and activates β-catenin [148], is overexpressed in more than 90% of HCCs and in around 75% of the peritumorous/precancerous liver tissue.
In addition to the dysregulation of the above pathways, HCV infection has been shown to induce or correlate with epigenetic changes that are likely to contribute to hepatocarcinogenesis. HCV-induced ROS have been shown to activate histone deacetylase in a fashion similar to hydrogen peroxide and to cause hypoacetylation of histones [149]. Hypomethylation of the IGF-2 locus in hepatitis C cirrhosis has been shown to predict HCC development [150]. Another study reported the hypermethylation of p16, p15, p14, pRB and the PTEN promoters in patients with sustained viral response in comparison to non-responders [151]. Finally, increased hTERT DNA levels have been found to predict HCC development [152].
Birke Bartosch123, Robert Thimme4, Hubert Blum4, Fabien Zoulim123Corresponding Author Informationemail address
Articles in Press
Jnl of Hepatology
published online 25 May 2009.
Although there is strong evidence that hepatitis C virus (HCV) is one of the leading causes of hepatocellular carcinoma (HCC), there is still much to understand regarding the mechanism of HCV-induced transformation. While liver fibrosis resulting from long-lasting chronic inflammation and liver regeneration resulting from immune-mediated cell death are likely factors that contribute to the development of HCC, the direct role of HCV proteins remains to be determined. In vitro studies have shown that HCV expression may interfere with cellular functions that are important for cell differentiation and cell growth. However, most studies were performed in artificial models which can only give clues for potential mechanisms that need to be confirmed in more relevant models. Furthermore, the difficulty to identify HCV proteins and infected liver cells in infected patients, contributes to the complexity of our current understanding. For these reasons, there is currently very little experimental evidence for a direct oncogenic role of HCV. Further studies are warranted to clarify these issues.
"HCV proteins interact with a number of host factors and signaling pathways and thus contribute to the progression from chronic hepatitis C to liver cirrhosis and HCC"
"Apart from chronic HCV infection other risk factors for HCC development are among others HBV infection, obesity in men, diabetes mellitus, heavy alcohol use and hereditary hemochromatosis. Successful clearance of chronic HCV infection has been shown to reduce the overall liver-related mortality and HCC incidence, providing further evidence for a causal role of HCV in this cancer"...."clinical data show a regression of lymphoma after successful treatment of HCV infection supporting the concept of HCV infection as a cause of lymphoma development in humans"
"In HCV-infected patients, host and environmental factors appear to be more important than viral factors in determining progression of the liver disease to cirrhosis and HCC. These factors include: older age at diagnosis (>55 years: 2- to 4-fold increased risk) [44], [45], duration of infection [30], male sex (2- to 3-fold increased risk) [46], severity of liver disease at presentation, co-morbidities such as porphyria cutanea tarda [47], heavy alcohol intake [3], [48], [49], [50], diabetes mellitus [51], [52], steatosis [53], [54], obesity [52], [55] and coinfections, especially with HBV [26], [56]. Slightly elevated serum bilirubin levels, decreased platelet counts and skin manifestations of liver disease, such as vascular spiders and/or palmar erythema correlate with the HCC risk"
"all HCV genotypes have been shown to interfere with glucose homeostasis, often at early stages in HCV infection"....."the initial or early stages of HCV infection are strongly associated with IR. In contrast, at late stages of disease and in particular in tumorigenesis, transformed cells have been shown to require more glucose and to upregulate insulin sensitivity and glucose uptake"
"in the context of chronic inflammation, the interplay between ER/oxidative stress, steatosis and IR induces a pro-oncogenic microenvironment that results in fibrogenesis and genomic instability. Even though HCV has been reported to have direct transforming properties, the liver microenvironment is thought to significantly modulate the transformation process because HCC develops in chronic HCV infection only over long periods of time."
"In chronic HCV infection, pro-carcinogenic cofactors are steatosis, oxidative stress and insulin resistance (IR). Thus chronic hepatitis C shares many similarities with non-alcoholic fatty liver disease (NAFLD), which may lead to non-alcoholic steatohepatitis (NASH) and HCC"
"a complex interplay between steatosis, ER/oxidative stress and IR (insulin resistance), whose underlying molecular mechanisms remain largely undefined, can lead to chronic liver inflammation, apoptosis and fibrogenesis that are central to the development of liver cirrhosis and HCC in patients with chronic hepatitis C."
