Public release date: 28-Dec-2010
Contact: Mika Ono
mikaono@scripps.edu
858-784-2052
Scripps Research Institute
Findings point toward a novel therapeutic target for antiviral drugs
JUPITER, FL, December 28, 2010 – Scientists from the Florida campus of The Scripps Research Institute have identified a molecular interaction between a structural hepatitis C virus protein (HCV) and a protein critical to viral replication. This new finding strongly suggests a novel method of inhibiting the production of the virus and a potential new therapeutic target for hepatitis C drug development.
The study was published in the January 2010 issue (Volume 92, Part 1) of the Journal of General Virology.
These new data underline the essential role of the viral protein known as "core" as a primary organizer of the infectious HCV particle assembly and support a new molecular understanding of the formation of the viral particle itself.
"While our finding that the HCV core interacts with the non-structural helicase protein was not totally unexpected, this had not really been confirmed until this study," said Scripps Florida Professor Donny Strosberg, who led the study. "But the most exciting part is that small molecule inhibitors of dimerization [the joining of two identical subunits] of core actually inhibit interaction between core and helicase, thus possibly preventing production of an infectious viral particle."
A Viral Plague
Hepatitis C virus infects between 130 and 170 million people worldwide and is the cause of an epidemic of liver cirrhosis and cancer. Because current HCV treatments are only partially effective, a number of alternative molecular mechanisms are actively being pursued as possible drug targets.
One of the critical problems of finding inhibitors for the hepatitis C virus is that it mutates at such prodigious rates. An RNA virus such as hepatitis C can mutate at a rate estimated as high as one million times that of DNA viruses such as the herpes virus.
With this in mind, Strosberg has been examining the core protein, the most conserved protein among all HCV genotypes. Core plays several essential roles in the viral cycle in the host cell. It is particularly important in the assembly of the hepatitis C nucleocapsid or capsid, an essential step in the formation of infectious viral particles; the nucleocapsid is the virus genome protected by a protein coat. By interacting with various structural and non-structural viral proteins, core plays an essential role in the HCV cycle during assembly and release of the infectious virus as well as disassembly of viral particles upon entering host cells. Core also interacts with a number of cellular proteins, possibly contributing to the disarmament of several host defense mechanisms and to the activation of oncogenic pathways.
Last year, Strosberg developed a novel quantitative test for monitoring these protein-protein interactions with the specific goal of identifying inhibitors of the core dimerization, which would block virus production. Strosberg and his colleagues uncovered peptides derived from the core protein of hepatitis C that inhibit not only dimerization of the core protein, but also production of the actual virus.
That earlier study led to the discovery of non-peptidic small organic molecules that strongly inhibited HCV production, one of which, SL201, was used in the new study.
In the new study, Strosberg and his colleagues focused on non-structural proteins that provide functions relating to HCV production, in particular NS3 helicase. The scientists' findings support a growing body of evidence that this protein participates in the assembly and production of infectious viral particles. The interaction of the core protein with this non-structural protein also confirms core as a key organizer of virus assembly and suggests it acts to facilitate the packaging and integration of the newly synthesized viral RNA.
###
The first author of the study, "Dimerization-Driven Interaction of Hepatitis C Virus Core Protein with Ns3 Helicase," is Guillaume Mousseau of Scripps Research. Additional authors include Smitha Kota, S. and Virginia Takahashi of Scripps Research, and David Frick of the University of Wisconsin, Milwaukee. For more information, see http://vir.sgmjournals.org/cgi/content/abstract/92/1/101.
The study was supported by the state of Florida, The Factor Foundation, and the National Institutes of Health.
About The Scripps Research Institute
The Scripps Research Institute is one of the world's largest independent, non-profit biomedical research organizations, at the forefront of basic biomedical science that seeks to comprehend the most fundamental processes of life. Scripps Research is internationally recognized for its discoveries in immunology, molecular and cellular biology, chemistry, neurosciences, autoimmune, cardiovascular, and infectious diseases, and synthetic vaccine development. An institution that evolved from the Scripps Metabolic Clinic founded by philanthropist Ellen Browning Scripps in 1924, Scripps Research currently employs approximately 3,000 scientists, postdoctoral fellows, scientific and other technicians, doctoral degree graduate students, and administrative and technical support personnel. Headquartered in La Jolla, California, the institute also includes Scripps Florida, whose researchers focus on basic biomedical science, drug discovery, and technology development. Scripps Florida is located in Jupiter, Florida. For more information, see http://www.scripps.edu/.
Source
December 28, 2010
Blacks Face Higher Death Risk from Early Liver Cancer
By John Gever, Senior Editor, MedPage Today
Published: December 21, 2010
Reviewed by Zalman S. Agus, MD; Emeritus Professor
University of Pennsylvania School of Medicine and
Dorothy Caputo, MA, RN, BC-ADM, CDE, Nurse Planner
African-Americans diagnosed with early-stage liver cancer face "persistently poor survival" relative to white patients, after accounting for other factors known to affect outcomes, researchers said.
Analyzing 12 years of data in the federal Surveillance, Epidemiology, and End Results (SEER) registry, researchers at the University of Michigan in Ann Arbor calculated a hazard ratio for death of 1.11 (95% CI 1.03 to 1.20) for black patients with stage I or II hepatocellular carcinoma (HCC) relative to white patients, after accounting for numerous factors, including the types of treatment provided.
"This finding is linked to two major issues that contribute to health-related disparities in minority populations," wrote Christopher Sonnenday, MD, MHS, and colleagues in the December issue of Archives of Surgery. "Black patients have poor access to treatment, and, even after obtaining treatment, they have relatively poor outcomes compared with white patients."
The study also showed that, after adjusting for the different treatment mixes seen among racial groups, survival rates were the same in Hispanic patients relative to whites, and were higher in Asian patients.
The latter finding, according to Sonnenday and colleagues, "may be explained by Asian patients tending to present with HCC resulting from chronic hepatitis B virus infection ... and by greater awareness of HCC owing to screening programs that target this population."
SEER data covering 13,244 cases of stage I or II HCC diagnosed from 1995 to 2006 were the basis for the findings. In addition to eventual outcomes, the data included tumor size at diagnosis, type of treatment provided, patients' race/ethnicity, age, and gender, and place of residence.
Patient-specific socioeconomic and educational data were not available, but the researchers made rough estimates using countywide averages for the patients' residences.
Sonnenday and colleagues found important differences in the types of treatment provided to the four racial-ethnic groups.
Overall, 32.8% of patients received "invasive therapy," defined as transplant, hepatectomy, or focused tumor ablation. In blacks and Hispanics, the figure was closer to 28%, while in whites it was 35%.
Adjusting for baseline tumor size, age, and the projected sociodemographic variables -- but not treatment type -- hazard ratios for death in the three nonwhite groups relative to whites were as follows:
• Blacks: 1.24 (95% CI 1.15 to 1.33)
• Hispanics: 1.08 (95% CI 1.01 to 1.15)
• Asian: 0.87 (95% CI 0.82 to 0.93)
Adding treatment type to the adjustments reduced these values in all three groups. In Hispanics, it declined to 0.97, which did not differ significantly from whites. In Asians, the fully adjusted hazard ratio was 0.84 (95% CI 0.79 to 0.89).
That the fully adjusted value for blacks of 1.11 remained significant was an indication that outcomes of aggressive treatment were not as good as in the other groups, Sonnenday and colleagues suggested.
They also considered the overall invasive-therapy rate of 32.8% to reflect "significant underuse of appropriate interventions for the most treatable stages of HCC."
But the racial differences in invasive therapy usage were also a concern, including a higher rate of hepatectomy in Asian patients and "a nearly twofold lower rate of liver transplant in blacks compared with white patients."
Sonnenday and colleagues described the latter as "clearly contingent on access to care," particularly in light of another recent study showing equal transplant rates in blacks and whites who get on waiting lists for the procedure.
A researcher at Johns Hopkins University in Baltimore concurred in an accompanying commentary that early-stage HCC patients are often undertreated.
Richard D. Schulik, MD, called the 32.8% figure for invasive therapy "alarming because stage I and II disease is typically treated with good outcomes."
But he warned that the study authors' other interpretations may not be valid.
They "assumed that patients ... had intrinsic differences that affected which therapy was selected for them," Schulick wrote.
"This assumption may not be as valid as the authors think because sometimes patients are treated according to how they enter the system for treatment."
Schulick wrote that in facilities that don't screen patients with a multidisciplinary team, the treatment choice may be heavily influenced by whether patients first see a surgical oncologist, a transplant surgeon, or an interventional radiologist.
He also emphasized the importance of study limitations listed by Sonnenday and colleagues, including the possibility that patients may have been inappropriately staged and the lack of patient-specific information on income, educational level, and other factors that were estimated crudely in the study.
The study was supported by the National Institutes of Health.
Study authors and Schulick declared they had no competing financial interests.
Primary source: Archives of Surgery
Source reference:
Mathur A, et al "Racial/ethnic disparities in access to care and survival for patients with early-stage hepatocellular carcinoma" Arch Surg 2010; 145: 1158-63.
Additional source: Archives of Surgery
Source reference:
Schulick R "Undertreatment of patients with early-stage hepatocellular carcinoma" Arch Surg 2010; 145: 1163-64.
Source
Published: December 21, 2010
Reviewed by Zalman S. Agus, MD; Emeritus Professor
University of Pennsylvania School of Medicine and
Dorothy Caputo, MA, RN, BC-ADM, CDE, Nurse Planner
African-Americans diagnosed with early-stage liver cancer face "persistently poor survival" relative to white patients, after accounting for other factors known to affect outcomes, researchers said.
Analyzing 12 years of data in the federal Surveillance, Epidemiology, and End Results (SEER) registry, researchers at the University of Michigan in Ann Arbor calculated a hazard ratio for death of 1.11 (95% CI 1.03 to 1.20) for black patients with stage I or II hepatocellular carcinoma (HCC) relative to white patients, after accounting for numerous factors, including the types of treatment provided.
"This finding is linked to two major issues that contribute to health-related disparities in minority populations," wrote Christopher Sonnenday, MD, MHS, and colleagues in the December issue of Archives of Surgery. "Black patients have poor access to treatment, and, even after obtaining treatment, they have relatively poor outcomes compared with white patients."
The study also showed that, after adjusting for the different treatment mixes seen among racial groups, survival rates were the same in Hispanic patients relative to whites, and were higher in Asian patients.
The latter finding, according to Sonnenday and colleagues, "may be explained by Asian patients tending to present with HCC resulting from chronic hepatitis B virus infection ... and by greater awareness of HCC owing to screening programs that target this population."
SEER data covering 13,244 cases of stage I or II HCC diagnosed from 1995 to 2006 were the basis for the findings. In addition to eventual outcomes, the data included tumor size at diagnosis, type of treatment provided, patients' race/ethnicity, age, and gender, and place of residence.
Patient-specific socioeconomic and educational data were not available, but the researchers made rough estimates using countywide averages for the patients' residences.
Sonnenday and colleagues found important differences in the types of treatment provided to the four racial-ethnic groups.
Overall, 32.8% of patients received "invasive therapy," defined as transplant, hepatectomy, or focused tumor ablation. In blacks and Hispanics, the figure was closer to 28%, while in whites it was 35%.
Adjusting for baseline tumor size, age, and the projected sociodemographic variables -- but not treatment type -- hazard ratios for death in the three nonwhite groups relative to whites were as follows:
• Blacks: 1.24 (95% CI 1.15 to 1.33)
• Hispanics: 1.08 (95% CI 1.01 to 1.15)
• Asian: 0.87 (95% CI 0.82 to 0.93)
Adding treatment type to the adjustments reduced these values in all three groups. In Hispanics, it declined to 0.97, which did not differ significantly from whites. In Asians, the fully adjusted hazard ratio was 0.84 (95% CI 0.79 to 0.89).
That the fully adjusted value for blacks of 1.11 remained significant was an indication that outcomes of aggressive treatment were not as good as in the other groups, Sonnenday and colleagues suggested.
They also considered the overall invasive-therapy rate of 32.8% to reflect "significant underuse of appropriate interventions for the most treatable stages of HCC."
But the racial differences in invasive therapy usage were also a concern, including a higher rate of hepatectomy in Asian patients and "a nearly twofold lower rate of liver transplant in blacks compared with white patients."
Sonnenday and colleagues described the latter as "clearly contingent on access to care," particularly in light of another recent study showing equal transplant rates in blacks and whites who get on waiting lists for the procedure.
A researcher at Johns Hopkins University in Baltimore concurred in an accompanying commentary that early-stage HCC patients are often undertreated.
Richard D. Schulik, MD, called the 32.8% figure for invasive therapy "alarming because stage I and II disease is typically treated with good outcomes."
But he warned that the study authors' other interpretations may not be valid.
They "assumed that patients ... had intrinsic differences that affected which therapy was selected for them," Schulick wrote.
"This assumption may not be as valid as the authors think because sometimes patients are treated according to how they enter the system for treatment."
Schulick wrote that in facilities that don't screen patients with a multidisciplinary team, the treatment choice may be heavily influenced by whether patients first see a surgical oncologist, a transplant surgeon, or an interventional radiologist.
He also emphasized the importance of study limitations listed by Sonnenday and colleagues, including the possibility that patients may have been inappropriately staged and the lack of patient-specific information on income, educational level, and other factors that were estimated crudely in the study.
The study was supported by the National Institutes of Health.
Study authors and Schulick declared they had no competing financial interests.
Primary source: Archives of Surgery
Source reference:
Mathur A, et al "Racial/ethnic disparities in access to care and survival for patients with early-stage hepatocellular carcinoma" Arch Surg 2010; 145: 1158-63.
Additional source: Archives of Surgery
Source reference:
Schulick R "Undertreatment of patients with early-stage hepatocellular carcinoma" Arch Surg 2010; 145: 1163-64.
Source
Pegylated Interferon-Lambda for Chronic Hepatitis C Virus Infection
Summary and Comment
In a phase Ib study, PEG-IFN-Lambda showed promise in both treatment-naive and relapsing patients.
The cornerstone for treating patients with hepatitis C virus (HCV) infection is pegylated interferon alfa (PEG-IFN-alpha ) plus ribavirin. However, adverse effects can reduce quality of life, lead to dose reductions, and even prompt discontinuation of therapy, with potential consequences for efficacy. Interferon-Lambda1, also known as interleukin-29, has shown in vitro antiviral activity similar to that of PEG-IFN-apha Unlike the widely distributed IFN-alpha receptors, expression of the IFN-Lambda1 receptor is more restricted, suggesting that a treatment that targets IFN-Lambda1 might be less prone to cause adverse effects.
In an industry-funded, open-label phase-Ib study, investigators tested the efficacy of PEG-IFN-Lambda in HCV genotype 1–infected patients. Twenty-four patients who had relapsed on PEG-IFN-alpha therapy received PEG-IFN-Lambda monotherapy (1.5 µg/kg or 3.0 µg/kg, either weekly or twice weekly), 25 patients who had relapsed on IFN-alpha therapy received PEG-IFN-Lambda (0.50–2.25 µg/kg weekly) plus ribavirin, and 7 treatment-naive patients received PEG-IFN-Lambda (1.5 µg/kg weekly) plus ribavirin. All treatments lasted 4 weeks; antiviral and safety assessments were made for at least another 4 weeks.
In two of the seven treatment-naive patients, HCV became undetectable by week 4. All PEG-IFN-Lambda doses induced virologic response, in a roughly dose-related pattern. Flu-like symptoms were uncommon; hematologic abnormalities were observed only in relation to use of ribavirin. The most commonly reported adverse symptoms were fatigue (29%), nausea (12%), and myalgias (11%). The incidence of adverse events was not dose related.
Comment: Current and future HCV therapies are likely to use interferon as the backbone. In this study, PEG-IFN-Lambda had strong antiviral activity with minimal adverse effects. If later-phase trials confirm the virologic response and low incidence of adverse effects, PEG-IFN-Lambda could become the foundation of future treatment regimens. Both treatment-naive and relapsing patients might then have treatment options that are more effective, better tolerated, and less likely to be discontinued than currently available therapies.
— David A. Johnson, MD
Published in Journal Watch Gastroenterology December 22, 2010
Citation(s):
Muir AJ et al. Phase 1b study of pegylated interferon lambda 1 with or without ribavirin in patients with chronic genotype 1 hepatitis C virus infection. Hepatology 2010 Sep; 52:822.
Medline abstract (Free)
In a phase Ib study, PEG-IFN-Lambda showed promise in both treatment-naive and relapsing patients.
The cornerstone for treating patients with hepatitis C virus (HCV) infection is pegylated interferon alfa (PEG-IFN-alpha ) plus ribavirin. However, adverse effects can reduce quality of life, lead to dose reductions, and even prompt discontinuation of therapy, with potential consequences for efficacy. Interferon-Lambda1, also known as interleukin-29, has shown in vitro antiviral activity similar to that of PEG-IFN-apha Unlike the widely distributed IFN-alpha receptors, expression of the IFN-Lambda1 receptor is more restricted, suggesting that a treatment that targets IFN-Lambda1 might be less prone to cause adverse effects.
In an industry-funded, open-label phase-Ib study, investigators tested the efficacy of PEG-IFN-Lambda in HCV genotype 1–infected patients. Twenty-four patients who had relapsed on PEG-IFN-alpha therapy received PEG-IFN-Lambda monotherapy (1.5 µg/kg or 3.0 µg/kg, either weekly or twice weekly), 25 patients who had relapsed on IFN-alpha therapy received PEG-IFN-Lambda (0.50–2.25 µg/kg weekly) plus ribavirin, and 7 treatment-naive patients received PEG-IFN-Lambda (1.5 µg/kg weekly) plus ribavirin. All treatments lasted 4 weeks; antiviral and safety assessments were made for at least another 4 weeks.
In two of the seven treatment-naive patients, HCV became undetectable by week 4. All PEG-IFN-Lambda doses induced virologic response, in a roughly dose-related pattern. Flu-like symptoms were uncommon; hematologic abnormalities were observed only in relation to use of ribavirin. The most commonly reported adverse symptoms were fatigue (29%), nausea (12%), and myalgias (11%). The incidence of adverse events was not dose related.
Comment: Current and future HCV therapies are likely to use interferon as the backbone. In this study, PEG-IFN-Lambda had strong antiviral activity with minimal adverse effects. If later-phase trials confirm the virologic response and low incidence of adverse effects, PEG-IFN-Lambda could become the foundation of future treatment regimens. Both treatment-naive and relapsing patients might then have treatment options that are more effective, better tolerated, and less likely to be discontinued than currently available therapies.
— David A. Johnson, MD
Published in Journal Watch Gastroenterology December 22, 2010
Citation(s):
Muir AJ et al. Phase 1b study of pegylated interferon lambda 1 with or without ribavirin in patients with chronic genotype 1 hepatitis C virus infection. Hepatology 2010 Sep; 52:822.
Medline abstract (Free)
Transient elastography and biomarkers for liver fibrosis assessment and follow-up of inactive hepatitis B carriers
Alimentary Pharmacology & Therapeutics
Early View (Articles online in advance of print)
L. Castéra1,2, P.-H. Bernard2, B. Le Bail3, J. Foucher1,2, P. Trimoulet4, W. Merrouche1, P. Couzigou1, V. de Lédinghen1
Article first published online: 23 DEC 2010
DOI: 10.1111/j.1365-2036.2010.04547.x
Abstract
Summary
Background Non invasive methods for fibrosis evaluation remain to be validated longitudinally in hepatitis B.
Aim To evaluate longitudinally transient elastography (TE) and biomarkers for liver fibrosis assessment and follow-up of hepatitis B virus (HBV) inactive carriers.
Methods Three hundred and twenty-nine consecutive HBeAg-negative HBV patients (201 inactive carriers) who underwent TE, Fibrotest and aspartate to platelet ratio index (APRI) the same day were studied.
Results TE (median 4.8 vs. 6.8 kPa, P < 0.0001), Fibrotest (0.16 vs. 0.35, P < 0.0001) and APRI values (0.28 vs. 0.43, P < 0.0001) were significantly lower in inactive carriers than in the remaining patients whereas they did not differ among inactive carriers according to HBV DNA levels. In 82 inactive carriers with repeated examinations, although differences were observed among individual patients, TE values did not differ significantly over time (median intra-patient changes at end of follow-up relative to baseline: −0.2 kPa, P = 0.12). Conversely, significant fluctuations were observed for Fibrotest (+0.03, P = 0.012) and APRI (−0.01, P < 0.05). Eleven inactive carriers (5.5%) had initial elevated TE values (>7.2 kPa) confirmed during follow-up in two with significant fibrosis (F2 and F3) on liver biopsy.
