Original article The pattern of pegylated interferon-α2b and ribavirin treatment failure in cirrhotic patients depends on hepatitis C virus genotype

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1 Antiviral Therapy 14: Original article The pattern of pegylated interferon-α2b and ribavirin treatment failure in cirrhotic patients depends on hepatitis C virus genotype Alessio Aghemo 1, Maria Grazia Rumi 1 *, Sara Monico 1, Gian Maria Prati 1, Roberta D Ambrosio 1, Maria Francesca Donato 1 and Massimo Colombo 1 1 AM Migliavacca Center for Liver Disease, First Division of Gastroenterology, Fondazione IRCCS Policlinico, Mangiagalli e Regina Elena, University of Milan, Milan, Italy *Corresponding author: mariagrazia.rumi@unimi.it Background: Failure of anti-hepatitis C therapy encompasses both primary non-response and post-treatment relapse. Treatment failure to pegylated interferon (PEG- IFN)-α2b and ribavirin (RBV) largely depends upon virus genotype, but the interaction between genotype, cirrhosis and pattern of treatment failure is unclear. We aimed to assess whether cirrhosis modifies the pattern of PEG-IFNα2b and RBV treatment failure. Methods: A total of 471 treatment-naive patients with histologically proven chronic hepatitis C virus (HCV) infection (106 with cirrhosis; 185 with HCV genotype 1 [HCV-1], 157 with HCV genotype 2 [HCV-2], 92 with HCV genotype 3 [HCV-3] and 37 with HCV genotype 4 [HCV-4]) were consecutively treated with PEG-IFN-α2b 1.5 µg weekly and weight-based RBV. Results: The sustained virological response (SVR) rates were 31% in HCV-1 and HCV-4, 80% in HCV-2 and 72% in HCV-3, and were lower in cirrhotic than in non- cirrhotic HCV-1 and HCV-4 (17% versus 36%; P=0.01), and HCV-3 (33% versus 79%; P=0.001), but not HCV-2 (69% versus 83%; P=0.1) patients. Treatment failure was the consequence of lower end-of-treatment response rates (37% versus 53%; P=0.06) plus higher post-treatment relapse rates (55% versus 31%; P=0.07) in cirrhotic HCV-1 and HCV-4 patients and higher rates of post-treatment relapse in HCV-2 (29% versus 10%; P=0.01) and HCV-3 cirrhotic patients (61% versus 12%; P<0.001). By multivariate analysis, HCV-1 and HCV-4 (odds ratio [OR] 7.44, 95% confidence interval [CI] ), and cirrhosis (OR 3.00, 95% CI ) were independent predictors of treatment failure. Conclusions: Cirrhosis is an important moderator of SVR, accounting for different patterns of treatment failure in patients infected with different genotypes. Introduction The combination of pegylated interferon (PEG-IFN)-α and ribavirin (RBV) is the current standard of care for patients with chronic hepatitis C, leading to persistent eradication of serum hepatitis C virus (HCV) RNA in approximately 50 85% of patients, depending upon HCV genotype [1 5]. Failure of eradicating serum HCV RNA with PEG-IFN-α and RBV might have more than one explanation. In fact, some patients fail to suppress HCV (primary non-response) and other patients, who have an initial response to treatment, fail to maintain viral suppression following treatment withdrawal (post-treatment relapse). Both registration trials and real world studies have shown that HCV genotype and the degree of liver fibrosis, which are the strongest pretherapy independent moderators of treatment outcome [3,6], play an important role in the outcome of PEG-IFN-α and RBV therapy. In addition, because of the low number of patients with cirrhosis enrolled in Phase III registration trials, the patterns and strength of the interaction between HCV genotype and liver fibrosis have never been evaluated; however, the understanding of this interaction could enable better individualization of PEG-IFN-α and RBV therapy. Different therapeutic strategies have been advocated in patients with a primary non-response to interferon (IFN) with respect to patients who relapse after an initial response to therapy. The advent of HCV protease (and polymerase) inhibitors might result in potentiation of primary response to PEG-IFN-α and RBV through early and persistent suppression of HCV replication [7] International Medical Press (print) (online) 577