"Similar to NAFLD, ER/oxidative stress, steatosis and IR are involved in the pathogenesis of chronic HCV infection, either as metabolic predisposition or directly induced by HCV (Fig. 1 and Table 2). An increased prevalence of steatosis and IR has been observed in patients with HCV infection and has prognostic implications, as it is associated with faster progression to cirrhosis and HCC as well as with a poorer response to treatment. In patients infected with HCV genotypes 1 and 2, steatosis often develops in the context of a pre-existing diabetes, IR or increased body mass index."..."HCV is thought to induce steatosis by interfering with lipid secretion and degradation and by increasing lipid synthesis"
9. Conclusions
Our current view is that the mechanism of HCV-induced HCC is multifactorial. However, because of the lack of adequate models, it has been difficult to demonstrate the specific roles of HCV proteins and the liver environment in the malignant transformation of hepatocytes. To identify and characterize these mechanisms, primary human hepatocyte cultures supporting chronic HCV infection would be most useful to examine the accumulation of transforming events, leading to the selection of transformed cells after several cell passages. An immunocompetent animal model, susceptible to chronic HCV infection, would be important to analyze not only the different viral proteins, but also the liver microenvironment involved in HCC development (including IR, steatosis, oxidative stress, cytokine expression in response to HCV expression, liver regeneration, fibrosis, etc.). The recent discovery of cellular co-receptors required for virus-entry and the better understanding to the molecular biology of HCV replication should open new avenues to address these important questions.
Associate Editor: K. Koike
1. Introduction
Chronic hepatitis C virus (HCV) infection is characterized by inflammatory lesions in the liver, often accompanied by intrahepatic lipid accumulation (steatosis) and progressive fibrosis of variable degrees, and long-term progression to cirrhosis and hepatocellular carcinoma (HCC) [1], [2]. HCC incidence has increased sharply over recent decades and has been attributed to chronic HCV infection. Chronic HCV infection, therefore, is a major risk factor for HCC development. Indeed, each year, 4-5% of patients with chronic hepatitis C develop HCC. Serological markers of HCV infection in patients with HCC range from 27% up to 80%, and HCV infection increases the risk for HCC development by an estimated 17-fold compared to healthy individuals [3], [4], [5], [6] (Table 1). Host, environmental and viral factors appear to play an important role in determining progression of chronic hepatitis C to liver cirrhosis and HCC, a process that frequently takes several decades (Fig. 1). The molecular mechanisms underlying HCC development remain ill-defined. So far, it has not been possible to correlate specific changes in gene expression patterns with HCC development. HCV does not integrate into its host genome and has a predominantly cytoplasmic life cycle [7]. Hepatocarcinogenesis, therefore, must involve several indirect mechanisms including the interplay between chronic inflammation, steatosis, fibrosis and oxidative stress and their pathological consequences. In addition, several HCV proteins have been shown to have direct oncogenic effects and to upregulate mitogenic processes. Increased cell proliferation in a setting of oxidative stress leads to accumulation of DNA damage and is thought to compromise gene and chromosome stability and to form the genomic basis for the malignant transformation of the hepatocyte. Here, we review the epidemiology of HCV-induced HCC and the potential underlying molecular mechanisms.
2. HCV infection: the virus and the disease
In the 1970s and 1980s, serological analyses developed for the detection of hepatitis A virus (HAV) and hepatitis B virus (HBV) infection, respectively, indicated that the majority of transfusion-transmitted hepatitis was not caused by either HAV or HBV and was therefore termed non-A, non-B hepatitis (NANBH). The etiological agent of NANBH was discovered in 1989 and was termed HCV. Based on its structural and functional organization HCV was classified into the family of the Flaviviridae, where it forms its own genus H epacivirus’ [8]. The HCV genome is a single-stranded, positive sense RNA of approx. 9600 nt in length [9] with genetic heterogeneity, resulting in its classification into six different genotypes. The HCV genome contains short non-coding regions (NCR) at each end. The coding sequence is translated into a polyprotein that is processed by viral and cellular proteases. The 5′-region of the genome encodes the structural proteins, including the nucleocapsid protein (core) and two envelope glycoproteins (E1 and E2) that form the viral particle, followed by a number of non-structural proteins (NSI), designated NS2 to NS5B in the 3′-region.