Conclusion Non-invasive tools, particularly TE, could be useful, in addition to HBV DNA and transaminase levels, for follow-up of HBV inactive carriers as well as better selection of patients who require a liver biopsy.
Source
Early View (Articles online in advance of print)
L. Castéra1,2, P.-H. Bernard2, B. Le Bail3, J. Foucher1,2, P. Trimoulet4, W. Merrouche1, P. Couzigou1, V. de Lédinghen1
Article first published online: 23 DEC 2010
DOI: 10.1111/j.1365-2036.2010.04547.x
Abstract
Summary
Background Non invasive methods for fibrosis evaluation remain to be validated longitudinally in hepatitis B.
Aim To evaluate longitudinally transient elastography (TE) and biomarkers for liver fibrosis assessment and follow-up of hepatitis B virus (HBV) inactive carriers.
Methods Three hundred and twenty-nine consecutive HBeAg-negative HBV patients (201 inactive carriers) who underwent TE, Fibrotest and aspartate to platelet ratio index (APRI) the same day were studied.
Results TE (median 4.8 vs. 6.8 kPa, P < 0.0001), Fibrotest (0.16 vs. 0.35, P < 0.0001) and APRI values (0.28 vs. 0.43, P < 0.0001) were significantly lower in inactive carriers than in the remaining patients whereas they did not differ among inactive carriers according to HBV DNA levels. In 82 inactive carriers with repeated examinations, although differences were observed among individual patients, TE values did not differ significantly over time (median intra-patient changes at end of follow-up relative to baseline: −0.2 kPa, P = 0.12). Conversely, significant fluctuations were observed for Fibrotest (+0.03, P = 0.012) and APRI (−0.01, P < 0.05). Eleven inactive carriers (5.5%) had initial elevated TE values (>7.2 kPa) confirmed during follow-up in two with significant fibrosis (F2 and F3) on liver biopsy.
Conclusion Non-invasive tools, particularly TE, could be useful, in addition to HBV DNA and transaminase levels, for follow-up of HBV inactive carriers as well as better selection of patients who require a liver biopsy.
Source
Current antiviral combination therapy for chronic hepatitis C patients who failed to interferon alfa-based treatment
Journal of Clinical Pharmacy and Therapeutics
Early View (Articles online in advance of print)
M. Trapero-Marugán MD1, J. Mendoza MD1, J. A. Moreno Monteagudo MD1, M. Chaparro MD1, L. García-Buey MD1, L. González-Moreno MD2, M. J. Borque BSc3, R. Moreno-Otero MD1
Article first published online: 22 DEC 2010
DOI: 10.1111/j.1365-2710.2010.01231.x
Abstract
Keywords:chronic hepatitis C; non-response;peginterferon; relapse; ribavirin; sustained virological response
Summary
What is known and Objective: Interferon-alfa-based therapy is effective in the treatment of Hepatitis C. However, some patients fail to respond and others relapse, after initially responding. Our objective was to assess the efficacy, safety and predictive factors for sustained virological response (SVR) to peginterferon plus ribavirin in chronic hepatitis C patients who failed to interferon-alfa (IFNα)-based therapy.
Methods: Seventy-five consecutive patients who failed to IFNα-based therapy were retreated with peginterferon plus ribavirin. Of these patients, 85% were infected by genotype 1. The primary endpoint was SVR.
Results and Discussion: Of 75 non-responder (n = 54) or relapser patients (n = 21), 50 were previously treated with IFNα-monotherapy and 25 with IFNα plus ribavirin. Global SVR rate was 41·3%: for patients re-treated with IFNα the response was 48% whilst for those retreated with IFNα plus ribavirin, it was 28%. For previous non-responders the SVR rate was 37% and for relapsers it was 52·4%.
What is new and Conclusion: Retreatment with peginterferon plus ribavirin is an effective option for some chronic hepatitis C non-responder or relapser patients. Higher SVR rate was achieved in relapsers and in those patients who received IFNα monotherapy previously.
Source
Early View (Articles online in advance of print)
M. Trapero-Marugán MD1, J. Mendoza MD1, J. A. Moreno Monteagudo MD1, M. Chaparro MD1, L. García-Buey MD1, L. González-Moreno MD2, M. J. Borque BSc3, R. Moreno-Otero MD1
Article first published online: 22 DEC 2010
DOI: 10.1111/j.1365-2710.2010.01231.x
Abstract
Keywords:chronic hepatitis C; non-response;peginterferon; relapse; ribavirin; sustained virological response
Summary
What is known and Objective: Interferon-alfa-based therapy is effective in the treatment of Hepatitis C. However, some patients fail to respond and others relapse, after initially responding. Our objective was to assess the efficacy, safety and predictive factors for sustained virological response (SVR) to peginterferon plus ribavirin in chronic hepatitis C patients who failed to interferon-alfa (IFNα)-based therapy.
Methods: Seventy-five consecutive patients who failed to IFNα-based therapy were retreated with peginterferon plus ribavirin. Of these patients, 85% were infected by genotype 1. The primary endpoint was SVR.
Results and Discussion: Of 75 non-responder (n = 54) or relapser patients (n = 21), 50 were previously treated with IFNα-monotherapy and 25 with IFNα plus ribavirin. Global SVR rate was 41·3%: for patients re-treated with IFNα the response was 48% whilst for those retreated with IFNα plus ribavirin, it was 28%. For previous non-responders the SVR rate was 37% and for relapsers it was 52·4%.
What is new and Conclusion: Retreatment with peginterferon plus ribavirin is an effective option for some chronic hepatitis C non-responder or relapser patients. Higher SVR rate was achieved in relapsers and in those patients who received IFNα monotherapy previously.
Source
Early Menopause is Associated with Lack of Response to Antiviral Therapy in Women with Chronic Hepatitis C
"This suggests that CHC in women should be treated early, disregarding the fact that liver disease is milder in women of reproductive age, as this condition will last only as long as the estrogen-exposed period."
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Article in Press
Gastroenterology Dec 2010 Ahead of print
"In this prospective study of 1000 consecutive patients with CHC, we found that menopause is independently associated with the severity of liver damage and with a remarkably lower likelihood of achieving SVR (46% vs 67%)......Recent studies suggest that post-menopausal women have accelerated progression of fibrosis,6,7 which is prevented by long-term estrogen exposure from HRT.7 In this large cohort of female HCV patients, we confirmed that liver inflammation and higher levels of GGT (a known surrogate marker of metabolic alterations and of TNF-α up-regulation)21, are independently associated with severe fibrosis. We also identified the length of estrogen deprivation as a strong independent risk factor for fibrosis: the longer the menopausal period, the higher the risk of severe fibrosis. It was 5-fold higher in women who had been menopausal for more than 10 years in comparison with early menopausal women.....
In conclusion, our data from a large cohort of European women with CHC show that menopause is associated with profound changes in TNF-α and IL-6 levels. These changes are greater than those resulting from HCV infection in women of reproductive age and result in a more pronounced inflammatory state, more rapid progression to fibrosis and a hitherto unrecognized resistance to antiviral therapy. This suggests that CHC in women should be treated early, disregarding the fact that liver disease is milder in women of reproductive age, as this condition will last only as long as the estrogen-exposed period. Alternative strategies should be tested for HCV-positive women presenting in early post-menopause. We are currently examining the combination of HRT with PEG IFN/Ribavirin therapy in a controlled randomized trial (EudraCT 2008-001260-36).
"Overall, SVR was achieved by 231 of 442 women (52.2%). SVR occurred in 121 of 263 menopausal women (46.0%) vs. 110 of 163 women of reproductive age (67.5%; OR 2.436, 95% CI 1.620-3.662; P < .0001). The rate of SVR was similar among women of reproductive age who were stratified....By multivariate analysis significant independent baseline predictors of SVR failure in women were the presence of menopause (OR 1.802; 95% CI 1.154-2.813, P = .01), cholesterol level (OR 0.997; 95% CI 0.943-0.991, P = .008), high GGT levels (OR 2.165; 95% CI 1.364- 3.436, P = .001) and genotype 1/4 (OR 3.861, 95% CI 2.433-6.134) P = .006)(Table 4, top).....These data support the hypothesis that menopause determines a switch to a systemic and hepatic pro-inflammatory state25 in which increased IL-6 and TNF-α production contribute to the observed resistance to IFN-based therapies. Women entering menopause would rapidly go from an estrogen-protected environment, where HCV-mediated inflammation is limited, to an estrogen-deprived one in which inflammation becomes less controlled and resistance to antiviral therapy increases remarkably, thus increasing the risk of developing severe fibrosis. Regardless of reproductive status, HCV-positive women had significantly higher serum levels of both TNF-α and IL-6 compared to controls (P < .0001 for all combinations) (Figure 1A). IL-6 levels in women of reproductive age were significantly lower than those in early post-menopausal women [2.6µ1.5 vs 8.9µ10.3 pg/ml; P = .007] and were lower, although to a lesser degree, than those in late post-menopausal women [2.6µ1.5 vs 4.8µ8,2 pg/ml; P = .017]."
Erica Villa1, Aimilia Karampatou1, Calogero Cammˆ2, Alfredo Di Leo3, Monica Luongo1, Anna Ferrari1, Salvatore Petta2, Luisa Losi1, Gloria Taliani4, Paolo Trande5, Barbara Lei1, Amalia Graziosi1, Veronica Bernabucci1, Rosina Critelli1, Paola Pazienza3, Maria Rendina3, Alessandro Antonelli6, Antonio Francavilla3 1. Dept of Gastroenterology, University of Modena and Reggio Emilia, Modena, Italy 2. Dept of Gastroenterology, University of Palermo, Palermo, Italy 3. Dept of Gastroenterology, University of Bari, Bari, Italy 4. Dept of Infectious Diseases, University of Rome "La Sapienza", Italy 5. Dept of Gastroenterology, AUSL, Modena, Italy 6. Department of Internal Medicine, University of Pisa, Italy
Received 20 August 2010; received in revised form 26 October 2010; accepted 2 December 2010. published online 17 December 2010. Accepted Manuscript
Abstract
Background & Aims
Chronic hepatitis c (CHC) and liver fibrosis progress more rapidly in men and menopausal women than in women of reproductive age. We investigated the associations among menopause, sustained virological response (SVR), and liver damage in patients with CHC.
Methods
We performed a prospective study of 1000 consecutive, treatment-naive patients ³18 years old with compensated liver disease from CHC. Liver biopsy samples were analyzed (for fibrosis, inflammation, and steatosis) before patients received standard antiviral therapy. From women (n = 442), we collected data on the presence, type, and timing of menopause; associated hormone and metabolic features; serum levels of interleukin-6 (IL-6); and hepatic tumor necrosis factor-α (TNF-α).
Results
Post-menopausal women achieved SVRs less frequently than women of reproductive age (46.0% vs. 67.5%, P<.0001) but as frequently as men (51.1%, P=.178). By multivariate regression analysis, independent significant predictors for women to not achieve an SVR were early menopause (odds ratio [OR]=8.055; 95% confidence interval [CI], 1.834-25.350), levels of γ-glutamyl transpeptidase (OR=2.165, 95% CI, 1.3643-.436), infection with hepatitis C virus (HCV) genotypes 1 or 4 (OR=3.861; 95% CI, 2.433-6.134), and cholesterol levels (OR=0.967; 95% CI 0.943-0.991). Early menopause was the only independent factor that predicted lack of an SVR among women with genotype 1 HCV infection (OR=3.933; 95% CI, 1.274-12.142). Baseline levels of liver inflammation, fibrosis, steatosis, serum IL-6 (P=.04), and hepatic TNF-α (P=.007) were significantly higher among post-menopausal than women of reproductive age.
Conclusions
Among women with CHC, early menopause was associated with a low likelihood of SVR, probably because of inflammatory factors that change at menopause.
"cirrhosis was present in 1.7% of non-menopausal women, 6.0% of early- and 11.0% of late-menopausal women (non menopausal vs late menopausal: P = .0014). Multivariate analysis showed that, in addition to necroinflammatory activity (OR 1.464, 95% CI 1.256-1.707, P < .001), low platelet levels (OR 0.976, 95% CI 0.966-0.986, P < .001) and elevated GGT (OR 1.010, 95% CI 1.003-1.018, P = .008), also a longer duration of estrogen deprivation (5 to 10 years: OR 4.078 (95% CI 1.013-16.409), P = .048; more than 10 years: OR 4.867 (95% CI 1.476-16.042), P = .009) was independently linked with severe fibrosis (Table 2)."
Introduction
Appraisal of the clinical course of chronic hepatitis C (CHC) has revealed several striking differences between men and women. The progression of fibrosis is more than twice as fast in men,1,2 even when potential confounding factors like age, duration of infection, or metabolic features are accounted for by multivariate analysis.1,3 There are conflicting data about SVR rates in women; some studies report that response rates are not significantly different from those of men,4 whereas others have identified female sex as an independent factor associated with SVR.5 This inconsistency might arise from the fact that the female cohorts have always been evaluated as whole, without taking into account differences in responses due to hormonal state.
The reduced rate of fibrosis among women disappears after menopause; in fact, post- menopausal women have accelerated progression of fibrosis,6,7 compared with men, which is slowed by long-term estrogen exposure with hormone replacement therapy (HRT).7 Post- menopausal women are also at higher risk of developing hepatocellular carcinoma (HCC); there is a more balanced ratio of men:women in later life that results from the higher incidence of HCC in older women.8,9
Antiviral therapy greatly improves the natural course of CHC when it results in a sustained virological response (SVR),10 even in patients with established cirrhosis.11 Negative predictive factors for SVR include older age,12-14 which might coincide with menopause. However, no studies have evaluated the impact of menopause itself on response to antiviral therapy.
We therefore evaluated the impact of menopause on SVR and on histological features in a prospective study. We collected data on the type and timing of menopause, parity, and the use and duration of hormone replacement therapy (HRT) in a cohort of 1000 patients with CHC undergoing standard antiviral therapy with pegylated interferon (PEG-IFN) and ribavirin (RBV).
Discussion
In this prospective study of 1000 consecutive patients with CHC, we found that menopause is independently associated with the severity of liver damage and with a remarkably lower likelihood of achieving SVR.
Various lines of evidence support a link between menopause and the severity of fibrosis in CHC patients. Recent studies suggest that post-menopausal women have accelerated progression of fibrosis,6,7 which is prevented by long-term estrogen exposure from HRT.7 In this large cohort of female HCV patients, we confirmed that liver inflammation and higher levels of GGT (a known surrogate marker of metabolic alterations and of TNF-α up-regulation)21, are independently associated with severe fibrosis. We also identified the length of estrogen deprivation as a strong independent risk factor for fibrosis: the longer the menopausal period, the higher the risk of severe fibrosis. It was 5-fold higher in women who had been menopausal for more than 10 years in comparison with early menopausal women.
We found menopause to be significantly correlated with necro-inflammation, steatosis and metabolic alterations (high cholesterol and glucose). Accordingly, a recent clinical study showed a higher prevalence of steatosis in post-menopausal CHC patients who were more than 55 years old.13 Experimental studies are in line with our clinical data on the association between menopause and liver damage, showing that menopause is associated with a pro-inflammatory state that can drive fibrosis progression and lead to HCC.8 In addition, some experimental data also indicate a potential role of estrogen insufficiency in steatogenesis.22,23
Menopause coincides with older age, which has been identified as a negative predictive factor for a SVR.13,14 However, the influence of menopause itself on response to antiviral therapy has not been investigated. We examined age and menopause in multivariate analysis and found that age was not independently correlated with SVR, whereas menopause was. Our findings indicate that menopause is associated with a remarkable and unrecognized resistance to antiviral therapy, especially in carriers of genotype 1 HCV. Moreover, our data indicate that early stages of menopause (estrogen deprivation for less than 5 years) correlate with failure of antiviral therapy.
This is particularly evident in women with genotype 1 HCV, who have less resistance to antiviral therapy with the passage of years after the onset of menopause. A possible explanation for this finding is that hepatic levels of TNF-α and circulating IL-6 are upregulated at the time of menopause. Another, unlikely, explanation is that occasional consumption of NSAIDs or unreported use of alcohol, causes levels of both cytokines to decrease after menopause, and hepatic expression of TNF-α levels become non-significantly different from those of women of reproductive age. (Figure 1C, panel a). A similar phenomenon has been described regarding bone loss and levels of IL-6, which affect bone loss less with time after menopause, becoming insignificant after 10 years.24 It is interesting that in men, who had levels of cytokines that were constant yet greater than those of women at all age groups tested, the OR of not achieving an SVR was not significantly different among those younger than 45 years or older than 55 (i.e. in 2 age groups similar to those of women of reproductive age or menopausal females).
Although our study was not designed to clarify the pathogenesis of this association, the results do suggest several hypotheses. We focused our attention on levels of TNF-α and IL-6; these cytokines undergo large changes during in menopause25 and in HCV infection, during which their levels are greatly up-regulated,26-29 and they are able to interfere with antiviral response. Our data show that the occurrence of menopause is associated with a large additional increase in circulating IL-6, a striking increase in hepatic TNF-α, and an increase in the expression of SOCS3 in the liver. While there are no reports linking serum IL-6 levels with resistance to IFN, TNF-α has been implicated as an independent factor associated with response to IFN,21, 29-30 and hepatic SOCS3 is reportedly the strongest factor influencing the outcome of interferon-based antiviral therapy,31-34 especially in patients with genotype 1.33 SOCS3 is induced by HCV core protein,35 and also by IL- 6 and TNF-α.31,32 These data support the hypothesis that menopause determines a switch to a systemic and hepatic pro-inflammatory state25 in which increased IL-6 and TNF-α production contribute to the observed resistance to IFN-based therapies. Women entering menopause would rapidly go from an estrogen-protected environment, where HCV-mediated inflammation is limited, to an estrogen-deprived one in which inflammation becomes less controlled and resistance to antiviral therapy increases remarkably, thus increasing the risk of developing severe fibrosis.
We have also confirmed that genotype 1 and higher GGT levels are independent negative predictors of SVR, both for the entire cohort and when women are considered as a group. Although hidden alcohol or drug abuse cannot be excluded in principle to explain GGT alteration, the bias, if present, was equally shared by all groups, the methodology of ascertainment being the same for all. More relevant, in this context, is the demonstrated relationship between GGTs levels and hepatic TNF-α mRNA, which in turn strongly related with non response in HCV patients21.
The study has some limitations. In particular we cannot rule-out that lack of data on vitamin D serum levels, a recently recognized factor influencing SVR achievement 36, could affect the interpretation of our results, as well as the absence of data about other adipokines/cytokines.
In conclusion, our data from a large cohort of European women with CHC show that menopause is associated with profound changes in TNF-α and IL-6 levels. These changes are greater than those resulting from HCV infection in women of reproductive age and result in a more pronounced inflammatory state, more rapid progression to fibrosis and a hitherto unrecognized resistance to antiviral therapy. This suggests that CHC in women should be treated early, disregarding the fact that liver disease is milder in women of reproductive age, as this condition will last only as long as the estrogen-exposed period. Alternative strategies should be tested for HCV-positive women presenting in early post-menopause. We are currently examining the combination of HRT with PEG IFN/Ribavirin therapy in a controlled randomized trial (EudraCT 2008-001260-36).
Results
Baseline features of all 1.000 patients according to sex are shown in supplementary table 1. Ninety- nine patients (9.9%) had a diagnosis of cirrhosis: cirrhosis was more frequent in men (12.3%) than women (6.7%) (P = .003), despite the latter being significantly older (52 vs. 48 years, P <.001).
Characteristics of Female Patients
At enrollment, 274 out of 442 women (62.0%) were menopausal. Table 1 reports the baseline characteristics of the female group stratified by reproductive status. In 220 women (80.2%) menopause was spontaneous while in 54 (19.8%) it resulted from surgery. Mean age at the time of menopause was 49.0 µ 2.7 years for spontaneous and 42.5 µ 6.9 years for surgical menopause (P < .0001). Length of estrogen deprivation was significantly longer in women with surgical menopause vs. those with spontaneous menopause (Table 1). Fifty-four of the women (19.7%) had a history of past estrogen or HRT starting soon after the onset of menopause and continuing for a median period of 5 years (range 1-20 years). In 51 of these women (94.5%), HRT was stopped a mean 8.2 µ 3.2 years (median 7 years) before standard antiviral therapy was started.
At baseline, menopausal women had significantly more frequent metabolic alterations (blood glucose, cholesterol) and significantly more histological liver damage than their counterparts who were of reproductive age (Table 1). An increase in the severity of fibrosis was evident between non-menopausal and early menopausal women [staging score: 1.4 (1.0) vs 2.0 (1.0), P = .002], and between early menopausal and late menopausal women [staging score: 2.0 (1.0) to 2.4 (1.2), P = .009]; cirrhosis was present in 1.7% of non-menopausal women, 6.0% of early- and 11.0% of late-menopausal women (non menopausal vs late menopausal: P = .0014). Multivariate analysis showed that, in addition to necroinflammatory activity (OR 1.464, 95% CI 1.256-1.707, P < .001), low platelet levels (OR 0.976, 95% CI 0.966-0.986, P < .001) and elevated GGT (OR 1.010, 95% CI 1.003-1.018, P = .008), also a longer duration of estrogen deprivation (5 to 10 years: OR 4.078 (95% CI 1.013-16.409), P = .048; more than 10 years: OR 4.867 (95% CI 1.476-16.042), P = .009) was independently linked with severe fibrosis (Table 2). Other factors influencing exposure to estrogen, including past pregnancies (OR 0.764, 95% CI .464-1.259, P = .29), more than 2 lifetime pregnancies (OR 0.914, 95%CI 0.607-1.377, P = .66), and HRT use (OR 0.454; 95%CI 0.059-3.508, P = .44) were unrelated to fibrosis severity. Menopausal women also had significantly higher liver necro-inflammatory activity, and a higher rate of steatosis compared with women of reproductive age (Table 1). Multivariate analysis for severe necro-inflammatory activity showed that stage of fibrosis (OR 3.610, 95%CI 1.785-7.301, P=<.001), GGT levels (OR 1.032,95%CI 1.003-1.062, P=.030) and longer duration of estrogen deprivation (5 to 10 years: OR 11.823, 95%CI 2.779-50.302, P= .001; more than 10 years: OR 9.292, 95% CI 2.531-34.111, P = .001) were independently linked with higher inflammatory activity (Table 3). Similarly age (OR 1.133, 95%CI 1.059-1.213, P=<.0001), baseline cholesterol (OR 0.981, 95%CI 1.026-1.333, P P<.0001), BMI (OR 1.170, , 95%CI 1.003-1.062, P=.019) and duration of estrogen deprivation (< than 5 years OR 3,726, 95%CI 1.219-11.385, P=.021; 5 to 10 years: OR 2,648, 95%CI 1.117- 6.276, P=.027; more than 10 years: OR 1.474, 95%CI 0.732-2.969, P=.278) were independently associated with steatosis by multivariate analyses.
Paired liver biopsies taken shortly before and after menopause were available for 19 women (median interval 3 years before and 2 years after); in other 20 women, paired biopsies were obtained while they were still of reproductive ages, at a median interval of 4 years. Analysis of the pairs of menopausal women and those of reproductive age revealed that inflammation had worsened in 8 women (42.1%), increasing by 2 points in 4 women, by 3 points in 3, and by 4 points in 1 of them. It was unchanged in 5 women (26.3%) and improved in 6 (31.6%), decreasing by 2 points in 2 women and 4 points in 4 of them. Fibrosis had progressed in 7 (36.8%), with fibrosis scores increasing by 1 point in 4 women, 2 points in 2, and 3 points in 1 of them. Two women improved by 1 point each and 10 women (52.6%) had no change in fibrosis stage. In the pairs of women who were both of reproductive age, inflammation had worsened in 6 women (30.0%), increasing by 1 point in 3 women and by 2 points in 3. It was unchanged in 5 women (25.0%) and improved in 9 (45.0%), decreasing by 1 point in 4 women and 2 points in 5 of them.
Fibrosis had progressed by 1 point in 7 (35.0%), improved by 1 point in 6 (30.0%) and unchanged in 7 (35.0%).
Results of antiviral treatment
Results are reported as Intention-to-treat analysis. Eight hundred thirty-eight patients completed the antiviral treatment program; forty-nine (4.9%) (34 men/15 women) withdrew because of side effects. SVR was achieved in 511 individuals (51.1%).
Significant independent predictors of SVR failure by multivariate analysis were genotype 1/4, necroinflammatory activity and GGT levels (Supplementary Table 2).
Influence of menopause on response to antiviral therapy
Overall, SVR was achieved by 231 of 442 women (52.2%). SVR occurred in 121 of 263 menopausal women (46.0%) vs. 110 of 163 women of reproductive age (67.5%; OR 2.436, 95% CI 1.620-3.662; P < .0001). The rate of SVR was similar among women of reproductive age who were stratified for parity [nulliparous vs parous 42 of 65 (64.6%) vs 69 of 103 (66.9%; OR 1.200, 95% CI 0.521-2.724; P = .668). Nulliparous menopausal women had worse SVR compared to postmenopausal women with any number of pregnancies [nulliparous vs parous 4 of 32 (12.5%) vs 130 of 242 (53.7%), (OR 0.132, 95% CI 0.029-0.607, P = .009).
Probability of SVR failure between men and unstratified women was similar (OR 0.827, 95% CI 0.615-1.113, P = .210). Instead, women of reproductive age had a significantly lower risk of not achieving SVR (women of reproductive age vs. men: OR 0.452, 0.295-0.693, P < .0001; menopausal women vs. men: OR: 1.212, 95% CI 0.853-1.722, P = .283). Among men, risk of not achieving an SVR was similar after stratification for age groups and comparable to that of women of reproductive age (<45 years) or menopausal (>55 years) women (men aged <45 years vs. men>55 years: OR 1.204, 95% CI 0.964-1.502, P = .101). Percentage of SVR in men younger than 45 years was 59.2% and in those older than 55, 50.0% (p=.114).
By multivariate analysis significant independent baseline predictors of SVR failure in women were the presence of menopause (OR 1.802; 95% CI 1.154-2.813, P = .01), cholesterol level (OR 0.997; 95% CI 0.943-0.991, P = .008), high GGT levels (OR 2.165; 95% CI 1.364- 3.436, P = .001) and genotype 1/4 (OR 3.861, 95% CI 2.433-6.134) P = .006)(Table 4, top). Substituting "duration of estrogen deprivation" for "menopause" in the multivariate model, the OR of SVR failure for women who were post-menopausal for less than 5 years was 8.055 (95% CI 1.834-25.390, P = .006): longer periods of estrogen deprivation were not significantly associated with SVR failure. In addition when replacing length of estrogen deprivation as categorical variable with the linear variable the latest remained significantly associated with lower SVR rate (OR 1.115; C.I. 1.048-1.185).
Restricting analysis to genotype 1-infected women, logistic regression analysis identified only menopause as an independent predictive factor for SVR failure (OR 2.908; 95% CI 1.544- 5.478, P = .001)(Table 4, bottom). Substituting "duration of estrogen deprivation" for "menopause" in the multivariate model revealed that the OR of SVR failure decreased in parallel with increasing time from the menopausal event: less than 5 years: 3.933 (95% CI: 1.274-12.142, P = .017); 5 to 10 years: 2.300 (95% CI 0.982-5.386, P = .055); more than 10 years 1.437 (0.743-2.781), P =.282. Similar to the entire population, in G1, when replacing length of estrogen deprivation as categorical variable with the linear variable the latest remained significantly associated with lower SVR rate (OR 1.088; C.I. 1.006-1.177).
Cytokines levels
Regardless of reproductive status, HCV-positive women had significantly higher serum levels of both TNF-α and IL-6 compared to controls (P < .0001 for all combinations) (Figure 1A). IL-6 levels in women of reproductive age were significantly lower than those in early post-menopausal women [2.6µ1.5 vs 8.9µ10.3 pg/ml; P = .007] and were lower, although to a lesser degree, than those in late post-menopausal women [2.6µ1.5 vs 4.8µ8,2 pg/ml; P = .017]. When only nulliparous women were considered, the difference in IL-6 levels between women of reproductive age and post-menopausal women was greater [3.2µ2.2 vs. 42.3µ18.2 pg/ml; P < .0001] and a small but significant difference was found also in TNF-α levels (27.0µ3.1 vs. 29.2µ2.5; P = .001)(Figure 1A). Among menopausal women, but not women of reproductive age, baseline levels of IL-6 correlated with higher baseline necro-inflammatory scores (menopausal: OR 3.571, 1.494-8.536, P=.004; reproductive age OR .727, .305-1.736, P=.473).
Analysis of the paired serum samples taken shortly before and after menopause revealed significantly different levels for IL-6 (before vs. after: 2.0µ1.2 vs. 9.2µ2.2, P = .03) but not for TNF-α (29.6µ2.4 vs. 28.7µ3.5, P = 0.395)(Figure 1B). The post-menopausal increase in IL-6 levels correlated with fibrosis progression (OR 3.333, 95%CI 1.293-8.591, P = .011) and with worsened necro-inflammatory score (OR 3.667, 95%CI 1.397-9.624, P=.004).
Levels of TNF-α and IL-6 in men were stratified to have age groups comparable to those of females stratified by hormonal status: <45 years (reproductive age); 50-55 years (early menopause); > 60 years (late menopause). Levels found for both cytokines did not differ significantly among the 3 age groups (TNF-α: 28.7µ16.2, 29.2µ21.2, 29.5µ7.5 and IL-6: 27.2µ35.0, 29.0µ42, 34µ51 respectively, NS for all combinations). Levels of TNF-α did not differ significantly between men and women (NS for all combinations), whereas levels of IL-6 were significantly higher among men (p<.0001 for all combinations but vs. nulliparous menopausal females: p=.025, p=.098, p=.393 for <45 years; 50-55 and > 60 year-old men respectively).
Immunohistochemical evaluation of TNF-α and SOCS3
Control liver tissue was negative or had very slight TNF-α staining. TNF-α positivity increased significantly after menopause, the highest values being present in biopsies taken shortly after the occurrence of menopause [liver biopsies scored +/++ for TNF-α: non menopausal vs menopausal: 21 of 93 (22%) vs 36 of 84 (43%), P = .007)] (Figure 1C, panel a).
No significant differences were observed in levels of TNF-α between 20 paired liver biopsies collected from women of reproductive ages (Figure 1C, panel b), whereas in the 19 paired liver specimens taken before and after menopause, staining intensity and the number of positive hepatocytes increased after menopause (Figure 1C, panel b; Figure 2, panel A). Differences in TNF-α scores correlated with circulating levels of IL-6 (2.5µ1.0 vs. 20.6µ41.8 pg/ml in specimens staining negative vs. positive for TNF-α, P = .045).
Staining for SOCS3 was absent from normal liver samples; it was also absent, or very weak, in samples from women of reproductive age, and strong to very strong in samples from post- menopausal women [reproductive age vs menopausal: 17 of 93 (18.2%) vs. 39 of 84 (46.6%), P < .0001]. TNF-α and SOCS3 were co-localized in 60% µ 10% of hepatocytes (Figure 2, Panel B).
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Gastroenterology Dec 2010 Ahead of print
"In this prospective study of 1000 consecutive patients with CHC, we found that menopause is independently associated with the severity of liver damage and with a remarkably lower likelihood of achieving SVR (46% vs 67%)......Recent studies suggest that post-menopausal women have accelerated progression of fibrosis,6,7 which is prevented by long-term estrogen exposure from HRT.7 In this large cohort of female HCV patients, we confirmed that liver inflammation and higher levels of GGT (a known surrogate marker of metabolic alterations and of TNF-α up-regulation)21, are independently associated with severe fibrosis. We also identified the length of estrogen deprivation as a strong independent risk factor for fibrosis: the longer the menopausal period, the higher the risk of severe fibrosis. It was 5-fold higher in women who had been menopausal for more than 10 years in comparison with early menopausal women.....
In conclusion, our data from a large cohort of European women with CHC show that menopause is associated with profound changes in TNF-α and IL-6 levels. These changes are greater than those resulting from HCV infection in women of reproductive age and result in a more pronounced inflammatory state, more rapid progression to fibrosis and a hitherto unrecognized resistance to antiviral therapy. This suggests that CHC in women should be treated early, disregarding the fact that liver disease is milder in women of reproductive age, as this condition will last only as long as the estrogen-exposed period. Alternative strategies should be tested for HCV-positive women presenting in early post-menopause. We are currently examining the combination of HRT with PEG IFN/Ribavirin therapy in a controlled randomized trial (EudraCT 2008-001260-36).
"Overall, SVR was achieved by 231 of 442 women (52.2%). SVR occurred in 121 of 263 menopausal women (46.0%) vs. 110 of 163 women of reproductive age (67.5%; OR 2.436, 95% CI 1.620-3.662; P < .0001). The rate of SVR was similar among women of reproductive age who were stratified....By multivariate analysis significant independent baseline predictors of SVR failure in women were the presence of menopause (OR 1.802; 95% CI 1.154-2.813, P = .01), cholesterol level (OR 0.997; 95% CI 0.943-0.991, P = .008), high GGT levels (OR 2.165; 95% CI 1.364- 3.436, P = .001) and genotype 1/4 (OR 3.861, 95% CI 2.433-6.134) P = .006)(Table 4, top).....These data support the hypothesis that menopause determines a switch to a systemic and hepatic pro-inflammatory state25 in which increased IL-6 and TNF-α production contribute to the observed resistance to IFN-based therapies. Women entering menopause would rapidly go from an estrogen-protected environment, where HCV-mediated inflammation is limited, to an estrogen-deprived one in which inflammation becomes less controlled and resistance to antiviral therapy increases remarkably, thus increasing the risk of developing severe fibrosis. Regardless of reproductive status, HCV-positive women had significantly higher serum levels of both TNF-α and IL-6 compared to controls (P < .0001 for all combinations) (Figure 1A). IL-6 levels in women of reproductive age were significantly lower than those in early post-menopausal women [2.6µ1.5 vs 8.9µ10.3 pg/ml; P = .007] and were lower, although to a lesser degree, than those in late post-menopausal women [2.6µ1.5 vs 4.8µ8,2 pg/ml; P = .017]."
Erica Villa1, Aimilia Karampatou1, Calogero Cammˆ2, Alfredo Di Leo3, Monica Luongo1, Anna Ferrari1, Salvatore Petta2, Luisa Losi1, Gloria Taliani4, Paolo Trande5, Barbara Lei1, Amalia Graziosi1, Veronica Bernabucci1, Rosina Critelli1, Paola Pazienza3, Maria Rendina3, Alessandro Antonelli6, Antonio Francavilla3 1. Dept of Gastroenterology, University of Modena and Reggio Emilia, Modena, Italy 2. Dept of Gastroenterology, University of Palermo, Palermo, Italy 3. Dept of Gastroenterology, University of Bari, Bari, Italy 4. Dept of Infectious Diseases, University of Rome "La Sapienza", Italy 5. Dept of Gastroenterology, AUSL, Modena, Italy 6. Department of Internal Medicine, University of Pisa, Italy
Received 20 August 2010; received in revised form 26 October 2010; accepted 2 December 2010. published online 17 December 2010. Accepted Manuscript
Abstract
Background & Aims
Chronic hepatitis c (CHC) and liver fibrosis progress more rapidly in men and menopausal women than in women of reproductive age. We investigated the associations among menopause, sustained virological response (SVR), and liver damage in patients with CHC.
Methods
We performed a prospective study of 1000 consecutive, treatment-naive patients ³18 years old with compensated liver disease from CHC. Liver biopsy samples were analyzed (for fibrosis, inflammation, and steatosis) before patients received standard antiviral therapy. From women (n = 442), we collected data on the presence, type, and timing of menopause; associated hormone and metabolic features; serum levels of interleukin-6 (IL-6); and hepatic tumor necrosis factor-α (TNF-α).
Results
Post-menopausal women achieved SVRs less frequently than women of reproductive age (46.0% vs. 67.5%, P<.0001) but as frequently as men (51.1%, P=.178). By multivariate regression analysis, independent significant predictors for women to not achieve an SVR were early menopause (odds ratio [OR]=8.055; 95% confidence interval [CI], 1.834-25.350), levels of γ-glutamyl transpeptidase (OR=2.165, 95% CI, 1.3643-.436), infection with hepatitis C virus (HCV) genotypes 1 or 4 (OR=3.861; 95% CI, 2.433-6.134), and cholesterol levels (OR=0.967; 95% CI 0.943-0.991). Early menopause was the only independent factor that predicted lack of an SVR among women with genotype 1 HCV infection (OR=3.933; 95% CI, 1.274-12.142). Baseline levels of liver inflammation, fibrosis, steatosis, serum IL-6 (P=.04), and hepatic TNF-α (P=.007) were significantly higher among post-menopausal than women of reproductive age.
Conclusions
Among women with CHC, early menopause was associated with a low likelihood of SVR, probably because of inflammatory factors that change at menopause.
"cirrhosis was present in 1.7% of non-menopausal women, 6.0% of early- and 11.0% of late-menopausal women (non menopausal vs late menopausal: P = .0014). Multivariate analysis showed that, in addition to necroinflammatory activity (OR 1.464, 95% CI 1.256-1.707, P < .001), low platelet levels (OR 0.976, 95% CI 0.966-0.986, P < .001) and elevated GGT (OR 1.010, 95% CI 1.003-1.018, P = .008), also a longer duration of estrogen deprivation (5 to 10 years: OR 4.078 (95% CI 1.013-16.409), P = .048; more than 10 years: OR 4.867 (95% CI 1.476-16.042), P = .009) was independently linked with severe fibrosis (Table 2)."
Introduction
Appraisal of the clinical course of chronic hepatitis C (CHC) has revealed several striking differences between men and women. The progression of fibrosis is more than twice as fast in men,1,2 even when potential confounding factors like age, duration of infection, or metabolic features are accounted for by multivariate analysis.1,3 There are conflicting data about SVR rates in women; some studies report that response rates are not significantly different from those of men,4 whereas others have identified female sex as an independent factor associated with SVR.5 This inconsistency might arise from the fact that the female cohorts have always been evaluated as whole, without taking into account differences in responses due to hormonal state.
The reduced rate of fibrosis among women disappears after menopause; in fact, post- menopausal women have accelerated progression of fibrosis,6,7 compared with men, which is slowed by long-term estrogen exposure with hormone replacement therapy (HRT).7 Post- menopausal women are also at higher risk of developing hepatocellular carcinoma (HCC); there is a more balanced ratio of men:women in later life that results from the higher incidence of HCC in older women.8,9
Antiviral therapy greatly improves the natural course of CHC when it results in a sustained virological response (SVR),10 even in patients with established cirrhosis.11 Negative predictive factors for SVR include older age,12-14 which might coincide with menopause. However, no studies have evaluated the impact of menopause itself on response to antiviral therapy.
We therefore evaluated the impact of menopause on SVR and on histological features in a prospective study. We collected data on the type and timing of menopause, parity, and the use and duration of hormone replacement therapy (HRT) in a cohort of 1000 patients with CHC undergoing standard antiviral therapy with pegylated interferon (PEG-IFN) and ribavirin (RBV).
Discussion
In this prospective study of 1000 consecutive patients with CHC, we found that menopause is independently associated with the severity of liver damage and with a remarkably lower likelihood of achieving SVR.
Various lines of evidence support a link between menopause and the severity of fibrosis in CHC patients. Recent studies suggest that post-menopausal women have accelerated progression of fibrosis,6,7 which is prevented by long-term estrogen exposure from HRT.7 In this large cohort of female HCV patients, we confirmed that liver inflammation and higher levels of GGT (a known surrogate marker of metabolic alterations and of TNF-α up-regulation)21, are independently associated with severe fibrosis. We also identified the length of estrogen deprivation as a strong independent risk factor for fibrosis: the longer the menopausal period, the higher the risk of severe fibrosis. It was 5-fold higher in women who had been menopausal for more than 10 years in comparison with early menopausal women.
We found menopause to be significantly correlated with necro-inflammation, steatosis and metabolic alterations (high cholesterol and glucose). Accordingly, a recent clinical study showed a higher prevalence of steatosis in post-menopausal CHC patients who were more than 55 years old.13 Experimental studies are in line with our clinical data on the association between menopause and liver damage, showing that menopause is associated with a pro-inflammatory state that can drive fibrosis progression and lead to HCC.8 In addition, some experimental data also indicate a potential role of estrogen insufficiency in steatogenesis.22,23
Menopause coincides with older age, which has been identified as a negative predictive factor for a SVR.13,14 However, the influence of menopause itself on response to antiviral therapy has not been investigated. We examined age and menopause in multivariate analysis and found that age was not independently correlated with SVR, whereas menopause was. Our findings indicate that menopause is associated with a remarkable and unrecognized resistance to antiviral therapy, especially in carriers of genotype 1 HCV. Moreover, our data indicate that early stages of menopause (estrogen deprivation for less than 5 years) correlate with failure of antiviral therapy.