HCV is considered hepatotropic, and only man and chimpanzees are susceptible to HCV infection and disease [10], [11]. While HCV RNA has been unequivocally detected in hepatocytes in liver biopsies from chronically infected patients and chimpanzees, the HCV genome seems to replicate also in cells of lymphoid origin and dendritic cells [12], [13], [14]. Circulating HCV particles have a diameter of 35–50nm and are frequently associated with either immune globulins or very low density lipoproteins (vLDL) [15]. Indeed, the vLDL biosynthesis machinery plays a pivotal role in the life cycle of HCV [15], [16], [17].
Risk factors for HCV transmission include transfusion of blood and blood products, transplantation of solid organs from infected donors, injecting drug use, unsafe therapeutic injections and occupational exposure to blood [18]. The rate of transmission after a needle-stick injury from HCV positive blood ranges from 0 to 10% in most studies. The rate of perinatal HCV transmission is 4–7% and occurs only when HCV RNA is detectable in maternal serum at delivery. Importantly, coinfection with HIV increases the rate of perinatal transmission 4- to 5-fold [18].
Persistence of HCV infection occurs in the majority of HCV-infected individuals. Indeed, acute hepatitis C resolves spontaneously only in about 10–40% of cases [19], [20]. Chronic hepatitis C is characterized by the persistence of elevated aminotransferase levels and HCV RNA in serum, but is otherwise generally asymptomatic. The rate of progression to severe liver disease is highly variable. Factors that promote clinical progression include alcohol intake, coinfection with HIV and/or HBV, male sex and older age at infection [2].
The estimated prevalence of HCV infection worldwide is 2.2% and active or passive vaccination is not available to date. Antiviral combination therapy with a pegylated interferon and ribavirin is usually administered only to patients with more advanced and progressive disease [19], [20], [21] due to cost, side effects and limited efficacy, especially in individuals infected with HCV genotype 1. Therefore, several novel antiviral agents are currently being evaluated including NS3-4A protease inhibitors, RNA dependent RNA polymerase inhibitors and different immune therapies [22].
3. Association between HCV infection and development of HCC or other malignancies
Chronic HCV infection is a major risk factor for HCC development and serological markers of HCV infection are found in up to 80% of patients with HCC in some areas of the world [23], [24] (Table 1). HCV infection is estimated to increase the risk for HCC development up to 17-fold [3], [4]. Host, environmental and viral factors appear to play an important role in determining progression of chronic hepatitis C to liver cirrhosis and HCC [2]. Some [25], [26], [27], [28], but not all clinical studies [29], [30], [31], [32], suggest that the risk of HCC development is associated with certain HCV genotypes, particularly genotype 1b. Apart from chronic HCV infection other risk factors for HCC development are among others HBV infection, obesity in men, diabetes mellitus, heavy alcohol use and hereditary hemochromatosis. Successful clearance of chronic HCV infection has been shown to reduce the overall liver-related mortality and HCC incidence, providing further evidence for a causal role of HCV in this cancer [33].
Apart from HCC, HCV is also a well-established risk factor of lymphoproliferative syndromes such as type II mixed cryoglobulinemia [34] and malignant lymphoma. Indeed, HCV infection increases the risk of B-cell non-Hodgkin lymphoma (B-NHL) 2- to 10-fold [35], [36] (Table 1). This association is particularly striking in southern Europe but much less in northern Europe and north America [35], [36], suggesting that differences in HCV prevalence in these geographic regions, in control populations and in methods of HCV detection may account for these findings. The mechanisms underlying HCV-related lymphoma development, including the contributing host and viral factors [37] remain to be identified. Interestingly, clinical data show a regression of lymphoma after successful treatment of HCV infection supporting the concept of HCV infection as a cause of lymphoma development in humans [38]. HCV infection has also been linked to the development of intrahepatic cholangiocarcinoma (ICC) [39], [40], [41]. It remains unclear, however, whether this association is independent from the underlying liver disease/cirrhosis.
Prospective and retrospective cohort studies of patients with HCV infection have shown the role of the duration of chronic hepatitis in HCC development and the link between HCC development and liver cirrhosis. These studies demonstrated the sequential occurrence of advanced liver fibrosis and the development of HCC. The incidence of HCC development was estimated to be between 3 and 5%/year in patients with liver cirrhosis [42], [43]. In HCV-infected patients, host and environmental factors appear to be more important than viral factors in determining progression of the liver disease to cirrhosis and HCC. These factors include: older age at diagnosis (>55 years: 2- to 4-fold increased risk) [44], [45], duration of infection [30], male sex (2- to 3-fold increased risk) [46], severity of liver disease at presentation, co-morbidities such as porphyria cutanea tarda [47], heavy alcohol intake [3], [48], [49], [50], diabetes mellitus [51], [52], steatosis [53], [54], obesity [52], [55] and coinfections, especially with HBV [26], [56]. Slightly elevated serum bilirubin levels, decreased platelet counts and skin manifestations of liver disease, such as vascular spiders and/or palmar erythema correlate with the HCC risk [26], [56]. Specific HLA class II alleles have also been associated with progression of chronic hepatitis C to decompensated cirrhosis or HCC. In this context, studies documented an association between DRB1∗1301/2 alleles and an asymptomatic HCV infection [44]. Further, an association between the HLA DQ02 allele and HCC development has been reported [44].
4. HCV-induced hepatocarcinogenesis
The mechanisms underlying the progression of HCV infection to HCC, which usually takes many years or decades, remain ill-defined [57], [58]. Transcriptomics and proteomics have helped to identify many genetic and epigenetic alterations associated with HCC clusters. However, the changes of gene expression identified in tumor cells are very heterogeneous, raising the question whether yet unidentified, specific changes at early, preneoplastic stages trigger the transformation process and whether differentiated hepatocytes or stem cells are at the origin of HCC [59], [60].
HCV is the only RNA virus with a predominantly cytoplasmic life cycle [7], [8]. All potentially pro-oncogenic events are therefore likely to be restricted to the cytoplasm, suggesting indirect mechanisms of hepatocarcinogenesis. While HCV infection leads to chronic inflammation, steatosis, fibrosis and oxidative DNA damage, several HCV proteins have been shown to have direct oncogenic effects and to upregulate mitogenesis [57], [61] (Table 2). The accumulation of oxidative stress and DNA damage in a setting of restricted cell cycle checkpoint control and/or accelerated cell division, is thought to compromise gene and chromosome stability and to form the genomic basis for the malignant transformation (Fig. 1). Indeed, in chronic HCV infection, changes in mitogen-activated protein kinase (MAPK) signaling, that regulates both cell metabolism and growth, are frequently detected [62], [63]. Markers of intracellular oxidative stress have also been found to be increased in patients with chronic HCV infection [64], [65] as well as HCV core transgenic mice [66], [67]. However, direct interactions of the various HCV proteins with host cell factors have also been shown to lead to changes in cellular signaling cascades involved in regulation of cell metabolism and division and seem to be sufficient to induce hepatocarcinogenesis [66], [68]. Overall, it is thought that the synergism between chronic inflammation and direct virus–host cell interactions triggers the malignant transformation of hepatocytes. The requirement for such a synergism would also explain the slow ‘multi-step’ transformation process that underlies human HCC development. Indeed, a considerable time lag between HCV infection and the development of cirrhosis and HCC is common and also explains the heterogeneity of genetic and epigenetic alterations observed in different HCCs [57], [58], [69].
5. HCV-induced changes in the hepatic glucose and lipid metabolism
Similar to NAFLD, ER/oxidative stress, steatosis and IR are involved in the pathogenesis of chronic HCV infection, either as metabolic predisposition or directly induced by HCV (Fig. 1 and Table 2). An increased prevalence of steatosis and IR has been observed in patients with HCV infection and has prognostic implications, as it is associated with faster progression to cirrhosis and HCC as well as with a poorer response to treatment. In patients infected with HCV genotypes 1 and 2, steatosis often develops in the context of a pre-existing diabetes, IR or increased body mass index. By comparison, in patients infected with HCV genotype 3, steatosis is directly induced by HCV, because it correlates with the viral load and reverses with response to antiviral treatment [71]. HCV is thought to induce steatosis by interfering with lipid secretion and degradation and by increasing lipid synthesis. The HCV core protein, which localizes to the surface of lipid droplets and mediates viral assembly in close association with the cellular fatty acid metabolism [16], as well as some HCV non-structural proteins, have been shown to interfere with very low density lipoprotein (vLDL) secretion [72], [73]. HCV infection also upregulates lipid synthesis [63], inhibits fatty acid oxidation [74], [75] and increases release of fatty acids from adipocytes [71]. Overall the effects of HCV proteins on lipid synthesis, secretion and oxidation seem to be most pronounced in HCV genotype 3 infection, but also occur in patients infected with other genotypes. Besides changes in the lipid metabolism, HCV core and several non-structural proteins, induce systemic oxidative stress and related signaling by various mechanisms [76]. With respect to IR, all HCV genotypes have been shown to interfere with glucose homeostasis, often at early stages in HCV infection [71]. The mechanism underlying IR and its severity seem again to be genotype dependent. HCV has been shown to interfere with insulin signaling by proteasomal degradation of IRS-1 and -2 either via SOCS proteins or the PI3K/Akt/mTOR pathway, as well as by IRS-1 inactivation via transforming growth factor (TGF)-α and PI3K/Akt [77]. Thus, the initial or early stages of HCV infection are strongly associated with IR. In contrast, at late stages of disease and in particular in tumorigenesis, transformed cells have been shown to require more glucose and to upregulate insulin sensitivity and glucose uptake [78].