This is particularly evident in women with genotype 1 HCV, who have less resistance to antiviral therapy with the passage of years after the onset of menopause. A possible explanation for this finding is that hepatic levels of TNF-α and circulating IL-6 are upregulated at the time of menopause. Another, unlikely, explanation is that occasional consumption of NSAIDs or unreported use of alcohol, causes levels of both cytokines to decrease after menopause, and hepatic expression of TNF-α levels become non-significantly different from those of women of reproductive age. (Figure 1C, panel a). A similar phenomenon has been described regarding bone loss and levels of IL-6, which affect bone loss less with time after menopause, becoming insignificant after 10 years.24 It is interesting that in men, who had levels of cytokines that were constant yet greater than those of women at all age groups tested, the OR of not achieving an SVR was not significantly different among those younger than 45 years or older than 55 (i.e. in 2 age groups similar to those of women of reproductive age or menopausal females).
Although our study was not designed to clarify the pathogenesis of this association, the results do suggest several hypotheses. We focused our attention on levels of TNF-α and IL-6; these cytokines undergo large changes during in menopause25 and in HCV infection, during which their levels are greatly up-regulated,26-29 and they are able to interfere with antiviral response. Our data show that the occurrence of menopause is associated with a large additional increase in circulating IL-6, a striking increase in hepatic TNF-α, and an increase in the expression of SOCS3 in the liver. While there are no reports linking serum IL-6 levels with resistance to IFN, TNF-α has been implicated as an independent factor associated with response to IFN,21, 29-30 and hepatic SOCS3 is reportedly the strongest factor influencing the outcome of interferon-based antiviral therapy,31-34 especially in patients with genotype 1.33 SOCS3 is induced by HCV core protein,35 and also by IL- 6 and TNF-α.31,32 These data support the hypothesis that menopause determines a switch to a systemic and hepatic pro-inflammatory state25 in which increased IL-6 and TNF-α production contribute to the observed resistance to IFN-based therapies. Women entering menopause would rapidly go from an estrogen-protected environment, where HCV-mediated inflammation is limited, to an estrogen-deprived one in which inflammation becomes less controlled and resistance to antiviral therapy increases remarkably, thus increasing the risk of developing severe fibrosis.
We have also confirmed that genotype 1 and higher GGT levels are independent negative predictors of SVR, both for the entire cohort and when women are considered as a group. Although hidden alcohol or drug abuse cannot be excluded in principle to explain GGT alteration, the bias, if present, was equally shared by all groups, the methodology of ascertainment being the same for all. More relevant, in this context, is the demonstrated relationship between GGTs levels and hepatic TNF-α mRNA, which in turn strongly related with non response in HCV patients21.
The study has some limitations. In particular we cannot rule-out that lack of data on vitamin D serum levels, a recently recognized factor influencing SVR achievement 36, could affect the interpretation of our results, as well as the absence of data about other adipokines/cytokines.
In conclusion, our data from a large cohort of European women with CHC show that menopause is associated with profound changes in TNF-α and IL-6 levels. These changes are greater than those resulting from HCV infection in women of reproductive age and result in a more pronounced inflammatory state, more rapid progression to fibrosis and a hitherto unrecognized resistance to antiviral therapy. This suggests that CHC in women should be treated early, disregarding the fact that liver disease is milder in women of reproductive age, as this condition will last only as long as the estrogen-exposed period. Alternative strategies should be tested for HCV-positive women presenting in early post-menopause. We are currently examining the combination of HRT with PEG IFN/Ribavirin therapy in a controlled randomized trial (EudraCT 2008-001260-36).
Results
Baseline features of all 1.000 patients according to sex are shown in supplementary table 1. Ninety- nine patients (9.9%) had a diagnosis of cirrhosis: cirrhosis was more frequent in men (12.3%) than women (6.7%) (P = .003), despite the latter being significantly older (52 vs. 48 years, P <.001).
Characteristics of Female Patients
At enrollment, 274 out of 442 women (62.0%) were menopausal. Table 1 reports the baseline characteristics of the female group stratified by reproductive status. In 220 women (80.2%) menopause was spontaneous while in 54 (19.8%) it resulted from surgery. Mean age at the time of menopause was 49.0 µ 2.7 years for spontaneous and 42.5 µ 6.9 years for surgical menopause (P < .0001). Length of estrogen deprivation was significantly longer in women with surgical menopause vs. those with spontaneous menopause (Table 1). Fifty-four of the women (19.7%) had a history of past estrogen or HRT starting soon after the onset of menopause and continuing for a median period of 5 years (range 1-20 years). In 51 of these women (94.5%), HRT was stopped a mean 8.2 µ 3.2 years (median 7 years) before standard antiviral therapy was started.
At baseline, menopausal women had significantly more frequent metabolic alterations (blood glucose, cholesterol) and significantly more histological liver damage than their counterparts who were of reproductive age (Table 1). An increase in the severity of fibrosis was evident between non-menopausal and early menopausal women [staging score: 1.4 (1.0) vs 2.0 (1.0), P = .002], and between early menopausal and late menopausal women [staging score: 2.0 (1.0) to 2.4 (1.2), P = .009]; cirrhosis was present in 1.7% of non-menopausal women, 6.0% of early- and 11.0% of late-menopausal women (non menopausal vs late menopausal: P = .0014). Multivariate analysis showed that, in addition to necroinflammatory activity (OR 1.464, 95% CI 1.256-1.707, P < .001), low platelet levels (OR 0.976, 95% CI 0.966-0.986, P < .001) and elevated GGT (OR 1.010, 95% CI 1.003-1.018, P = .008), also a longer duration of estrogen deprivation (5 to 10 years: OR 4.078 (95% CI 1.013-16.409), P = .048; more than 10 years: OR 4.867 (95% CI 1.476-16.042), P = .009) was independently linked with severe fibrosis (Table 2). Other factors influencing exposure to estrogen, including past pregnancies (OR 0.764, 95% CI .464-1.259, P = .29), more than 2 lifetime pregnancies (OR 0.914, 95%CI 0.607-1.377, P = .66), and HRT use (OR 0.454; 95%CI 0.059-3.508, P = .44) were unrelated to fibrosis severity. Menopausal women also had significantly higher liver necro-inflammatory activity, and a higher rate of steatosis compared with women of reproductive age (Table 1). Multivariate analysis for severe necro-inflammatory activity showed that stage of fibrosis (OR 3.610, 95%CI 1.785-7.301, P=<.001), GGT levels (OR 1.032,95%CI 1.003-1.062, P=.030) and longer duration of estrogen deprivation (5 to 10 years: OR 11.823, 95%CI 2.779-50.302, P= .001; more than 10 years: OR 9.292, 95% CI 2.531-34.111, P = .001) were independently linked with higher inflammatory activity (Table 3). Similarly age (OR 1.133, 95%CI 1.059-1.213, P=<.0001), baseline cholesterol (OR 0.981, 95%CI 1.026-1.333, P P<.0001), BMI (OR 1.170, , 95%CI 1.003-1.062, P=.019) and duration of estrogen deprivation (< than 5 years OR 3,726, 95%CI 1.219-11.385, P=.021; 5 to 10 years: OR 2,648, 95%CI 1.117- 6.276, P=.027; more than 10 years: OR 1.474, 95%CI 0.732-2.969, P=.278) were independently associated with steatosis by multivariate analyses.
Paired liver biopsies taken shortly before and after menopause were available for 19 women (median interval 3 years before and 2 years after); in other 20 women, paired biopsies were obtained while they were still of reproductive ages, at a median interval of 4 years. Analysis of the pairs of menopausal women and those of reproductive age revealed that inflammation had worsened in 8 women (42.1%), increasing by 2 points in 4 women, by 3 points in 3, and by 4 points in 1 of them. It was unchanged in 5 women (26.3%) and improved in 6 (31.6%), decreasing by 2 points in 2 women and 4 points in 4 of them. Fibrosis had progressed in 7 (36.8%), with fibrosis scores increasing by 1 point in 4 women, 2 points in 2, and 3 points in 1 of them. Two women improved by 1 point each and 10 women (52.6%) had no change in fibrosis stage. In the pairs of women who were both of reproductive age, inflammation had worsened in 6 women (30.0%), increasing by 1 point in 3 women and by 2 points in 3. It was unchanged in 5 women (25.0%) and improved in 9 (45.0%), decreasing by 1 point in 4 women and 2 points in 5 of them.
Fibrosis had progressed by 1 point in 7 (35.0%), improved by 1 point in 6 (30.0%) and unchanged in 7 (35.0%).
Results of antiviral treatment
Results are reported as Intention-to-treat analysis. Eight hundred thirty-eight patients completed the antiviral treatment program; forty-nine (4.9%) (34 men/15 women) withdrew because of side effects. SVR was achieved in 511 individuals (51.1%).
Significant independent predictors of SVR failure by multivariate analysis were genotype 1/4, necroinflammatory activity and GGT levels (Supplementary Table 2).
Influence of menopause on response to antiviral therapy
Overall, SVR was achieved by 231 of 442 women (52.2%). SVR occurred in 121 of 263 menopausal women (46.0%) vs. 110 of 163 women of reproductive age (67.5%; OR 2.436, 95% CI 1.620-3.662; P < .0001). The rate of SVR was similar among women of reproductive age who were stratified for parity [nulliparous vs parous 42 of 65 (64.6%) vs 69 of 103 (66.9%; OR 1.200, 95% CI 0.521-2.724; P = .668). Nulliparous menopausal women had worse SVR compared to postmenopausal women with any number of pregnancies [nulliparous vs parous 4 of 32 (12.5%) vs 130 of 242 (53.7%), (OR 0.132, 95% CI 0.029-0.607, P = .009).
Probability of SVR failure between men and unstratified women was similar (OR 0.827, 95% CI 0.615-1.113, P = .210). Instead, women of reproductive age had a significantly lower risk of not achieving SVR (women of reproductive age vs. men: OR 0.452, 0.295-0.693, P < .0001; menopausal women vs. men: OR: 1.212, 95% CI 0.853-1.722, P = .283). Among men, risk of not achieving an SVR was similar after stratification for age groups and comparable to that of women of reproductive age (<45 years) or menopausal (>55 years) women (men aged <45 years vs. men>55 years: OR 1.204, 95% CI 0.964-1.502, P = .101). Percentage of SVR in men younger than 45 years was 59.2% and in those older than 55, 50.0% (p=.114).
By multivariate analysis significant independent baseline predictors of SVR failure in women were the presence of menopause (OR 1.802; 95% CI 1.154-2.813, P = .01), cholesterol level (OR 0.997; 95% CI 0.943-0.991, P = .008), high GGT levels (OR 2.165; 95% CI 1.364- 3.436, P = .001) and genotype 1/4 (OR 3.861, 95% CI 2.433-6.134) P = .006)(Table 4, top). Substituting "duration of estrogen deprivation" for "menopause" in the multivariate model, the OR of SVR failure for women who were post-menopausal for less than 5 years was 8.055 (95% CI 1.834-25.390, P = .006): longer periods of estrogen deprivation were not significantly associated with SVR failure. In addition when replacing length of estrogen deprivation as categorical variable with the linear variable the latest remained significantly associated with lower SVR rate (OR 1.115; C.I. 1.048-1.185).
Restricting analysis to genotype 1-infected women, logistic regression analysis identified only menopause as an independent predictive factor for SVR failure (OR 2.908; 95% CI 1.544- 5.478, P = .001)(Table 4, bottom). Substituting "duration of estrogen deprivation" for "menopause" in the multivariate model revealed that the OR of SVR failure decreased in parallel with increasing time from the menopausal event: less than 5 years: 3.933 (95% CI: 1.274-12.142, P = .017); 5 to 10 years: 2.300 (95% CI 0.982-5.386, P = .055); more than 10 years 1.437 (0.743-2.781), P =.282. Similar to the entire population, in G1, when replacing length of estrogen deprivation as categorical variable with the linear variable the latest remained significantly associated with lower SVR rate (OR 1.088; C.I. 1.006-1.177).
Cytokines levels
Regardless of reproductive status, HCV-positive women had significantly higher serum levels of both TNF-α and IL-6 compared to controls (P < .0001 for all combinations) (Figure 1A). IL-6 levels in women of reproductive age were significantly lower than those in early post-menopausal women [2.6µ1.5 vs 8.9µ10.3 pg/ml; P = .007] and were lower, although to a lesser degree, than those in late post-menopausal women [2.6µ1.5 vs 4.8µ8,2 pg/ml; P = .017]. When only nulliparous women were considered, the difference in IL-6 levels between women of reproductive age and post-menopausal women was greater [3.2µ2.2 vs. 42.3µ18.2 pg/ml; P < .0001] and a small but significant difference was found also in TNF-α levels (27.0µ3.1 vs. 29.2µ2.5; P = .001)(Figure 1A). Among menopausal women, but not women of reproductive age, baseline levels of IL-6 correlated with higher baseline necro-inflammatory scores (menopausal: OR 3.571, 1.494-8.536, P=.004; reproductive age OR .727, .305-1.736, P=.473).
Analysis of the paired serum samples taken shortly before and after menopause revealed significantly different levels for IL-6 (before vs. after: 2.0µ1.2 vs. 9.2µ2.2, P = .03) but not for TNF-α (29.6µ2.4 vs. 28.7µ3.5, P = 0.395)(Figure 1B). The post-menopausal increase in IL-6 levels correlated with fibrosis progression (OR 3.333, 95%CI 1.293-8.591, P = .011) and with worsened necro-inflammatory score (OR 3.667, 95%CI 1.397-9.624, P=.004).
Levels of TNF-α and IL-6 in men were stratified to have age groups comparable to those of females stratified by hormonal status: <45 years (reproductive age); 50-55 years (early menopause); > 60 years (late menopause). Levels found for both cytokines did not differ significantly among the 3 age groups (TNF-α: 28.7µ16.2, 29.2µ21.2, 29.5µ7.5 and IL-6: 27.2µ35.0, 29.0µ42, 34µ51 respectively, NS for all combinations). Levels of TNF-α did not differ significantly between men and women (NS for all combinations), whereas levels of IL-6 were significantly higher among men (p<.0001 for all combinations but vs. nulliparous menopausal females: p=.025, p=.098, p=.393 for <45 years; 50-55 and > 60 year-old men respectively).
Immunohistochemical evaluation of TNF-α and SOCS3
Control liver tissue was negative or had very slight TNF-α staining. TNF-α positivity increased significantly after menopause, the highest values being present in biopsies taken shortly after the occurrence of menopause [liver biopsies scored +/++ for TNF-α: non menopausal vs menopausal: 21 of 93 (22%) vs 36 of 84 (43%), P = .007)] (Figure 1C, panel a).
No significant differences were observed in levels of TNF-α between 20 paired liver biopsies collected from women of reproductive ages (Figure 1C, panel b), whereas in the 19 paired liver specimens taken before and after menopause, staining intensity and the number of positive hepatocytes increased after menopause (Figure 1C, panel b; Figure 2, panel A). Differences in TNF-α scores correlated with circulating levels of IL-6 (2.5µ1.0 vs. 20.6µ41.8 pg/ml in specimens staining negative vs. positive for TNF-α, P = .045).
Staining for SOCS3 was absent from normal liver samples; it was also absent, or very weak, in samples from women of reproductive age, and strong to very strong in samples from post- menopausal women [reproductive age vs menopausal: 17 of 93 (18.2%) vs. 39 of 84 (46.6%), P < .0001]. TNF-α and SOCS3 were co-localized in 60% µ 10% of hepatocytes (Figure 2, Panel B).
Source
Maintenance Peginterferon Therapy and Other Factors Associated with Hepatocellular Carcinoma in Patients with Advanced Hepatitis C
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Article in Press
Gastroenterology Dec 2010
Anna S. Lok, 1 James E. Everhart, 2 Elizabeth C. Wright, 3 Adrian M. Di Bisceglie, 4 Hae-Young Kim, 5 Richard K. Sterling, 6 Gregory T. Everson, 7 Karen L. Lindsay, 8 William M. Lee, 9 Herbert L. Bonkovsky, 10,11 Jules L. Dienstag, 12,13 Marc G. Ghany, 14 Chihiro Morishima, 15 Timothy R. Morgan, 16,17 and the HALT-C Trial Group
FROM JULES of NATAP: this was presented at AASLD where I went to the mic after Dr Lok presented the data and reminded the large audience I previously questioned and doubted the original findings that peg maintenence therapy was not useful when they were first presented several years ago. My questioning of their original findings were not well received but now upon further followup it appears maintenance therapy can provide benefit for patients with cirrhosis. The utility of maintenance therapy may still have application for patients who are nonresponders to the new HCV oral protease inhibitors expected to be approved in 2011, to prevent further disease progression until better, more effective multi-drug oral therapy combinations become available.
"extended follow-up of the HALT-C Trial cohort showed a modest benefit of long-term peginterferon therapy in reducing the incidence of HCC in patients with hepatitis C and cirrhosis but not in those with advanced precirrhotic fibrosis, an effect that took several years after completion of therapy to become apparent. Although the results were corroborated in multiple sub-analyses, the clinical implications are not clear. Given the marginal beneficial effect and its restriction to only part of the HALT-C Trial cohort, that no overall long-term benefit on mortality was observed in this cohort,29 and given the side effects of peginterferon, necessitating dose reduction or discontinuation in a substantial number of patients, the utility of maintenance peginterferon therapy to prevent HCC in patients with HCV-related cirrhosis is doubtful.....When patients with cirrhosis at baseline were analyzed separately, however, significantly fewer of those randomized to receive peginterferon had a diagnosis of HCC than those randomized to no treatment, 6.8% (14/207) vs.15.5% (34/220), respectively (HR for HCC in patients randomized to receive peginterferon 0.45 [95% CI 0.24-0.83], P = 0.01). Interferon has been shown to have antitumor effects in some malignancies and may have other beneficial effects including antiproliferative and antiangiogenic effects.19, 20 Chronic inflammation has been postulated to play a role in hepatitis B virus- (HBV) and HCV-related HCC. The observation that reduction in HCC incidence in the HALT-C Trial cohort was greater among the treated patients experiencing a reduction in HAI suggests that interferon may have acted through this pathway"
"The Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) Trial.......Analysis of the HALT-C Trial results after 3.5 years of treatment revealed that peginterferon did not reduce the overall risk of liver disease progression.4 A subsequent report focusing on HCC development also showed no difference between the treated and control groups during and immediately after the period of maintenance peginterferon therapy.5 We continued to follow the HALT-C Trial cohort off therapy for a total of up to 8.7 years to monitor for the development of decompensated liver disease and HCC. During the extended follow-up period, the number of patients with HCC increased progressively......
.......Although treated patients were less likely to have HCC than controls, 7.2% (37/515) vs. 9.6% (51/533), this difference was not statistically significant (hazard ratio [HR] for HCC in patients randomized to peginterferon treatment 0.77, 95% confidence interval [CI] 0.51-1.18, P = 0.24) (Figure 1A). ......When patients with cirrhosis at baseline were analyzed separately, however, significantly fewer of those randomized to receive peginterferon had a diagnosis of HCC than those randomized to no treatment, 6.8% (14/207) vs.15.5% (34/220), respectively (HR for HCC in patients randomized to receive peginterferon 0.45 [95% CI 0.24-0.83], P = 0.01). The cumulative incidence of HCC at 3, 5 and 7 years in the peginterferon group was 2.6%, 5.1%, and 7.8%, and, in the untreated control group, 4.0%, 11.1%, and 24.2%, respectively (log-rank test, P = 0.009). The absolute risk difference was 1.5% (95% CI -2.1%-5.0%) at 3 years, 6.1% (95% CI 0.4%-11.7%) at 5 years, and 16.4% (95% CI 2.3%-24.1%) at 7 years; the incidence of HCC in the two treatment groups did not diverge until after year 4 (Figure 1B)."
.......Because of the unexpected difference among patients with cirrhosis between our previous and the current findings, we performed additional analyses to verify the consistency of the results. A beneficial effect of peginterferon was observed even when the analysis was restricted to patients with definite HCC (i.e., after exclusion of 20 patients with presumed HCC as defined by the protocol). In addition, a dose-response effect of peginterferon was apparent, as suggested by three findings. First, among patients randomized to receive peginterferon, multivariate analysis showed that those who stopped treatment before 2 years did not have a reduction in the risk of HCC after adjusting for laboratory markers of advanced liver disease. Second, among patients receiving peginterferon, those experiencing a ≥2-point reduction in HAI had an approximately 3-fold reduction in HCC compared to those who did not have a reduction in HAI on follow-up liver biopsy. Third, in a regression analysis of factors associated with the development of HCC, including treatment assignment, peginterferon therapy was associated with an approximate 50% reduction in the incidence of HCC over a median of 6.1 year observation period among patients with cirrhosis at baseline even when adjusted for other risk factors including age, race, smoking history, and laboratory tests associated with advanced liver disease. Nevertheless, while analysis by the two pre-specified strata of histological severity (bridging fibrosis and cirrhosis) was warranted, why the effect of peginterferon was confined to patients with cirrhosis is unclear, and the possibility that this is a chance finding cannot be excluded."