6. From liver inflammation to liver cancer
The interdependence between steatosis, IR and oxidative stress is important for disease progression in NAFLD as well as in hepatitis C and induces tissue damage and inflammation with activation of hepatic stellate cells (HSCs). Activated HSCs become responsive to both proliferative and fibrogenic cytokines and undergo epithelial to mesenchymal trans-differentiation (EMT) into contractile myo-fibroblast-like cells, that synthesize extracellular matrix (ECM) components, which accumulate over time to form fibrous scars or fibrosis. Ultimately, regenerating hepatocytes become enclosed by scar tissue and form nodules that define cirrhosis. HSCs are activated by products and effectors of oxidative stress and growth factors, cytokines, adipokines and chemokines, secreted by hepatocytes, Kupffer and inflammatory cells that infiltrate the liver in response to infection. The cytokine TGF-β, a potent inhibitor of epithelial cell growth and tumor suppressor, is a key regulator of EMT and also has pro-oncogenic functions. Importantly, recent findings indicate that TGF-β induces EMT not only in HSCs but possibly also in hepatocytes [79]. TGF-β signaling is upregulated in fibrosis in HCV-infected patients and stimulates ECM deposition and accumulation. IR may link fibrosis and steatosis, since it stimulates HSCs to deposit ECM. Several signaling cascades are involved in fibrogenesis, including SMADs, PI3K-Akt and various MAPK pathways, such as p38 and JNK. While SMADs are indispensable for EMT, TGF-β signaling via SMAD interacts with other signaling pathways to mediate pro-oncogenic EMT. JNK activation by the pro-inflammatory cytokine interleukin (IL)-1β can shift TGF-β signaling from tumor suppression to oncogenesis with increased fibrogenesis, cell motility and transactivation of cell cycle regulatory genes [79], [80]. Thus, in the context of chronic inflammation, the interplay between ER/oxidative stress, steatosis and IR induces a pro-oncogenic microenvironment that results in fibrogenesis and genomic instability. Even though HCV has been reported to have direct transforming properties, the liver microenvironment is thought to significantly modulate the transformation process because HCC develops in chronic HCV infection only over long periods of time.
7. Direct oncogenic effects of HCV
Apart from complex interactions among themselves, HCV proteins interact with a number of host factors and signaling pathways and thus contribute to the progression from chronic hepatitis C to liver cirrhosis and HCC (Table 2). By modulating gene transcription and translation as well as post-translational events, the HCV proteins interfere with innate immunity to favor viral persistence and liver inflammation; they alter cell signaling, apoptosis, membrane physiology and protein trafficking, induce oxidative stress, genomic instability as well as malignant transformation. Among the HCV proteins core, NS3, NS4B and NS5A have all been shown to have transforming potential when transiently or stably expressed in cell culture, or in transgenic mice expressing the different viral proteins or the HCV polyprotein [81], [82], [83], [84].