Abstract
Background & Aims
Interferon reportedly decreases the incidence of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C. The Hepatitis C anti-viral long-term treatment against cirrhosis (HALT-C) trial showed that 4 years of maintenance therapy with peginterferon does not reduce liver disease progression. We investigated whether peginterferon decreases the incidence of HCC in the HALT-C cohort over a longer post-treatment follow-up period.
Methods
The study included 1,048 patients with chronic Hepatitis C (Ishak fibrosis scores ≥3) who did not have a sustained virological response (SVR) to therapy. They were randomly assigned to groups given a half-dose of peginterferon or no treatment (controls) for 3.5 years and followed for a median 6.1 (maximum 8.7) years.
Results
Eighty-eight patients developed HCC (68 definite, 20 presumed): 37/515 that were given peginterferon (7.2%) and 51/533 controls (9.6%; P=0.24). There was a significantly lower incidence of HCC among patients given peginterferon therapy who had cirrhosis, but not fibrosis, based on analysis of baseline biopsy samples. After 7 years, the cumulative incidences of HCC in treated and control patients with cirrhosis were 7.8% and 24.2%, respectively (hazard ratio [HR]=0.45; 95% confidence interval [CI]: 0.24Ð0.83); in treated and control patients with fibrosis they were 8.3% and 6.8%, respectively (HR=1.44; 95% CI: 0.77Ð2.69). Treated patients with a ≥2-point decrease in the histologic activity index, based on a follow-up biopsy, had a lower incidence of HCC than those with unchanged or increased scores (2.9% vs. 9.4%; P=0.03).
Conclusions
Extended analysis of the HALT-C cohort showed that long-term peginterferon therapy does not reduce the incidence of HCC among patients with advanced hepatitis C who did not achieve SVRs. Patients with cirrhosis who received peginterferon treatment had a lower risk for HCC than controls.
INTRODUCTION
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most common cause of cancer death in the world.1 In the United States, hepatitis C is the greatest contributor to both the occurrence of HCC and its observed increased incidence over the last 20 years. Among persons with chronic hepatitis C, risk factors for the development of HCC are incompletely understood. HCC has been found primarily, but not exclusively, in patients with cirrhosis. In addition to the degree of liver fibrosis, biochemical markers of advanced liver disease (e.g., low platelet count, low albumin), presence of esophageal varices, diabetes, obesity, and use of tobacco and excessive alcohol have been associated with an increased risk for HCC. The risk of HCC is decreased in patients with chronic hepatitis C who achieved a sustained virological response (SVR) to interferon/ribavirin treatment;2, 3 however, the effectiveness of interferon therapy in reducing HCC among patients with hepatitis C virus (HCV)-related cirrhosis who did not achieve an SVR is unclear.
The Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) Trial was a randomized, multi-center trial to determine whether 3.5 years of half-dose pegylated interferon (peginterferon) treatment reduced liver disease progression among patients with hepatitis C and advanced fibrosis who were nonresponders to peginterferon and ribavirin. Analysis of the HALT-C Trial results after 3.5 years of treatment revealed that peginterferon did not reduce the overall risk of liver disease progression.4 A subsequent report focusing on HCC development also showed no difference between the treated and control groups during and immediately after the period of maintenance peginterferon therapy.5 We continued to follow the HALT-C Trial cohort off therapy for a total of up to 8.7 years to monitor for the development of decompensated liver disease and HCC. During the extended follow-up period, the number of patients with HCC increased progressively. The aims of the current analysis were: 1) to determine the incidence of HCC among the HALT-C Trial cohort after a longer duration of follow-up, 2) to identify factors associated with the development of HCC in the HALT-C Trial cohort, and 3) to ascertain whether maintenance peginterferon therapy had any delayed, post- treatment effect in preventing HCC.
DISCUSSION
Previously, we reported that maintenance peginterferon in the HALT-C Trial was not effective in preventing clinical or histological outcomes.4 It has been suggested that the limited duration of follow-up in these reports may not have been sufficient to observe a clinical benefit of peginterferon as chemoprevention for HCC;8 in this vein, retrospective studies have suggested that a benefit of interferon in reducing the occurrence of HCC may not be observed for 5 years or more.9 Supporting this notion is a recent meta-analysis demonstrating that patients with hepatitis C who received interferon therapy had a significantly lower incidence of HCC but significant heterogeneity in the results limited confidence in this conclusion, and the duration of follow-up was an independent contributor to the heterogeneity.3 The process of hepatocarcinogenesis is well recognized to take place over many years, and, even after HCC has developed, many months to years may elapse before the tumor can be detected clinically. The HALT-C Trial investigators acknowledged the possibility that the initially planned follow-up period of 4 years from randomization (6 months after completion of randomized treatment) may have been too short to detect a benefit.10 Therefore, the protocol was amended to extend the duration of follow-up.
With continued observation of this large cohort of patients with advanced hepatitis C for up to 8.7 (median 6.1) years, we found that the number of patients with HCC increased to 88 from 53 (including 5 prevalent cases diagnosed in the first year) in an earlier report.5 Continued follow-up of the HALT-C Trial cohort for up to 5 years after the end of therapy did not show a statistically significant benefit of low dose peginterferon in reducing the incidence of HCC in patients with advanced hepatitis C who did not achieve SVR. In sub-group analysis, however, patients with cirrhosis at baseline assigned to treatment had lower risk of HCC.
Because of the unexpected difference among patients with cirrhosis between our previous and the current findings, we performed additional analyses to verify the consistency of the results. A beneficial effect of peginterferon was observed even when the analysis was restricted to patients with definite HCC (i.e., after exclusion of 20 patients with presumed HCC as defined by the protocol). In addition, a dose-response effect of peginterferon was apparent, as suggested by three findings. First, among patients randomized to receive peginterferon, multivariate analysis showed that those who stopped treatment before 2 years did not have a reduction in the risk of HCC after adjusting for laboratory markers of advanced liver disease. Second, among patients receiving peginterferon, those experiencing a ≥2-point reduction in HAI had an approximately 3-fold reduction in HCC compared to those who did not have a reduction in HAI on follow-up liver biopsy. Third, in a regression analysis of factors associated with the development of HCC, including treatment assignment, peginterferon therapy was associated with an approximate 50% reduction in the incidence of HCC over a median of 6.1 year observation period among patients with cirrhosis at baseline even when adjusted for other risk factors including age, race, smoking history, and laboratory tests associated with advanced liver disease. Nevertheless, while analysis by the two pre-specified strata of histological severity (bridging fibrosis and cirrhosis) was warranted, why the effect of peginterferon was confined to patients with cirrhosis is unclear, and the possibility that this is a chance finding cannot be excluded.
A small randomized controlled trial by Nishiguchi et al11 was the first to suggest that interferon may reduce the risk of HCC in patients with HCV-related cirrhosis. This report was followed by many other reports, mostly from Japan and Europe. The majority of these studies were retrospective or cohort studies comparing the incidence of HCC in patients who had received interferon for 6-12 months with historical or concurrent patients who had not received treatment. The median duration of follow-up ranged from 2 to 15 years. Most studies showed a benefit of treatment, although the effect was predominantly seen in patients who achieved SVR. In addition, three independent meta-analyses suggested that interferon therapy decreased the incidence of HCC in patients with chronic hepatitis C3, 12, 13 In the most recent of these meta- analyses, the relative risk (RR) of HCC for treated vs. untreated patients was 0.43 (95% CI 0.33-0.56, P<0.00001) and for patients with SVR vs. without SVR was 0.35 (95% CI 0.26-0.46, P<0.0001); however, only four of the studies included were randomized controlled trials.
Few studies have compared the incidence of HCC in interferon nonresponders and untreated patients. In two large retrospective studies, the incidence of HCC in these two groups was similar. HCC developed in 14% (20/148) of nonresponders and 12% (19/144) of untreated patients in the study by Imai et al14 and in 11% (39/342) of nonresponders and 10% (54/562) of untreated patients in the study by Yu et al.15 As was true for the 3.5-year randomized phase of the HALT-C Trial, two other studies of long-term, low-dose peginterferon (COPILOT and EPIC3) in patients with advanced fibrosis or cirrhosis showed no benefit of treatment on overall clinical outcomes or HCC.16, 17 To date, however, only preliminary data on outcomes up to 4 years have been reported in these two studies.
The mechanisms by which peginterferon might prevent HCC are not clear. Certainly, achieving an SVR is associated with a reduction in HCC. Indeed, patients in HALT-C trial who achieved SVR after 48 weeks of combination therapy had lower risk of HCC compared to lead-in phase nonresponders, HR 0.19, 95% CI 0.04-0.80.18 Patients who had >2 log decline in HCV RNA after 1.5 years of low-dose peginterferon also had a lower incidence of HCC compared to those with <2 log decline but the difference was not significant and only 15.4% of the patients receiving low-dose peginterferon had >2 log decrease in HCV RNA after 18 months of treatment. Interferon has been shown to have antitumor effects in some malignancies and may have other beneficial effects including antiproliferative and antiangiogenic effects.19, 20 Chronic inflammation has been postulated to play a role in hepatitis B virus- (HBV) and HCV-related HCC. The observation that reduction in HCC incidence in the HALT-C Trial cohort was greater among the treated patients experiencing a reduction in HAI suggests that interferon may have acted through this pathway; however, absence of benefit among control patients with a reduction in hepatic inflammation indicates that interferon may exert its effects through other mechanisms and that a reduction in HAI on follow-up biopsies in interferon-treated patients may be a surrogate of other interferon effects.
The reasons for the apparent delay in the chemopreventive effect of peginterferon are also not known, but we speculate that the delay is related to the slow growth rate of HCC, estimated to have a median doubling time of 117 days.21 In fact, the Gompertzian model would predict that an HCC may have been present for several years before reaching a detectable size.22 Therefore, the HCCs diagnosed in the first 3 to 5 years may well have been present at enrollment into the HALT-C Trial. If, indeed, peginterferon prevented HCC, several years would have been required to detect this effect. In this study, the divergence in HCC incidence between the treated and control patients with cirrhosis at baseline was not apparent until after year 4. Unfortunately, with the termination of this study, we could not determine whether the protective effect disappears or increases with longer follow-up.
Several other interesting observations emerged during this study. Severity of liver disease and portal hypertension, as reflected by more abnormal biochemical tests, lower platelet counts, and presence of esophageal varices were associated with an increased risk of HCC. On the other hand, several studies have shown that patients with increased BMI have a higher rate of HCC;23, 24 while patients in the HALT-C Trial with higher BMI had a significantly lower incidence of HCC. An association between obesity and HCC in other studies may be related to the inclusion of patients with cirrhosis secondary to non-alcoholic fatty liver disease or the inclusion of patients with earlier stage liver disease in whom obesity and steatosis may have accelerated fibrosis progression thereby increasing the risk of HCC. The HALT-C Trial excluded patients with severe steatohepatitis and all patients had advanced fibrosis or cirrhosis. Alcohol and smoking have been reported to be associated with HCC. In the HALT-C Trial, although the average life-time alcohol consumption prior to entry into the HALT-C Trial was high, neither duration of regular alcohol consumption nor life-time alcohol consumption was associated with an increased risk of HCC. Smoking is a risk factor for many cancers, but its association with HCC is not as well described. Several prior reports suggested an association between smoking and histological severity of liver disease,25 and a few reports have appeared of an association between smoking and HCC.24, 26 In this study, smoking was a significant risk factor for HCC, even after adjustment for other risk factors. These data highlight the importance of counseling patients with liver disease on smoking cessation. Esophageal varices and more recently increase in hepatic venous pressure gradient had been shown to be predictors of HCC.27, 28 We found that both presence of varices at baseline and incident varices were associated with increased risk of HCC underscoring the need to evaluate for portal hypertension in patients with advanced liver disease.
In an earlier analysis of the incidence of HCC in the HALT-C Trial cohort, we found that 17% of patients with HCC had no evidence of cirrhosis on serial histology.5 In the current analysis, 8% of HCC developed in patients who had no evidence of cirrhosis. The lower rate of HCC in noncirrhotic patients in the current analysis is related to the subsequent finding of cirrhosis on explant livers in some of the earlier patients and a higher probability that patients diagnosed with HCC during the last two years of the HALT-C study had progressed from bridging fibrosis to cirrhosis. These data confirmed that HCC can occur in non-cirrhotic patients but the rate is low and the cost-effectiveness of HCC surveillance in these patients is unclear.
In conclusion, extended follow-up of the HALT-C Trial cohort showed a modest benefit of long-term peginterferon therapy in reducing the incidence of HCC in patients with hepatitis C and cirrhosis but not in those with advanced precirrhotic fibrosis, an effect that took several years after completion of therapy to become apparent. Although the results were corroborated in multiple sub-analyses, the clinical implications are not clear. Given the marginal beneficial effect and its restriction to only part of the HALT-C Trial cohort, that no overall long-term benefit on mortality was observed in this cohort,29 and given the side effects of peginterferon, necessitating dose reduction or discontinuation in a substantial number of patients, the utility of maintenance peginterferon therapy to prevent HCC in patients with HCV-related cirrhosis is doubtful.
RESULTS
Patient Characteristics
Of the 1,050 patients randomized, 90 met predefined criteria for HCC (presumed or definite), but two were excluded because they only met criteria for presumed HCC at the time of HCC diagnosis, did not receive HCC treatment, and did not meet criteria for definite HCC after more than 2 years of follow-up. Therefore, 1,048 patients were included in this analysis. The median duration of follow-up from randomization was 6.1 (range 0-8.7) years.
Table 1 summarizes the characteristics of the patients at the time of enrollment into the HALT-C Trial. Compared to patients without HCC, those in whom HCC subsequently developed were older, had lower body mass index (BMI), had laboratory values indicating more advanced liver disease, higher AFP, higher HAI, and higher fibrosis score, and were more likely to have esophageal varices and to have been smokers.
Incidence of HCC
Of the 88 patients who met HALT-C Trial criteria for HCC, 68 had definite HCC (66 were histologically confirmed), while 20 had presumed HCC (Table 2). Sixty-six (75%) of these 88 patients had stage 1 or 2 HCC according to the United Network of Organ Sharing classification. Although treated patients were less likely to have HCC than controls, 7.2% (37/515) vs. 9.6% (51/533), this difference was not statistically significant (hazard ratio [HR] for HCC in patients randomized to peginterferon treatment 0.77, 95% confidence interval [CI] 0.51-1.18, P = 0.24) (Figure 1A).
When patients with cirrhosis at baseline were analyzed separately, however, significantly fewer of those randomized to receive peginterferon had a diagnosis of HCC than those randomized to no treatment, 6.8% (14/207) vs.15.5% (34/220), respectively (HR for HCC in patients randomized to receive peginterferon 0.45 [95% CI 0.24-0.83], P = 0.01). The cumulative incidence of HCC at 3, 5 and 7 years in the peginterferon group was 2.6%, 5.1%, and 7.8%, and, in the untreated control group, 4.0%, 11.1%, and 24.2%, respectively (log-rank test, P = 0.009). The absolute risk difference was 1.5% (95% CI -2.1%-5.0%) at 3 years, 6.1% (95% CI 0.4%-11.7%) at 5 years, and 16.4% (95% CI 2.3%-24.1%) at 7 years; the incidence of HCC in the two treatment groups did not diverge until after year 4 (Figure 1B).
Among patients with noncirrhotic fibrosis at baseline, similar numbers of treated and control patients were found to have a diagnosis of HCC, 7.5% (23/308) vs. 5.4% (17/313), respectively (HR for HCC in these patients randomized to receive peginterferon 1.44 (95% CI, 0.77-2.69, P = 0.26). Seven of these 40 patients had no evidence of cirrhosis on any of the follow-up biopsies, surgically resected tumors or liver explants. In noncirrhotics, the cumulative incidence of HCC at 3, 5 and 7 years in the peginterferon group was 2.1%, 6.4%, and 8.3% and in the control group, 1.4%, 2.9%, and 6.8%, respectively; the incidence of HCC in the two treatment groups was similar throughout the duration of the study (Figure 1B). Cox regression with interaction confirmed an interaction between treatment group and fibrosis stratum (P = 0.01).
To determine whether the observed results could have been related to uneven distribution of patients with presumed HCC in the two treatment groups, we repeated the analysis after exclusion of the 20 patients who only met criteria for presumed HCC. The results were similar, HR for HCC in the treated group 0.43 (95% CI 0.21-0.89, P = 0.02) for patients with cirrhosis at baseline and 1.86 (95% CI 0.91-3.78, P = 0.09) for patients with bridging fibrosis at baseline.
Because a beneficial effect of peginterferon was not observed during the first 4 years, we repeated the analysis on 994 patients who were still alive, had not undergone liver transplantation, and did not have HCC up to 1400 days after randomization (roughly 3 months after completion of the randomized treatment phase or 3.75 years from randomization). The cumulative incidence of HCC at 5 and 7 years in the peginterferon vs. control groups was significant, 0.7% vs. 4.0% and 3.6% vs. 18.1%, respectively (log-rank test, P = 0.01) among patients with baseline cirrhosis and insignificant, 2.5% vs. 0.8% and 4.4% vs. 4.8%, respectively (log-rank test, P = 0.69) among patients with baseline bridging fibrosis (Figure 1C). The hazard ratios were 0.33 (95% CI 0.13-0.83, P = 0.02) for patients with baseline cirrhosis and 1.19 (95% CI 0.52-2.69, P = 0.69) for patients with baseline bridging fibrosis. For all patients combined, the HR was 0.64 (95% CI 0.36-1.14; P = 0.13).
Patients might succumb from a liver-related death or undergo liver transplantation and no longer be at risk for HCC; therefore, a competing risk analysis to estimate the effect of liver transplantation or liver-related death on the incidence of HCC was conducted. Similar numbers of patients in the peginterferon and control groups underwent liver transplantation or had liver- related deaths (31 and 35 among patients with cirrhosis at baseline and 14 and 13 among patients with bridging fibrosis at baseline, respectively). The differences in the incidence of HCC between the peginterferon and control groups did not change after adjusting for these competing risks (HR 0.45, 95% CI 0.24-0.83, P = 0.01 for patients with cirrhosis at baseline and HR 1.44, 95% CI 0.77-2.69, P = 0.26 for patients with bridging fibrosis at baseline).
Impact of Duration of Peginterferon Treatment on Incidence of HCC
To further confirm the potential beneficial effect of peginterferon in reducing the incidence of HCC, we assessed the association between adherence to maintenance peginterferon and the incidence of HCC. For this analysis, treated patients were stratified into two groups: patients who were still receiving peginterferon (regardless of dose) ≥2 years after randomization and those who had stopped peginterferon <2 years after randomization but continued to be followed. Patients who received peginterferon for ≥2 years had a significantly lower incidence of HCC compared to those who were treated for <2 years (HR 0.36, 95% CI 0.16-0.82, P = 0.02) (Table 3); however, this difference was observed only in the patients with cirrhosis at baseline (HR 0.10, 95% CI 0.03-0.44, P = 0.002). Because patients who tolerated peginterferon treatment had less advanced liver disease (supplementary table), we performed a multivariate analysis to determine whether the duration of peginterferon treatment was associated, independent of disease severity, with a reduced risk of HCC, which was, in fact, the case (see below).
Viral Suppression and Incidence of HCC
Of the 468 patients randomized to maintenance peginterferon and were evaluated for virologic response at year 1.5, 30/396 (7.6%), 1/36 (2.8%), and 1/36 (2.8%) patients with <2 log, 2-4 log, and ≥4 log decrease in HCV RNA were subsequently diagnosed to have HCC, HR for the patients with ≥2 vs. <2 log decrease in HCV RNA 0.38 (95% CI 0.09-1.59, P = 0.18).