The HCV core protein is a highly conserved, basic protein that multimerizes, probably in conjunction with microtubules [85] to form the viral nucleocapsid and to package the viral RNA genome. It is localized at the cytoplasmic surface of the ER and on lipid droplets, and the latter observation is likely related to the induction of liver steatosis observed in HCV-infected patients as well as in transgenic mice overexpressing HCV core [86], [87], [88]. Core has also been shown to localize to the outer membranes of mitochondria [89] and is involved in changes of apoptosis and lipid metabolism as well as in malignant transformation. Among many interactions with cellular factors, core has been shown to induce ROS production via interaction with heat shock protein Hsp60 [90], to bind the tumor suppressor proteins p53 [91], [92], p73 [93] and pRb [94]. Core also inhibits the expression of the cyclin-dependent kinase (CDK) inhibitor p21/Waf [95]. p21 is a transcriptional target of p53 and blocks the cyclin/CDK complexes involved in cell-cycle control and tumor formation. Core induces activation of the Raf1/MAPK pathway [96], [97], protects cells from serum starvation and growth arrest and drives cells into proliferation. NF-κB transcription has been shown to be activated [98], [99], [100] and repressed [101] by HCV core. HCV core has also been shown to activate the Wnt/b-catenin pathway, which controls cell proliferation, DNA synthesis and cell-cycle progression [102]. Furthermore, HCV core variants have been shown to interact with SMAD3 and to inhibit the TGF-β pathway [103]. TGF-β signaling not only controls cell proliferation, differentiation and apoptosis but also stimulates liver regeneration and fibrogenesis through its actions on the extracellular matrix (see above). TGF-β levels are frequently increased in patients with chronic HCV infection and correlate with the degree of fibrosis [104], [105]. Finally, HCV core protein associates with cellular membranes [88], [106] and lipid vesicles [106], binds to apolipoprotein II [107], [108] and reduces microsomal triglyceride transfer protein activity [108], resulting in impaired assembly and secretion of vLDL, steatosis and oxidative stress. These in vitro findings are likely to be relevant for HCV pathogenesis because transgenic mice expressing HCV core protein also develop steatosis [108], [109] and HCC [67], [68].
Overexpression of E2 inhibits eIF2α phosphorylation by the dsRNA-activated protein kinase (PKR) or the ER-stress signaling kinase PERK [110], [111]. Similarly, overexpression of NS4A, NS4B, or NS4A-4B has been reported to induce an ER stress-mediated unfolded protein response, to reduce ER-to-Golgi trafficking, to inhibit protein synthesis, and to cause cytopathic effects [112], [113], [114], [115], [116]. NS2 has been shown to inhibit the cellular proapoptotic molecule CIDE-B [117] and to downregulate transcription [118].
NS3-4A serine protease has been reported to interact with p53 to repress p21 function, to block activation of the transcription factors IRF-3 and NF-κB and to antagonize the innate antiviral defenses by interfering with RIG-1, MDA5 and TLR3 mediated signal transduction [119], [120], [121]. Indeed, RIG-I inactivation has been shown to render Huh-7 cells permissive to HCV replication [122], [123].
NS5A has been shown to interact with the geranylgeranylated cellular protein FBL2 [124], an F-box motif containing protein that is probably involved in targeting cellular proteins of yet unknown identity for ubiquitylation and degradation. A number of studies suggest that NS5A is also involved in IFN resistance [8] and one possible mechanism may be its ability to induce expression of the type I interferon anatagonist IL-8 [125]. In addition, NS5A has been described to contain an ‘interferon sensitivity determining region’ (ISDR), that has been described to mediate inhibition of PKR, an activator of the innate immunity [83], [126], [127]. The accumulation of mutations in this region is thought to correlate with treatment efficacy [128], [129]. Importantly, overexpression of NS5A has been reported to induce a number of effects in cells, including oxidative stress, activation of signaling pathways such as STAT-3, PI3K, and NF-κB and altered transcriptional regulation including p21 [130], [131], [132] and of pRB [133]. Other NS5A interaction partners include apoliporotein A1, the major protein found on HDLs, the tumor suppressor p53, Grb-2, an adaptor protein involved in mitogen signaling, SRCAP, an adenosine triphosphatase (ATPase) that activates cellular transcription, karyopherin β3, a protein involved in nuclear trafficking, Cdk1/2, cyclin-dependent and Fyn, Hck, Lck, and Lyn, Src-family kinases [8], [134], [135], [136], [137]. It has also been reported that NS5A expression in the context of the HCV polyprotein results in the inhibition of the transcription factor Forkhead as well as in the phosphorylation and inactivation of the GSK-3, leading to accumulation of β-catenin and stimulation of β-catenin-dependent transcription [138]. Finally, NS5A dependent activation of upstream binding factor, a Pol I DNA binding transcription factor, which occurs as a result of up-regulation of both cyclin D1 and CDK4, leads to enhancement of rRNA transcription activation [139].
8. Genetic and epigenetic changes in HCV-induced hepato-carcinogenesis
HCCs are genetically very heterogeneous tumors. This is not unexpected, given the number of etiological factors implicated in its development, the complexity of hepatocyte physiology and the advanced stage at which HCCs are usually diagnosed. Genome-wide analysis of genetic alterations occurring in HCC revealed two major mechanisms of hepatocarcinogenesis. In the first, genetic alterations are acquired in the context of elevated oxidative stress caused by the vicious circle between chronic inflammation, IR and steatosis; in the second, transformation is induced by β-catenin mutations that dysregulate the Wnt pathway [140]. So far, it has not been possible to correlate chromosome instability with a consistent pattern of proto-oncogene activation in HCC, but several growth factor signaling pathways are frequently affected, including insulin-growth factor (IGF)-, hepatocyte growth factor-, Wnt-, TGF-α/EGF- and TGF-β-signaling [58], [141]. The interplay between these pathways and their respective roles and contributions to HCC development remain to be elucidated, however. One of the most commonly affected pathways involved in cell cycle check control is the p53 pathway, that limits cell survival and proliferation in response to telomere shortening and oncogene activation in order to ensure genome integrity. Loss of p53 by e.g. deletion, mutation, degradation or direct inhibition during progression of chronic hepatitis C to cirrhosis likely results in proliferation of hepatocytes with shortened telomeres or chromosomal damage and to predispose to hepatocarcinogenesis. Indeed, p21 expression, a downstream target of p53 that blocks cell entry into the S phase, is increased in cirrhosis when presumably significant numbers of hepatocytes with genome damage have accumulated, but is lost in premalignant liver lesions and HCC [142]. Besides p53, the retinoblastoma (Rb) pathway is another major checkpoint that limits cell proliferation in response to DNA damage, telomere shortening and oncogene activation. In human HCC, the Rb pathway is defective in more than 80% of cases [143]. Moreover, gankyrin, an inhibitor of p53 and Rb check point function is overexpressed in the vast majority of HCCs [144]. Expression of insulin-like growth factor IGF-2 is frequent and thought to be an early event in hepatocarcinogenesis, present in more than 60% of dysplastic nodules and HCC [145]. IGF-2 receptor impairs cell proliferation by promoting degradation of the IGF-2 mitogen and by activation of TGF-β signaling [146].
β-Catenin pathway activation is very common in hepatocarcinogenesis and detectable in more than 50% of HCCs. It can directly induce hepatocyte transformation without the need for multiple genetic/epigenetic alterations [147]. β-Catenin activation is mainly induced by β-catenin gene mutations and/or Wnt signaling pathway alterations [143]. Wnt/frizzled/β-catenin signaling is mediated by a complex interaction between a Wnt ligand (Wnt) and a Frizzled receptor (Fzd), mostly in cooperation with the low density lipoprotein receptor (LDLR)-related proteins LRP-5 or -6. Normally, the Wnt/β-catenin pathway is involved in cell growth and proliferation as well as developmental control and cell adhesion. Cellular levels of β-catenin are tightly regulated by proteasome-dependent degradation, which is in turn controlled by the activity of the APC and Axin1 proteins, and the glycogen synthase kinase-3b (GSK-3b). A recent report indicates that Fzd-7, which stabilizes and activates β-catenin [148], is overexpressed in more than 90% of HCCs and in around 75% of the peritumorous/precancerous liver tissue.
In addition to the dysregulation of the above pathways, HCV infection has been shown to induce or correlate with epigenetic changes that are likely to contribute to hepatocarcinogenesis. HCV-induced ROS have been shown to activate histone deacetylase in a fashion similar to hydrogen peroxide and to cause hypoacetylation of histones [149]. Hypomethylation of the IGF-2 locus in hepatitis C cirrhosis has been shown to predict HCC development [150]. Another study reported the hypermethylation of p16, p15, p14, pRB and the PTEN promoters in patients with sustained viral response in comparison to non-responders [151]. Finally, increased hTERT DNA levels have been found to predict HCC development [152].
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