Association of Decrease in HAI with Decreased Incidence of HCC
In an earlier analysis of the HALT-C Trial data, we showed that maintenance peginterferon reduced HAI compared to no treatment.4 To determine whether the effect of peginterferon on prevention of HCC was related to its effect in decreasing hepatic inflammation, we analyzed 426 patients in the peginterferon group and 416 in the control group who had repeat liver biopsies at 1.5 years. Treated patients who had an unchanged or increased HAI had a higher incidence of HCC than those who had a decrease in HAI by ≥2 points on the year-1.5 biopsy: 9.4% vs. 2.9% (HR 2.98 and 95% CI 1.14-7.81, P = 0.03) (Table 4). The incidence of HCC in the control group was similar regardless of changes in HAI on the year-1.5 biopsy and comparable to that in the treatment group that did not have a decrease in HAI.
Multivariate Analyses for Predictors of HCC
We undertook stepwise Cox regression analysis to determine whether maintenance peginterferon had an independent effect in preventing HCC. Selected factors that were significant on univariate analysis: age, BMI, platelet, albumin, AST, alkaline phosphatase, total bilirubin, prothrombin time, esophageal varices, HAI, having ever smoked cigarettes, and other relevant factors including gender, black race, cirrhosis on baseline biopsy and treatment assignment were included in the model. To avoid over-fitting, several related laboratory markers that were significant on univariate analysis (Table 1) were not included in the model. Patients with cirrhosis on baseline biopsy assigned to peginterferon treatment had half the rate of HCC outcomes as those randomized to no treatment (HR 0.50, 95% CI 0.27-0.95) (Table 5). In contrast, the rate of HCC outcomes in patients with fibrosis on baseline biopsy, when analyzed separately, was similar regardless of treatment assignment (p = 0.38). Older age, lower BMI, lower platelet, higher AST, higher alkaline phosphatase, and history of smoking were also associated with increased risk of HCC. When the analysis was limited to patients in the treatment group, ability to remain on low-dose peginterferon for ≥2 years was an independent predictor that HCC would not develop (HR 0.39, 95% CI 0.16-0.92).
Because presence of esophageal varices at randomization was a strong risk factor for HCC in a previous analysis of HALT-C Trial outcomes,5 we examined the effect of their presence and development more closely, based on results from the baseline and year-3.5 upper endoscopies. Of the 781 patients who underwent a second endoscopy, 185 had varices noted at randomization, 148 had varices first noted at the second endoscopy (incident varices), and 448 did not have varices on either examination. Relative to patients without varices on either examination, subsequent HCC was more likely for patients with varices at randomization (HR 2.54, 95% CI 1.32-4.88) and for patients with incident varices (HR 2.68, 95% CI 1.35-5.32).
Source
Article in Press
Gastroenterology Dec 2010
Anna S. Lok, 1 James E. Everhart, 2 Elizabeth C. Wright, 3 Adrian M. Di Bisceglie, 4 Hae-Young Kim, 5 Richard K. Sterling, 6 Gregory T. Everson, 7 Karen L. Lindsay, 8 William M. Lee, 9 Herbert L. Bonkovsky, 10,11 Jules L. Dienstag, 12,13 Marc G. Ghany, 14 Chihiro Morishima, 15 Timothy R. Morgan, 16,17 and the HALT-C Trial Group
FROM JULES of NATAP: this was presented at AASLD where I went to the mic after Dr Lok presented the data and reminded the large audience I previously questioned and doubted the original findings that peg maintenence therapy was not useful when they were first presented several years ago. My questioning of their original findings were not well received but now upon further followup it appears maintenance therapy can provide benefit for patients with cirrhosis. The utility of maintenance therapy may still have application for patients who are nonresponders to the new HCV oral protease inhibitors expected to be approved in 2011, to prevent further disease progression until better, more effective multi-drug oral therapy combinations become available.
"extended follow-up of the HALT-C Trial cohort showed a modest benefit of long-term peginterferon therapy in reducing the incidence of HCC in patients with hepatitis C and cirrhosis but not in those with advanced precirrhotic fibrosis, an effect that took several years after completion of therapy to become apparent. Although the results were corroborated in multiple sub-analyses, the clinical implications are not clear. Given the marginal beneficial effect and its restriction to only part of the HALT-C Trial cohort, that no overall long-term benefit on mortality was observed in this cohort,29 and given the side effects of peginterferon, necessitating dose reduction or discontinuation in a substantial number of patients, the utility of maintenance peginterferon therapy to prevent HCC in patients with HCV-related cirrhosis is doubtful.....When patients with cirrhosis at baseline were analyzed separately, however, significantly fewer of those randomized to receive peginterferon had a diagnosis of HCC than those randomized to no treatment, 6.8% (14/207) vs.15.5% (34/220), respectively (HR for HCC in patients randomized to receive peginterferon 0.45 [95% CI 0.24-0.83], P = 0.01). Interferon has been shown to have antitumor effects in some malignancies and may have other beneficial effects including antiproliferative and antiangiogenic effects.19, 20 Chronic inflammation has been postulated to play a role in hepatitis B virus- (HBV) and HCV-related HCC. The observation that reduction in HCC incidence in the HALT-C Trial cohort was greater among the treated patients experiencing a reduction in HAI suggests that interferon may have acted through this pathway"
"The Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) Trial.......Analysis of the HALT-C Trial results after 3.5 years of treatment revealed that peginterferon did not reduce the overall risk of liver disease progression.4 A subsequent report focusing on HCC development also showed no difference between the treated and control groups during and immediately after the period of maintenance peginterferon therapy.5 We continued to follow the HALT-C Trial cohort off therapy for a total of up to 8.7 years to monitor for the development of decompensated liver disease and HCC. During the extended follow-up period, the number of patients with HCC increased progressively......
.......Although treated patients were less likely to have HCC than controls, 7.2% (37/515) vs. 9.6% (51/533), this difference was not statistically significant (hazard ratio [HR] for HCC in patients randomized to peginterferon treatment 0.77, 95% confidence interval [CI] 0.51-1.18, P = 0.24) (Figure 1A). ......When patients with cirrhosis at baseline were analyzed separately, however, significantly fewer of those randomized to receive peginterferon had a diagnosis of HCC than those randomized to no treatment, 6.8% (14/207) vs.15.5% (34/220), respectively (HR for HCC in patients randomized to receive peginterferon 0.45 [95% CI 0.24-0.83], P = 0.01). The cumulative incidence of HCC at 3, 5 and 7 years in the peginterferon group was 2.6%, 5.1%, and 7.8%, and, in the untreated control group, 4.0%, 11.1%, and 24.2%, respectively (log-rank test, P = 0.009). The absolute risk difference was 1.5% (95% CI -2.1%-5.0%) at 3 years, 6.1% (95% CI 0.4%-11.7%) at 5 years, and 16.4% (95% CI 2.3%-24.1%) at 7 years; the incidence of HCC in the two treatment groups did not diverge until after year 4 (Figure 1B)."
.......Because of the unexpected difference among patients with cirrhosis between our previous and the current findings, we performed additional analyses to verify the consistency of the results. A beneficial effect of peginterferon was observed even when the analysis was restricted to patients with definite HCC (i.e., after exclusion of 20 patients with presumed HCC as defined by the protocol). In addition, a dose-response effect of peginterferon was apparent, as suggested by three findings. First, among patients randomized to receive peginterferon, multivariate analysis showed that those who stopped treatment before 2 years did not have a reduction in the risk of HCC after adjusting for laboratory markers of advanced liver disease. Second, among patients receiving peginterferon, those experiencing a ≥2-point reduction in HAI had an approximately 3-fold reduction in HCC compared to those who did not have a reduction in HAI on follow-up liver biopsy. Third, in a regression analysis of factors associated with the development of HCC, including treatment assignment, peginterferon therapy was associated with an approximate 50% reduction in the incidence of HCC over a median of 6.1 year observation period among patients with cirrhosis at baseline even when adjusted for other risk factors including age, race, smoking history, and laboratory tests associated with advanced liver disease. Nevertheless, while analysis by the two pre-specified strata of histological severity (bridging fibrosis and cirrhosis) was warranted, why the effect of peginterferon was confined to patients with cirrhosis is unclear, and the possibility that this is a chance finding cannot be excluded."
Abstract
Background & Aims
Interferon reportedly decreases the incidence of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C. The Hepatitis C anti-viral long-term treatment against cirrhosis (HALT-C) trial showed that 4 years of maintenance therapy with peginterferon does not reduce liver disease progression. We investigated whether peginterferon decreases the incidence of HCC in the HALT-C cohort over a longer post-treatment follow-up period.
Methods
The study included 1,048 patients with chronic Hepatitis C (Ishak fibrosis scores ≥3) who did not have a sustained virological response (SVR) to therapy. They were randomly assigned to groups given a half-dose of peginterferon or no treatment (controls) for 3.5 years and followed for a median 6.1 (maximum 8.7) years.
Results
Eighty-eight patients developed HCC (68 definite, 20 presumed): 37/515 that were given peginterferon (7.2%) and 51/533 controls (9.6%; P=0.24). There was a significantly lower incidence of HCC among patients given peginterferon therapy who had cirrhosis, but not fibrosis, based on analysis of baseline biopsy samples. After 7 years, the cumulative incidences of HCC in treated and control patients with cirrhosis were 7.8% and 24.2%, respectively (hazard ratio [HR]=0.45; 95% confidence interval [CI]: 0.24Ð0.83); in treated and control patients with fibrosis they were 8.3% and 6.8%, respectively (HR=1.44; 95% CI: 0.77Ð2.69). Treated patients with a ≥2-point decrease in the histologic activity index, based on a follow-up biopsy, had a lower incidence of HCC than those with unchanged or increased scores (2.9% vs. 9.4%; P=0.03).
Conclusions
Extended analysis of the HALT-C cohort showed that long-term peginterferon therapy does not reduce the incidence of HCC among patients with advanced hepatitis C who did not achieve SVRs. Patients with cirrhosis who received peginterferon treatment had a lower risk for HCC than controls.
INTRODUCTION
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most common cause of cancer death in the world.1 In the United States, hepatitis C is the greatest contributor to both the occurrence of HCC and its observed increased incidence over the last 20 years. Among persons with chronic hepatitis C, risk factors for the development of HCC are incompletely understood. HCC has been found primarily, but not exclusively, in patients with cirrhosis. In addition to the degree of liver fibrosis, biochemical markers of advanced liver disease (e.g., low platelet count, low albumin), presence of esophageal varices, diabetes, obesity, and use of tobacco and excessive alcohol have been associated with an increased risk for HCC. The risk of HCC is decreased in patients with chronic hepatitis C who achieved a sustained virological response (SVR) to interferon/ribavirin treatment;2, 3 however, the effectiveness of interferon therapy in reducing HCC among patients with hepatitis C virus (HCV)-related cirrhosis who did not achieve an SVR is unclear.
The Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) Trial was a randomized, multi-center trial to determine whether 3.5 years of half-dose pegylated interferon (peginterferon) treatment reduced liver disease progression among patients with hepatitis C and advanced fibrosis who were nonresponders to peginterferon and ribavirin. Analysis of the HALT-C Trial results after 3.5 years of treatment revealed that peginterferon did not reduce the overall risk of liver disease progression.4 A subsequent report focusing on HCC development also showed no difference between the treated and control groups during and immediately after the period of maintenance peginterferon therapy.5 We continued to follow the HALT-C Trial cohort off therapy for a total of up to 8.7 years to monitor for the development of decompensated liver disease and HCC. During the extended follow-up period, the number of patients with HCC increased progressively. The aims of the current analysis were: 1) to determine the incidence of HCC among the HALT-C Trial cohort after a longer duration of follow-up, 2) to identify factors associated with the development of HCC in the HALT-C Trial cohort, and 3) to ascertain whether maintenance peginterferon therapy had any delayed, post- treatment effect in preventing HCC.
DISCUSSION
Previously, we reported that maintenance peginterferon in the HALT-C Trial was not effective in preventing clinical or histological outcomes.4 It has been suggested that the limited duration of follow-up in these reports may not have been sufficient to observe a clinical benefit of peginterferon as chemoprevention for HCC;8 in this vein, retrospective studies have suggested that a benefit of interferon in reducing the occurrence of HCC may not be observed for 5 years or more.9 Supporting this notion is a recent meta-analysis demonstrating that patients with hepatitis C who received interferon therapy had a significantly lower incidence of HCC but significant heterogeneity in the results limited confidence in this conclusion, and the duration of follow-up was an independent contributor to the heterogeneity.3 The process of hepatocarcinogenesis is well recognized to take place over many years, and, even after HCC has developed, many months to years may elapse before the tumor can be detected clinically. The HALT-C Trial investigators acknowledged the possibility that the initially planned follow-up period of 4 years from randomization (6 months after completion of randomized treatment) may have been too short to detect a benefit.10 Therefore, the protocol was amended to extend the duration of follow-up.
With continued observation of this large cohort of patients with advanced hepatitis C for up to 8.7 (median 6.1) years, we found that the number of patients with HCC increased to 88 from 53 (including 5 prevalent cases diagnosed in the first year) in an earlier report.5 Continued follow-up of the HALT-C Trial cohort for up to 5 years after the end of therapy did not show a statistically significant benefit of low dose peginterferon in reducing the incidence of HCC in patients with advanced hepatitis C who did not achieve SVR. In sub-group analysis, however, patients with cirrhosis at baseline assigned to treatment had lower risk of HCC.
Because of the unexpected difference among patients with cirrhosis between our previous and the current findings, we performed additional analyses to verify the consistency of the results. A beneficial effect of peginterferon was observed even when the analysis was restricted to patients with definite HCC (i.e., after exclusion of 20 patients with presumed HCC as defined by the protocol). In addition, a dose-response effect of peginterferon was apparent, as suggested by three findings. First, among patients randomized to receive peginterferon, multivariate analysis showed that those who stopped treatment before 2 years did not have a reduction in the risk of HCC after adjusting for laboratory markers of advanced liver disease. Second, among patients receiving peginterferon, those experiencing a ≥2-point reduction in HAI had an approximately 3-fold reduction in HCC compared to those who did not have a reduction in HAI on follow-up liver biopsy. Third, in a regression analysis of factors associated with the development of HCC, including treatment assignment, peginterferon therapy was associated with an approximate 50% reduction in the incidence of HCC over a median of 6.1 year observation period among patients with cirrhosis at baseline even when adjusted for other risk factors including age, race, smoking history, and laboratory tests associated with advanced liver disease. Nevertheless, while analysis by the two pre-specified strata of histological severity (bridging fibrosis and cirrhosis) was warranted, why the effect of peginterferon was confined to patients with cirrhosis is unclear, and the possibility that this is a chance finding cannot be excluded.
A small randomized controlled trial by Nishiguchi et al11 was the first to suggest that interferon may reduce the risk of HCC in patients with HCV-related cirrhosis. This report was followed by many other reports, mostly from Japan and Europe. The majority of these studies were retrospective or cohort studies comparing the incidence of HCC in patients who had received interferon for 6-12 months with historical or concurrent patients who had not received treatment. The median duration of follow-up ranged from 2 to 15 years. Most studies showed a benefit of treatment, although the effect was predominantly seen in patients who achieved SVR. In addition, three independent meta-analyses suggested that interferon therapy decreased the incidence of HCC in patients with chronic hepatitis C3, 12, 13 In the most recent of these meta- analyses, the relative risk (RR) of HCC for treated vs. untreated patients was 0.43 (95% CI 0.33-0.56, P<0.00001) and for patients with SVR vs. without SVR was 0.35 (95% CI 0.26-0.46, P<0.0001); however, only four of the studies included were randomized controlled trials.
Few studies have compared the incidence of HCC in interferon nonresponders and untreated patients. In two large retrospective studies, the incidence of HCC in these two groups was similar. HCC developed in 14% (20/148) of nonresponders and 12% (19/144) of untreated patients in the study by Imai et al14 and in 11% (39/342) of nonresponders and 10% (54/562) of untreated patients in the study by Yu et al.15 As was true for the 3.5-year randomized phase of the HALT-C Trial, two other studies of long-term, low-dose peginterferon (COPILOT and EPIC3) in patients with advanced fibrosis or cirrhosis showed no benefit of treatment on overall clinical outcomes or HCC.16, 17 To date, however, only preliminary data on outcomes up to 4 years have been reported in these two studies.
The mechanisms by which peginterferon might prevent HCC are not clear. Certainly, achieving an SVR is associated with a reduction in HCC. Indeed, patients in HALT-C trial who achieved SVR after 48 weeks of combination therapy had lower risk of HCC compared to lead-in phase nonresponders, HR 0.19, 95% CI 0.04-0.80.18 Patients who had >2 log decline in HCV RNA after 1.5 years of low-dose peginterferon also had a lower incidence of HCC compared to those with <2 log decline but the difference was not significant and only 15.4% of the patients receiving low-dose peginterferon had >2 log decrease in HCV RNA after 18 months of treatment. Interferon has been shown to have antitumor effects in some malignancies and may have other beneficial effects including antiproliferative and antiangiogenic effects.19, 20 Chronic inflammation has been postulated to play a role in hepatitis B virus- (HBV) and HCV-related HCC. The observation that reduction in HCC incidence in the HALT-C Trial cohort was greater among the treated patients experiencing a reduction in HAI suggests that interferon may have acted through this pathway; however, absence of benefit among control patients with a reduction in hepatic inflammation indicates that interferon may exert its effects through other mechanisms and that a reduction in HAI on follow-up biopsies in interferon-treated patients may be a surrogate of other interferon effects.
The reasons for the apparent delay in the chemopreventive effect of peginterferon are also not known, but we speculate that the delay is related to the slow growth rate of HCC, estimated to have a median doubling time of 117 days.21 In fact, the Gompertzian model would predict that an HCC may have been present for several years before reaching a detectable size.22 Therefore, the HCCs diagnosed in the first 3 to 5 years may well have been present at enrollment into the HALT-C Trial. If, indeed, peginterferon prevented HCC, several years would have been required to detect this effect. In this study, the divergence in HCC incidence between the treated and control patients with cirrhosis at baseline was not apparent until after year 4. Unfortunately, with the termination of this study, we could not determine whether the protective effect disappears or increases with longer follow-up.
Several other interesting observations emerged during this study. Severity of liver disease and portal hypertension, as reflected by more abnormal biochemical tests, lower platelet counts, and presence of esophageal varices were associated with an increased risk of HCC. On the other hand, several studies have shown that patients with increased BMI have a higher rate of HCC;23, 24 while patients in the HALT-C Trial with higher BMI had a significantly lower incidence of HCC. An association between obesity and HCC in other studies may be related to the inclusion of patients with cirrhosis secondary to non-alcoholic fatty liver disease or the inclusion of patients with earlier stage liver disease in whom obesity and steatosis may have accelerated fibrosis progression thereby increasing the risk of HCC. The HALT-C Trial excluded patients with severe steatohepatitis and all patients had advanced fibrosis or cirrhosis. Alcohol and smoking have been reported to be associated with HCC. In the HALT-C Trial, although the average life-time alcohol consumption prior to entry into the HALT-C Trial was high, neither duration of regular alcohol consumption nor life-time alcohol consumption was associated with an increased risk of HCC. Smoking is a risk factor for many cancers, but its association with HCC is not as well described. Several prior reports suggested an association between smoking and histological severity of liver disease,25 and a few reports have appeared of an association between smoking and HCC.24, 26 In this study, smoking was a significant risk factor for HCC, even after adjustment for other risk factors. These data highlight the importance of counseling patients with liver disease on smoking cessation. Esophageal varices and more recently increase in hepatic venous pressure gradient had been shown to be predictors of HCC.27, 28 We found that both presence of varices at baseline and incident varices were associated with increased risk of HCC underscoring the need to evaluate for portal hypertension in patients with advanced liver disease.
In an earlier analysis of the incidence of HCC in the HALT-C Trial cohort, we found that 17% of patients with HCC had no evidence of cirrhosis on serial histology.5 In the current analysis, 8% of HCC developed in patients who had no evidence of cirrhosis. The lower rate of HCC in noncirrhotic patients in the current analysis is related to the subsequent finding of cirrhosis on explant livers in some of the earlier patients and a higher probability that patients diagnosed with HCC during the last two years of the HALT-C study had progressed from bridging fibrosis to cirrhosis. These data confirmed that HCC can occur in non-cirrhotic patients but the rate is low and the cost-effectiveness of HCC surveillance in these patients is unclear.
In conclusion, extended follow-up of the HALT-C Trial cohort showed a modest benefit of long-term peginterferon therapy in reducing the incidence of HCC in patients with hepatitis C and cirrhosis but not in those with advanced precirrhotic fibrosis, an effect that took several years after completion of therapy to become apparent. Although the results were corroborated in multiple sub-analyses, the clinical implications are not clear. Given the marginal beneficial effect and its restriction to only part of the HALT-C Trial cohort, that no overall long-term benefit on mortality was observed in this cohort,29 and given the side effects of peginterferon, necessitating dose reduction or discontinuation in a substantial number of patients, the utility of maintenance peginterferon therapy to prevent HCC in patients with HCV-related cirrhosis is doubtful.
RESULTS
Patient Characteristics
Of the 1,050 patients randomized, 90 met predefined criteria for HCC (presumed or definite), but two were excluded because they only met criteria for presumed HCC at the time of HCC diagnosis, did not receive HCC treatment, and did not meet criteria for definite HCC after more than 2 years of follow-up. Therefore, 1,048 patients were included in this analysis. The median duration of follow-up from randomization was 6.1 (range 0-8.7) years.
Table 1 summarizes the characteristics of the patients at the time of enrollment into the HALT-C Trial. Compared to patients without HCC, those in whom HCC subsequently developed were older, had lower body mass index (BMI), had laboratory values indicating more advanced liver disease, higher AFP, higher HAI, and higher fibrosis score, and were more likely to have esophageal varices and to have been smokers.
Incidence of HCC
Of the 88 patients who met HALT-C Trial criteria for HCC, 68 had definite HCC (66 were histologically confirmed), while 20 had presumed HCC (Table 2). Sixty-six (75%) of these 88 patients had stage 1 or 2 HCC according to the United Network of Organ Sharing classification. Although treated patients were less likely to have HCC than controls, 7.2% (37/515) vs. 9.6% (51/533), this difference was not statistically significant (hazard ratio [HR] for HCC in patients randomized to peginterferon treatment 0.77, 95% confidence interval [CI] 0.51-1.18, P = 0.24) (Figure 1A).
When patients with cirrhosis at baseline were analyzed separately, however, significantly fewer of those randomized to receive peginterferon had a diagnosis of HCC than those randomized to no treatment, 6.8% (14/207) vs.15.5% (34/220), respectively (HR for HCC in patients randomized to receive peginterferon 0.45 [95% CI 0.24-0.83], P = 0.01). The cumulative incidence of HCC at 3, 5 and 7 years in the peginterferon group was 2.6%, 5.1%, and 7.8%, and, in the untreated control group, 4.0%, 11.1%, and 24.2%, respectively (log-rank test, P = 0.009). The absolute risk difference was 1.5% (95% CI -2.1%-5.0%) at 3 years, 6.1% (95% CI 0.4%-11.7%) at 5 years, and 16.4% (95% CI 2.3%-24.1%) at 7 years; the incidence of HCC in the two treatment groups did not diverge until after year 4 (Figure 1B).
Among patients with noncirrhotic fibrosis at baseline, similar numbers of treated and control patients were found to have a diagnosis of HCC, 7.5% (23/308) vs. 5.4% (17/313), respectively (HR for HCC in these patients randomized to receive peginterferon 1.44 (95% CI, 0.77-2.69, P = 0.26). Seven of these 40 patients had no evidence of cirrhosis on any of the follow-up biopsies, surgically resected tumors or liver explants. In noncirrhotics, the cumulative incidence of HCC at 3, 5 and 7 years in the peginterferon group was 2.1%, 6.4%, and 8.3% and in the control group, 1.4%, 2.9%, and 6.8%, respectively; the incidence of HCC in the two treatment groups was similar throughout the duration of the study (Figure 1B). Cox regression with interaction confirmed an interaction between treatment group and fibrosis stratum (P = 0.01).
To determine whether the observed results could have been related to uneven distribution of patients with presumed HCC in the two treatment groups, we repeated the analysis after exclusion of the 20 patients who only met criteria for presumed HCC. The results were similar, HR for HCC in the treated group 0.43 (95% CI 0.21-0.89, P = 0.02) for patients with cirrhosis at baseline and 1.86 (95% CI 0.91-3.78, P = 0.09) for patients with bridging fibrosis at baseline.
Because a beneficial effect of peginterferon was not observed during the first 4 years, we repeated the analysis on 994 patients who were still alive, had not undergone liver transplantation, and did not have HCC up to 1400 days after randomization (roughly 3 months after completion of the randomized treatment phase or 3.75 years from randomization). The cumulative incidence of HCC at 5 and 7 years in the peginterferon vs. control groups was significant, 0.7% vs. 4.0% and 3.6% vs. 18.1%, respectively (log-rank test, P = 0.01) among patients with baseline cirrhosis and insignificant, 2.5% vs. 0.8% and 4.4% vs. 4.8%, respectively (log-rank test, P = 0.69) among patients with baseline bridging fibrosis (Figure 1C). The hazard ratios were 0.33 (95% CI 0.13-0.83, P = 0.02) for patients with baseline cirrhosis and 1.19 (95% CI 0.52-2.69, P = 0.69) for patients with baseline bridging fibrosis. For all patients combined, the HR was 0.64 (95% CI 0.36-1.14; P = 0.13).
Patients might succumb from a liver-related death or undergo liver transplantation and no longer be at risk for HCC; therefore, a competing risk analysis to estimate the effect of liver transplantation or liver-related death on the incidence of HCC was conducted. Similar numbers of patients in the peginterferon and control groups underwent liver transplantation or had liver- related deaths (31 and 35 among patients with cirrhosis at baseline and 14 and 13 among patients with bridging fibrosis at baseline, respectively). The differences in the incidence of HCC between the peginterferon and control groups did not change after adjusting for these competing risks (HR 0.45, 95% CI 0.24-0.83, P = 0.01 for patients with cirrhosis at baseline and HR 1.44, 95% CI 0.77-2.69, P = 0.26 for patients with bridging fibrosis at baseline).
Impact of Duration of Peginterferon Treatment on Incidence of HCC
To further confirm the potential beneficial effect of peginterferon in reducing the incidence of HCC, we assessed the association between adherence to maintenance peginterferon and the incidence of HCC. For this analysis, treated patients were stratified into two groups: patients who were still receiving peginterferon (regardless of dose) ≥2 years after randomization and those who had stopped peginterferon <2 years after randomization but continued to be followed. Patients who received peginterferon for ≥2 years had a significantly lower incidence of HCC compared to those who were treated for <2 years (HR 0.36, 95% CI 0.16-0.82, P = 0.02) (Table 3); however, this difference was observed only in the patients with cirrhosis at baseline (HR 0.10, 95% CI 0.03-0.44, P = 0.002). Because patients who tolerated peginterferon treatment had less advanced liver disease (supplementary table), we performed a multivariate analysis to determine whether the duration of peginterferon treatment was associated, independent of disease severity, with a reduced risk of HCC, which was, in fact, the case (see below).
Viral Suppression and Incidence of HCC
Of the 468 patients randomized to maintenance peginterferon and were evaluated for virologic response at year 1.5, 30/396 (7.6%), 1/36 (2.8%), and 1/36 (2.8%) patients with <2 log, 2-4 log, and ≥4 log decrease in HCV RNA were subsequently diagnosed to have HCC, HR for the patients with ≥2 vs. <2 log decrease in HCV RNA 0.38 (95% CI 0.09-1.59, P = 0.18).
Association of Decrease in HAI with Decreased Incidence of HCC
In an earlier analysis of the HALT-C Trial data, we showed that maintenance peginterferon reduced HAI compared to no treatment.4 To determine whether the effect of peginterferon on prevention of HCC was related to its effect in decreasing hepatic inflammation, we analyzed 426 patients in the peginterferon group and 416 in the control group who had repeat liver biopsies at 1.5 years. Treated patients who had an unchanged or increased HAI had a higher incidence of HCC than those who had a decrease in HAI by ≥2 points on the year-1.5 biopsy: 9.4% vs. 2.9% (HR 2.98 and 95% CI 1.14-7.81, P = 0.03) (Table 4). The incidence of HCC in the control group was similar regardless of changes in HAI on the year-1.5 biopsy and comparable to that in the treatment group that did not have a decrease in HAI.
Multivariate Analyses for Predictors of HCC
We undertook stepwise Cox regression analysis to determine whether maintenance peginterferon had an independent effect in preventing HCC. Selected factors that were significant on univariate analysis: age, BMI, platelet, albumin, AST, alkaline phosphatase, total bilirubin, prothrombin time, esophageal varices, HAI, having ever smoked cigarettes, and other relevant factors including gender, black race, cirrhosis on baseline biopsy and treatment assignment were included in the model. To avoid over-fitting, several related laboratory markers that were significant on univariate analysis (Table 1) were not included in the model. Patients with cirrhosis on baseline biopsy assigned to peginterferon treatment had half the rate of HCC outcomes as those randomized to no treatment (HR 0.50, 95% CI 0.27-0.95) (Table 5). In contrast, the rate of HCC outcomes in patients with fibrosis on baseline biopsy, when analyzed separately, was similar regardless of treatment assignment (p = 0.38). Older age, lower BMI, lower platelet, higher AST, higher alkaline phosphatase, and history of smoking were also associated with increased risk of HCC. When the analysis was limited to patients in the treatment group, ability to remain on low-dose peginterferon for ≥2 years was an independent predictor that HCC would not develop (HR 0.39, 95% CI 0.16-0.92).
Because presence of esophageal varices at randomization was a strong risk factor for HCC in a previous analysis of HALT-C Trial outcomes,5 we examined the effect of their presence and development more closely, based on results from the baseline and year-3.5 upper endoscopies. Of the 781 patients who underwent a second endoscopy, 185 had varices noted at randomization, 148 had varices first noted at the second endoscopy (incident varices), and 448 did not have varices on either examination. Relative to patients without varices on either examination, subsequent HCC was more likely for patients with varices at randomization (HR 2.54, 95% CI 1.32-4.88) and for patients with incident varices (HR 2.68, 95% CI 1.35-5.32).
Source
Pharmacodynamics of PEG-IFN alpha-2a and HCV response as a function of IL28B polymorphism in HIV/HCV co-infected patients
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JAIDS Journal of Acquired Immune Deficiency Syndromes Publish Ahead of Print Dec 19 2010
Evaldo Stanislau Affonso de Araœjo1, Harel Dahari2, Scott J Cotler2, Thomas J Layden2, Avidan U Neumann3, Carlos Eduardo Melo1, Antonio Alci Barone1 1University of Sao Paulo Hospital das Cl’nicas, Sao Paulo, Brazil; 2Department of Medicine, University of Illinois at Chicago, Chicago, USA and3Bar Ilan University, Life Sciences Faculty, Ramat Gan, Israel.
"In conclusion, in HIV/HCV co-infected patients, the IL28B-CC genotype was most strongly associated with a higher first phase viral decline and greater average PEG-IFN effectiveness during the first week of therapy, ∈7max.
Pharmacodynamic analysis showed that genotype-CC conferred increased sensitivity to PEG-IFN, as shown by a lower PEG-IFN-α-2a EC50. These kinetic findings raise the possibility that the IL28B CC-genotype favorably affects viral response by augmenting IFN-λ mediated activation of the IFN signaling cascade, leading to increased effectiveness in blocking virion production/release. Notably, as we approach a new era of combination therapy with PEG-IFN and direct antiviral agents, a better understanding of factors associated with PEG-IFN-related viral kinetics will provide the basis to develop optimal treatment strategies for HCV28, 29. Larger and more detailed studies are needed to confirm these new observations in HIV/HCV co-infected patients."
Abstract
We examined the association between IL28B single-nucleotide-polymorphism rs12979860, hepatitis C virus (HCV) kinetic and pegylated-interferon-alpha-2a pharmacodynamic parameters in HIV/HCV-co-infected patients from South America. Twenty-six subjects received PEG-IFN-alpha-2a+ribavirin. Serum HCV-RNA and interferon concentrations were measured frequently during the first 12-weeks of therapy and analyzed using mathematical models. African Americans and Whites had a similar distribution of IL28B genotypes (p=0.5). The CC genotype was overrepresented (p=0.015) in patients infected with HCV genotype-3 compared to genotype-1. In both genotype-1 and genotype-3, the first-phase-viral decline and the average PEG-IFN- alpha-2a effectiveness during the first week of therapy were larger (trend P≤0.12) in genotype-CC compared with genotypes-TC/TT. In genotype-1 patients, the second- slower phase of viral decline (days 2-29) and infected-cells-loss rate, σ, were larger (p=0.02 and 0.11, respectively) in genotype-CC than in genotypes-TC/TT. These associations were not observed in genotype-3 patients.
Introduction
Coinfection with human immunodeficiency virus (HIV) and HCV affects approximately 10 million people worldwide1 and up to 100,000 persons in Brazil2. Antiviral therapy for hepatitis C virus (HCV) consisting of pegylated-interferon-α (PEG-IFN) and ribavirin (standard of care, SOC) has potential adverse effects, and response rates are lower in HCV/HIV co-infected than in HCV-monoinfected patients3-9. Consequently, there is considerable interest in identifying better predictors of treatment response. A seminal study showed that single nucleotide polymorphisms (SNPs) in the IL28B gene region were associated with race/ethnicity and correlated with response to pegylated interferon-alpha (PEG-IFN) and ribavirin therapy in HCV mono-infected patients10-13.
Recently, early HCV kinetics (e.g., first and second phases of viral decline) have been evaluated as a function of IL28B SNPs in HCV mono-infected patients, although pharmacodynamic parameters are lacking in these analyses14, 15. To the best of our knowledge, there is no such information in HIV/HCV co-infected individuals. To determine the relationship between IL28B polymorphisms and viral/host parameters in HIV/HCV co-infected patients, we compared IL28B genotypes with recent results of viral response16 and estimates of viral kinetic and pharmacodynamic parameters17 in 26 patients with HIV and HCV who were treated with PEG-IFN-α-2a and ribavirin.
Discussion
A detailed HCV kinetic analysis provided new and important information regarding the impact of IL28B genotype on response to PEG-IFN plus RBV in HIV/HCV co-infected patients. Overall, the CC-genotype was most strongly associated with a higher first phase viral decline and maximum PEG-IFN effectiveness during the first week of therapy, ∈7max. These findings indicate that PEG-IFN has greater efficacy in blocking HCV production/release in patients with the favorable IL28B CC-genotype. Our results are in agreement with recent findings in HCV-mono-infection patients14, 15. The PEG-IFN-α-2a EC50 was lower in genotype-CC compared with genotype-TC/TT, providing further evidence that the CC-genotype confers a higher sensitivity to IFN treatment. Thus far, the mechanism of action of the IL28B polymorphism has not been identified. The location of the genetic polymorphism upstream of the IFN-λ gene18, 19 raises the possibility that the IL28B genotype mediates endogenous production of IFN- λ, which contributes to the first phase response by stimulating IFN signaling.
The second phase viral decline slope also was associated with genotype-CC in genotype 1 patients (Table 1). The overall correlation between the CC-genotype and the infection death/loss rate, σ, was less prominent (trend, p=0.11). This discrepancy could be explained by the fact that 6 out of 21 of patients who finished 48 weeks of therapy in our study17 had a triphasic viral decline pattern, consisting of a first phase (1-2 days) with a rapid virus load decline followed by a "shoulder phase" (8 - 28 days), in which virus levels decay slowly or remain constant, and a third phase of renewed viral decay20, 21. Calculating the slower phase slope from the measured data includes the "shoulder phase" in these triphasic patients. In contrast, by using a mathematical model that includes hepatocytes proliferation, estimated σ reflects the final slope and excludes the shoulder phase22. Interestingly, five of the six triphasic patients had genotype-TT/TC and only one had genotype-CC (not shown). In addition, we recently showed that drug effectiveness, ∈, can significantly affect the serum second phase slope decline22, and that when ∈ ~ 1 the slower phase slope is close to σ. Indeed, when the analysis was performed only in patients with first phase decline > 1 log (i.e., ∈>90%), the association between IL28B genotype-CC and the slower phase slope was lost (p=0.5, not shown), in agreement with recent results in HCV mono-infection patients15. Thus, although the slower phase slope, and as a consequence RVR rates, are correlated with the IL28B genotype, the driving effect is the difference in the IFN anti- viral effectiveness in blocking virion production (first phase decline). The data suggest that the IL28B polymorphism has less of an effect on the infected-cell-loss rate that has been attributed to immune mediated clearance of infected cells23.
Among viral response parameters, we found that RVR and cEVR rates are significantly associated with genotype-CC (Table S1), in agreement with recent results in HCV (genotypes 1/2/3) monoinfected patients15, 24. Among HCV genotype-1 subjects in our study, 10 subjects (out of 11) who had genotype CT/TT failed to achieve an SVR in agreement with the strong association recently shown in larger HIV/HCV co-infected cohorts by Rallon et al.25 and Pineda et al26. However, the weak association between genotype-CC and SVR (p=0.3; Table S1) in HCV genotype-1 infected subjects in our study may be related to the small sample size and due the discontinuation of five patients at week 12 of therapy as previously explained17. The faster first and second phase viral declines observed in IL28B genotype-1-CC patients and the higher first phase viral decline in genotype-3-CC patients provide evidence that the IL28B genotype favorably impacts on viral kinetics.
Evaluation of baseline characteristics showed genotype-CC subjects had a higher body weight. Larger studies are needed to evaluate whether genotype CC might overcome the deleterious impact of higher body weight on SVR. In addition, there was a significantly higher proportion of IL28B genotype-CC in patients infected with HCV- genotype 3 than in patients infected with HCV-genotype 1 (Table S1), in agreement with recent studies that include HIV/HCV-coinfected25, and HCV-monoinfected individuals27. We previously reported higher first and faster second phase viral declines in genotype-3 compared to genotype-1 HIV/HCV co-infected patients16. In the current study, we identified a higher prevalence of IL28B CC-genotype in HCV genotype-3 cases, but no association between IL28B genotype and the second phase viral decline or the loss rate of HCV-infected cells, σ. Since the second-slope phase and/or σ correlate with the outcome of therapy23, these findings suggest that the relatively rapid second phase viral decline in genotype-3 patients is related to factors other than the genetic polymorphism. Our observations might explain the lack of association between IL28B genotype and SVR in genotype-3 patients recently reported by Rallon et al.25 In contrast, higher ∈7average and σ were associated (trend, p=0.1) with CC- genotype in HCV genotype-1 infected patients. The trends observed in genotype-1 patients may reflect the small sample size and is anticipated to be significant in larger studies.
Previous studies of HCV mono-infected patients conducted in the United States reported a higher proportion of IL28B genotype-TC/TT in African Americans than in non-Hispanic Caucasians10, 14. In contrast, we did not identify a significant (p=0.5) difference in IL28B genotype frequencies between African Americans and White patients from Brazil (Table 1). The lack of an association between race and IL28B genotype in our South American patient population might partly explain the lack of association between race/ethnicity and viral kinetic parameters or viral response patterns in our recent reports16, 17. Indeed, preliminary results indicate that ~80% (of 353) of HCV-mono-infected individuals in Brazil are TC or TT with a similar distribution of CC/TC/TT genotypes between African Americans and Whites (Araujo et al. manuscript in preparation). Larger studies are needed to provide a comprehensive evaluation of IL28B genotypes and viral kinetics by race/ethnicity in South America, where patient ancestry may differ from that in the United States.
In conclusion, in HIV/HCV co-infected patients, the IL28B-CC genotype was most strongly associated with a higher first phase viral decline and greater average PEG-IFN effectiveness during the first week of therapy, ∈7max. Pharmacodynamic analysis showed that genotype-CC conferred increased sensitivity to PEG-IFN, as shown by a lower PEG-IFN-α-2a EC50. These kinetic findings raise the possibility that the IL28B CC-genotype favorably affects viral response by augmenting IFN-λ mediated activation of the IFN signaling cascade, leading to increased effectiveness in blocking virion production/release. Notably, as we approach a new era of combination therapy with PEG-IFN and direct antiviral agents, a better understanding of factors associated with PEG-IFN-related viral kinetics will provide the basis to develop optimal treatment strategies for HCV28, 29. Larger and more detailed studies are needed to confirm these new observations in HIV/HCV co-infected patients.
Patients and Methods
Data form twenty six HIV/HCV co-infected patients who were treated with PEG-IFN- α2a (180 µg/week) plus weight based ribavirin (11 mg/kg/day) and provided informed written consent for DNA and HCV RNA kinetic testing are included here. Detailed baseline characteristics, viral response and viral kinetic and pharmacodynamic parameters, estimated via mathematical modeling, were recently studied16, 17. The SNP near the IL28B gene, rs12979860, was examined using the 5' nuclease assay with allele specific PCR probes as recently described10. Genotyping was conducted in a blinded fashion. We used nonparametric methods analyses to compare parameters with IL28B genotype-CC vs genotype-TC/TT. To compare categorical variables, we used the two- tailed Fisher Exact and Pearson Chi-Square Tests. The level of statistical significance was set at (p≤ 0.05). All tests were performed by SPSS v.17 Chicago, IL. Parameters are presented as median and interquartile (IQR) [Table 1 and Supplemental Digital Content 1, http://links.lww.com/QAI/A112].
Results
There were no differences in the distribution of IL28B genotypes by age, gender or race/ethnicity (Table S1). Notably, the distribution of IL28B genotypes was similar between Whites and African Americans in this sample of patients from Brazil. Patients with genotype-CC were significantly heavier (mean 71 kg) than patients with genotype-TC/TT (mean 65 kg) [p=0.012]. Baseline virus levels were similar across IL28B genotypes. A higher proportion (p=0.015) of HCV genotype-3 patients had IL28B genotype-CC (8 out of 11) compared to those with HCV genotype-1 (3 out of 15) [Table S1].
Viral response and IL28B polymorphism.
The rapid virologic response rate (RVR, HCV RNA undetectable at week 4) was greater in patients with genotype-CC compared to those with genotype-TC/TT (p=0.04). The CC genotype was even more strongly associated (p=0.004 in Table S1) with the complete early virologic response rate, cEVR (HCV RNA undetectable at week 12). End treatment response and sustained virological response (SVR) rates were higher in patients with genotype-CC but did not reach significance (p=0.5 and 0.2, respectively) probably due to the discontinuation of 5 patients by week 12 of therapy (Table S1). The response rates among HCV genotype (1 vs 3) and IL28B genotype (CC vs CT/TT) are shown in Table S1.
Viral kinetics and IL28B polymorphism.
Overall, the first phase viral decline from baseline to nadir viral load (see Vmin in Table 1) was significantly (p=0.005) higher in patients with genotype-CC (median (IQR) 1.7 (0.6)) than in patients with genotype- TC/TT (0.92 (0.8), Table 1 and Fig. 1A). The slower-second-phase slopes calculated from day 7 to day 15 or from day 2 to day 29 also were significantly faster (p=0.046 and p=0.01, respectively) in patients with genotype-CC (median (IQR) 1.1 (1.1) and 0.7(0.6) log/wk, respectively) than in patients with genotype-TC/TT (0.4(0.5) and 0.3(0.4) log/wk, respectively, Table 1 and Figs. 1 A&B). Confining the analysis to HCV genotype- 1 patients confirmed the associations between IL28B genotype-CC and higher first phase (p=0.08) viral decline and second phase calculated between d2-d29 (p=0.02) viral decline slope (Table 1 and Figs. 1 C&D). Interestingly, in HCV genotype-3 subjects, while there was a trend toward a higher first phase viral decline (p=0.1) with genotype-CC, the second-slower phase of viral decline was not associated (p=0.7 for both d7-d15 and d2-d29) with IL28B genotype-CC (Table 1 and Figs. 1 E&F).
Viral kinetic and pharmacodynamic parameters and IL28B polymorphism.
The maximum PEG-IFN effectiveness during the first week of therapy, ∈7max, the average PEG-IFN effectiveness during the first week of therapy, ∈7average , and the maximum PEG- IFN effectiveness from week 4 to week 12 of therapy, ∈max, were significantly (p=0.008, 0.008 and 0.044, respectively) higher in genotype-CC (median (IQR) 94% (11%), 92%(13%) and 96%(6%), respectively) than in patients with genotype-TC/TT (81% (45%), 77%(45%) and 87%(33%), respectively; Table 1). The PEG-IFN concentration at which the PEG-IFN-α-2a effectiveness in blocking viral production is half its maximum, EC50, was significantly (p=0.02) lower in genotype-CC (median (IQR) 1.3(1.8)) than in genotype-TC or TT (3.3(11.5)). When the analysis was confined to HCV genotype-1 cases, EC50 was lower (but not significant p=0.6) in genotype-CC, but there was a trend toward a correlation between genotype-CC and both ∈7average and σ (P=0.11, Table 1). In contrast, in HCV-genotype-3 patients, there were trends toward higher ∈7average and lower EC50 (p=0.1) with genotype-CC, while an association was less evident for σ (P=0.7, Table 1) among IL28B genotypes.
Figure 1. HCV kinetics per IL28B genotypes per HCV genotypes.
(A) (C) and (E) early viral kinetics from initiation of therapy until day 7, in patients infected with HCV genotype-1/3 (GT-1/3), genotype-1 (GT-1) or genotype-3 (GT-3), respectively. (B) (D) and (F) viral kinetics from initiation of therapy until week 12, in patients infected with HCV genotype-1/3 (GT-1/3), genotype-1 (GT-1) or genotype-3 (GT-3), respectively. Statistical differences in the first and slower (second) phases of viral decline are shown in Table 1. Gray filled symbols represent undetectable HCV RNA (<10 IU/ml) in all patients at week 12. Vertical lines represent standard error of the mean (note that in (E) and (F) vertical lines in genotype-TC curve (circles) are missing since the curve represents one patient).
Source
JAIDS Journal of Acquired Immune Deficiency Syndromes Publish Ahead of Print Dec 19 2010
Evaldo Stanislau Affonso de Araœjo1, Harel Dahari2, Scott J Cotler2, Thomas J Layden2, Avidan U Neumann3, Carlos Eduardo Melo1, Antonio Alci Barone1 1University of Sao Paulo Hospital das Cl’nicas, Sao Paulo, Brazil; 2Department of Medicine, University of Illinois at Chicago, Chicago, USA and3Bar Ilan University, Life Sciences Faculty, Ramat Gan, Israel.
"In conclusion, in HIV/HCV co-infected patients, the IL28B-CC genotype was most strongly associated with a higher first phase viral decline and greater average PEG-IFN effectiveness during the first week of therapy, ∈7max.
Pharmacodynamic analysis showed that genotype-CC conferred increased sensitivity to PEG-IFN, as shown by a lower PEG-IFN-α-2a EC50. These kinetic findings raise the possibility that the IL28B CC-genotype favorably affects viral response by augmenting IFN-λ mediated activation of the IFN signaling cascade, leading to increased effectiveness in blocking virion production/release. Notably, as we approach a new era of combination therapy with PEG-IFN and direct antiviral agents, a better understanding of factors associated with PEG-IFN-related viral kinetics will provide the basis to develop optimal treatment strategies for HCV28, 29. Larger and more detailed studies are needed to confirm these new observations in HIV/HCV co-infected patients."
Abstract
We examined the association between IL28B single-nucleotide-polymorphism rs12979860, hepatitis C virus (HCV) kinetic and pegylated-interferon-alpha-2a pharmacodynamic parameters in HIV/HCV-co-infected patients from South America. Twenty-six subjects received PEG-IFN-alpha-2a+ribavirin. Serum HCV-RNA and interferon concentrations were measured frequently during the first 12-weeks of therapy and analyzed using mathematical models. African Americans and Whites had a similar distribution of IL28B genotypes (p=0.5). The CC genotype was overrepresented (p=0.015) in patients infected with HCV genotype-3 compared to genotype-1. In both genotype-1 and genotype-3, the first-phase-viral decline and the average PEG-IFN- alpha-2a effectiveness during the first week of therapy were larger (trend P≤0.12) in genotype-CC compared with genotypes-TC/TT. In genotype-1 patients, the second- slower phase of viral decline (days 2-29) and infected-cells-loss rate, σ, were larger (p=0.02 and 0.11, respectively) in genotype-CC than in genotypes-TC/TT. These associations were not observed in genotype-3 patients.
Introduction
Coinfection with human immunodeficiency virus (HIV) and HCV affects approximately 10 million people worldwide1 and up to 100,000 persons in Brazil2. Antiviral therapy for hepatitis C virus (HCV) consisting of pegylated-interferon-α (PEG-IFN) and ribavirin (standard of care, SOC) has potential adverse effects, and response rates are lower in HCV/HIV co-infected than in HCV-monoinfected patients3-9. Consequently, there is considerable interest in identifying better predictors of treatment response. A seminal study showed that single nucleotide polymorphisms (SNPs) in the IL28B gene region were associated with race/ethnicity and correlated with response to pegylated interferon-alpha (PEG-IFN) and ribavirin therapy in HCV mono-infected patients10-13.
Recently, early HCV kinetics (e.g., first and second phases of viral decline) have been evaluated as a function of IL28B SNPs in HCV mono-infected patients, although pharmacodynamic parameters are lacking in these analyses14, 15. To the best of our knowledge, there is no such information in HIV/HCV co-infected individuals. To determine the relationship between IL28B polymorphisms and viral/host parameters in HIV/HCV co-infected patients, we compared IL28B genotypes with recent results of viral response16 and estimates of viral kinetic and pharmacodynamic parameters17 in 26 patients with HIV and HCV who were treated with PEG-IFN-α-2a and ribavirin.
Discussion
A detailed HCV kinetic analysis provided new and important information regarding the impact of IL28B genotype on response to PEG-IFN plus RBV in HIV/HCV co-infected patients. Overall, the CC-genotype was most strongly associated with a higher first phase viral decline and maximum PEG-IFN effectiveness during the first week of therapy, ∈7max. These findings indicate that PEG-IFN has greater efficacy in blocking HCV production/release in patients with the favorable IL28B CC-genotype. Our results are in agreement with recent findings in HCV-mono-infection patients14, 15. The PEG-IFN-α-2a EC50 was lower in genotype-CC compared with genotype-TC/TT, providing further evidence that the CC-genotype confers a higher sensitivity to IFN treatment. Thus far, the mechanism of action of the IL28B polymorphism has not been identified. The location of the genetic polymorphism upstream of the IFN-λ gene18, 19 raises the possibility that the IL28B genotype mediates endogenous production of IFN- λ, which contributes to the first phase response by stimulating IFN signaling.
The second phase viral decline slope also was associated with genotype-CC in genotype 1 patients (Table 1). The overall correlation between the CC-genotype and the infection death/loss rate, σ, was less prominent (trend, p=0.11). This discrepancy could be explained by the fact that 6 out of 21 of patients who finished 48 weeks of therapy in our study17 had a triphasic viral decline pattern, consisting of a first phase (1-2 days) with a rapid virus load decline followed by a "shoulder phase" (8 - 28 days), in which virus levels decay slowly or remain constant, and a third phase of renewed viral decay20, 21. Calculating the slower phase slope from the measured data includes the "shoulder phase" in these triphasic patients. In contrast, by using a mathematical model that includes hepatocytes proliferation, estimated σ reflects the final slope and excludes the shoulder phase22. Interestingly, five of the six triphasic patients had genotype-TT/TC and only one had genotype-CC (not shown). In addition, we recently showed that drug effectiveness, ∈, can significantly affect the serum second phase slope decline22, and that when ∈ ~ 1 the slower phase slope is close to σ. Indeed, when the analysis was performed only in patients with first phase decline > 1 log (i.e., ∈>90%), the association between IL28B genotype-CC and the slower phase slope was lost (p=0.5, not shown), in agreement with recent results in HCV mono-infection patients15. Thus, although the slower phase slope, and as a consequence RVR rates, are correlated with the IL28B genotype, the driving effect is the difference in the IFN anti- viral effectiveness in blocking virion production (first phase decline). The data suggest that the IL28B polymorphism has less of an effect on the infected-cell-loss rate that has been attributed to immune mediated clearance of infected cells23.
Among viral response parameters, we found that RVR and cEVR rates are significantly associated with genotype-CC (Table S1), in agreement with recent results in HCV (genotypes 1/2/3) monoinfected patients15, 24. Among HCV genotype-1 subjects in our study, 10 subjects (out of 11) who had genotype CT/TT failed to achieve an SVR in agreement with the strong association recently shown in larger HIV/HCV co-infected cohorts by Rallon et al.25 and Pineda et al26. However, the weak association between genotype-CC and SVR (p=0.3; Table S1) in HCV genotype-1 infected subjects in our study may be related to the small sample size and due the discontinuation of five patients at week 12 of therapy as previously explained17. The faster first and second phase viral declines observed in IL28B genotype-1-CC patients and the higher first phase viral decline in genotype-3-CC patients provide evidence that the IL28B genotype favorably impacts on viral kinetics.
Evaluation of baseline characteristics showed genotype-CC subjects had a higher body weight. Larger studies are needed to evaluate whether genotype CC might overcome the deleterious impact of higher body weight on SVR. In addition, there was a significantly higher proportion of IL28B genotype-CC in patients infected with HCV- genotype 3 than in patients infected with HCV-genotype 1 (Table S1), in agreement with recent studies that include HIV/HCV-coinfected25, and HCV-monoinfected individuals27. We previously reported higher first and faster second phase viral declines in genotype-3 compared to genotype-1 HIV/HCV co-infected patients16. In the current study, we identified a higher prevalence of IL28B CC-genotype in HCV genotype-3 cases, but no association between IL28B genotype and the second phase viral decline or the loss rate of HCV-infected cells, σ. Since the second-slope phase and/or σ correlate with the outcome of therapy23, these findings suggest that the relatively rapid second phase viral decline in genotype-3 patients is related to factors other than the genetic polymorphism. Our observations might explain the lack of association between IL28B genotype and SVR in genotype-3 patients recently reported by Rallon et al.25 In contrast, higher ∈7average and σ were associated (trend, p=0.1) with CC- genotype in HCV genotype-1 infected patients. The trends observed in genotype-1 patients may reflect the small sample size and is anticipated to be significant in larger studies.
Previous studies of HCV mono-infected patients conducted in the United States reported a higher proportion of IL28B genotype-TC/TT in African Americans than in non-Hispanic Caucasians10, 14. In contrast, we did not identify a significant (p=0.5) difference in IL28B genotype frequencies between African Americans and White patients from Brazil (Table 1). The lack of an association between race and IL28B genotype in our South American patient population might partly explain the lack of association between race/ethnicity and viral kinetic parameters or viral response patterns in our recent reports16, 17. Indeed, preliminary results indicate that ~80% (of 353) of HCV-mono-infected individuals in Brazil are TC or TT with a similar distribution of CC/TC/TT genotypes between African Americans and Whites (Araujo et al. manuscript in preparation). Larger studies are needed to provide a comprehensive evaluation of IL28B genotypes and viral kinetics by race/ethnicity in South America, where patient ancestry may differ from that in the United States.
In conclusion, in HIV/HCV co-infected patients, the IL28B-CC genotype was most strongly associated with a higher first phase viral decline and greater average PEG-IFN effectiveness during the first week of therapy, ∈7max. Pharmacodynamic analysis showed that genotype-CC conferred increased sensitivity to PEG-IFN, as shown by a lower PEG-IFN-α-2a EC50. These kinetic findings raise the possibility that the IL28B CC-genotype favorably affects viral response by augmenting IFN-λ mediated activation of the IFN signaling cascade, leading to increased effectiveness in blocking virion production/release. Notably, as we approach a new era of combination therapy with PEG-IFN and direct antiviral agents, a better understanding of factors associated with PEG-IFN-related viral kinetics will provide the basis to develop optimal treatment strategies for HCV28, 29. Larger and more detailed studies are needed to confirm these new observations in HIV/HCV co-infected patients.
Patients and Methods
Data form twenty six HIV/HCV co-infected patients who were treated with PEG-IFN- α2a (180 µg/week) plus weight based ribavirin (11 mg/kg/day) and provided informed written consent for DNA and HCV RNA kinetic testing are included here. Detailed baseline characteristics, viral response and viral kinetic and pharmacodynamic parameters, estimated via mathematical modeling, were recently studied16, 17. The SNP near the IL28B gene, rs12979860, was examined using the 5' nuclease assay with allele specific PCR probes as recently described10. Genotyping was conducted in a blinded fashion. We used nonparametric methods analyses to compare parameters with IL28B genotype-CC vs genotype-TC/TT. To compare categorical variables, we used the two- tailed Fisher Exact and Pearson Chi-Square Tests. The level of statistical significance was set at (p≤ 0.05). All tests were performed by SPSS v.17 Chicago, IL. Parameters are presented as median and interquartile (IQR) [Table 1 and Supplemental Digital Content 1, http://links.lww.com/QAI/A112].
Results
There were no differences in the distribution of IL28B genotypes by age, gender or race/ethnicity (Table S1). Notably, the distribution of IL28B genotypes was similar between Whites and African Americans in this sample of patients from Brazil. Patients with genotype-CC were significantly heavier (mean 71 kg) than patients with genotype-TC/TT (mean 65 kg) [p=0.012]. Baseline virus levels were similar across IL28B genotypes. A higher proportion (p=0.015) of HCV genotype-3 patients had IL28B genotype-CC (8 out of 11) compared to those with HCV genotype-1 (3 out of 15) [Table S1].
Viral response and IL28B polymorphism.
The rapid virologic response rate (RVR, HCV RNA undetectable at week 4) was greater in patients with genotype-CC compared to those with genotype-TC/TT (p=0.04). The CC genotype was even more strongly associated (p=0.004 in Table S1) with the complete early virologic response rate, cEVR (HCV RNA undetectable at week 12). End treatment response and sustained virological response (SVR) rates were higher in patients with genotype-CC but did not reach significance (p=0.5 and 0.2, respectively) probably due to the discontinuation of 5 patients by week 12 of therapy (Table S1). The response rates among HCV genotype (1 vs 3) and IL28B genotype (CC vs CT/TT) are shown in Table S1.
Viral kinetics and IL28B polymorphism.
Overall, the first phase viral decline from baseline to nadir viral load (see Vmin in Table 1) was significantly (p=0.005) higher in patients with genotype-CC (median (IQR) 1.7 (0.6)) than in patients with genotype- TC/TT (0.92 (0.8), Table 1 and Fig. 1A). The slower-second-phase slopes calculated from day 7 to day 15 or from day 2 to day 29 also were significantly faster (p=0.046 and p=0.01, respectively) in patients with genotype-CC (median (IQR) 1.1 (1.1) and 0.7(0.6) log/wk, respectively) than in patients with genotype-TC/TT (0.4(0.5) and 0.3(0.4) log/wk, respectively, Table 1 and Figs. 1 A&B). Confining the analysis to HCV genotype- 1 patients confirmed the associations between IL28B genotype-CC and higher first phase (p=0.08) viral decline and second phase calculated between d2-d29 (p=0.02) viral decline slope (Table 1 and Figs. 1 C&D). Interestingly, in HCV genotype-3 subjects, while there was a trend toward a higher first phase viral decline (p=0.1) with genotype-CC, the second-slower phase of viral decline was not associated (p=0.7 for both d7-d15 and d2-d29) with IL28B genotype-CC (Table 1 and Figs. 1 E&F).
Viral kinetic and pharmacodynamic parameters and IL28B polymorphism.
The maximum PEG-IFN effectiveness during the first week of therapy, ∈7max, the average PEG-IFN effectiveness during the first week of therapy, ∈7average , and the maximum PEG- IFN effectiveness from week 4 to week 12 of therapy, ∈max, were significantly (p=0.008, 0.008 and 0.044, respectively) higher in genotype-CC (median (IQR) 94% (11%), 92%(13%) and 96%(6%), respectively) than in patients with genotype-TC/TT (81% (45%), 77%(45%) and 87%(33%), respectively; Table 1). The PEG-IFN concentration at which the PEG-IFN-α-2a effectiveness in blocking viral production is half its maximum, EC50, was significantly (p=0.02) lower in genotype-CC (median (IQR) 1.3(1.8)) than in genotype-TC or TT (3.3(11.5)). When the analysis was confined to HCV genotype-1 cases, EC50 was lower (but not significant p=0.6) in genotype-CC, but there was a trend toward a correlation between genotype-CC and both ∈7average and σ (P=0.11, Table 1). In contrast, in HCV-genotype-3 patients, there were trends toward higher ∈7average and lower EC50 (p=0.1) with genotype-CC, while an association was less evident for σ (P=0.7, Table 1) among IL28B genotypes.
Figure 1. HCV kinetics per IL28B genotypes per HCV genotypes.
(A) (C) and (E) early viral kinetics from initiation of therapy until day 7, in patients infected with HCV genotype-1/3 (GT-1/3), genotype-1 (GT-1) or genotype-3 (GT-3), respectively. (B) (D) and (F) viral kinetics from initiation of therapy until week 12, in patients infected with HCV genotype-1/3 (GT-1/3), genotype-1 (GT-1) or genotype-3 (GT-3), respectively. Statistical differences in the first and slower (second) phases of viral decline are shown in Table 1. Gray filled symbols represent undetectable HCV RNA (<10 IU/ml) in all patients at week 12. Vertical lines represent standard error of the mean (note that in (E) and (F) vertical lines in genotype-TC curve (circles) are missing since the curve represents one patient).
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