2 A Aghemo et al. By contrast, extension of the standard of care therapy might improve HCV eradication rates by reducing the risk of post-treatment relapse [8 11]. To assess whether HCV genotype and liver fibrosis interact as outcome moderators of anti-hcv therapy, we analysed the rates and patterns of treatment failure of treatment-naive patients with chronic hepatitis C, who consecutively underwent treatment with PEG-IFN-α2b and RBV at the AM Migliavacca Center for Liver Disease (University of Milan, Milan, Italy). Methods Patients Between September 2001 and April 2006, 583 previously untreated adult patients with chronic hepatitis C were consecutively evaluated for antiviral treatment at the liver centre (AM Migliavacca Center for Liver Disease). Of these, 68 patients with persistently normal alanine aminotransferase (ALT) levels were left untreated as instructed by the Italian National Health System (INHS) recommendations, 23 refused treatment and 21 were not eligible because of advanced age (>70 years) or comorbidities that contraindicated IFN therapy. A total of 471 (81%) patients gave their written consent to PEG-IFN-α2b and RBV combination therapy according to the National Institutes of Health (NIH) recommendations [12]. Chronic hepatitis C was defined by at least 1 year serum positivity for HCV RNA, ALT levels >1.5 the upper limit of normal and by a liver biopsy (performed in the year preceding treatment) consistent with chronic hepatitis C. Patients were excluded if they had coinfection with hepatitis B virus or HIV, decompensated liver disease, drug dependence or >40 g/day alcohol intake. Patients with poorly controlled diabetes, severe depression, autoimmune diseases or concomitant malignant neoplastic diseases were also excluded. Patients with high titres of nonorgan-specific autoantibodies were included if their liver biopsy did not show the histological hallmarks of autoimmune liver disease and was consistent with chronic hepatitis C. Patients gave their written informed consent to receive therapy and gave permission for use of their medical records. Treatment All patients were treated with PEG-IFN-α2b ( PEGINTRON ; Schering Plough, Kenilworth, NJ, USA) at doses of 1.5 µg/kg once per week subcutaneously. RBV (REBETOL ; Schering Plough) was dosed according to baseline weight (that is, 800 mg for patients who were <65 kg, 1,000 mg for kg and 1,200 mg for >85 kg). Treatment duration was 24 weeks for patients with HCV genotype 2 (HCV-2) and HCV genotype 3 (HCV-3) and 48 weeks for patients infected with HCV genotype 1 (HCV-1) and HCV genotype 4 (HCV-4). From September 2001 to December 2003, therapy was discontinued in HCV-1 and HCV-4 patients if HCV RNA was still detectable at week 24 of treatment. Starting January 2004, therapy was discontinued if quantitative HCV RNA testing at week 12 decreased by <2 log 10 IU/ml compared with baseline values, and at week 24 if HCV RNA was still detectable in patients whose HCV RNA had decreased by >2 log 10 IU/ml at week 12. All patients were evaluated for safety and tolerance of treatment every 4 weeks during the treatment period. PEG-IFNα2b was reduced to 1 µg/kg per week whenever neutrophil counts decreased to < cells/l and it was withdrawn when neutrophil counts decreased to < cells/l. RBV doses were tapered by 200 mg/day when haemoglobin decreased to <100 g/l and it was discontinued in patients with <85 g/l haemoglobin. During the study period, the use of growth factors, such as erythropoietin and granulocyte colony-stimulating factor, were not allowed according to the INHS regulations. Patients were considered adherent to the assigned dose regimen if they received at least 80% of both drugs for at least 80% of the treatment duration (the 80/80/80 rule) [13]. Measurements Serum ALT and aspartate aminotransferase (AST) activities were measured by an automated method at 37 C (normal value 37 IU/l), fasting glucose, serum iron, iron-binding capacity and ferritin were measured using standard methods. Serum HCV RNA was assessed by in-house nested reverse transcriptase (RT)- PCR, using specific primers from the 5 non-coding region, at baseline and during treatment at weeks 4, 12, 24 and 48, and after therapy at weeks 4, 12 and 24. The limit of the assay sensitivity was approximately 25 IU/ml HCV RNA [14]. HCV was genotyped by nested RT-PCR using universal biotinylated primers in the 5 non-coding region (Line Probe Assay INNO-LIPA HCV 2; Innogenetics, Zwijndrecht, Belgium). Serum HCV RNA was quantified by Versant HCV RNA 3.0 assays (bdna 3.0; Bayer Corporation, Emeryville, CA, USA), with a sensitivity limit of 615 IU/ml and a dynamic range of 615 7,700,000 IU/ml. Commercially available enzyme immunoassays were used to determine serum hepatitis B surface antigen and antibodies to hepatitis B core antigens, hepatitis A virus and HIV. Antibodies to nuclear, smooth muscle, mithochondrial, liver and kidney microsomal antigens were assayed on rat liver and kidney cryostat sections by immunofluorescence. Antinuclear antibodies were confirmed on Hep2 cells. Body mass index was calculated for all patients at baseline International Medical Press

3 HCV genotype and cirrhosis Table 1. Demographic and clinical characteristics of the 471 patients enrolled, stratified by HCV genotype Characteristic Overall (n=471) HCV-1 (n=185) HCV-2 (n=157) HCV-3 (n=92) HCV-4 (n=37) Caucasian, n (%) 451 (96) 184 (99) 157 (100) 92 (100) 20 (54) Male gender, n (%) 269 (57) 106 (57) 68 (43) 60 (65) 35 (95) Mean age, years (±sd) 51 (12.44) 52 (12.23) 57.7 (10.23) 41 (7.44) 44 (7.06) Mean body weight, kg (±sd) 69.4 (12.5) 69.3 (12.3) 66.8 (11.7) 70.6 (12.5) 77.5 (13.0) Mean BMI, kg/m 2 (±sd) 24.1 (3.2) 24.1 (3.2) 24.0 (3.4) 23.9 (3.3) 25.0 (3.1) BMI>27 kg/m 2, n (%) 81 (17) 29 (16) 28 (18) 17 (18) 7 (19) Infection modality Parenteral, n (%) 84 (18) 25 (14) 45 (29) 12 (13) 2 (5) IVDA, n (%) 76 (16) 20 (11) 2 (1) 44 (48) 10 (27) Sporadic, n (%) 311 (66) 140 (75) 110 (70) 36 (39) 25 (68) Median disease duration, 120 (7 720) 120 (10 720) 120 (7 624) 132 (12 384) 96 (12 312) months (range) Mean ALT, IU/l (±sd) (112) (96.5) (149) (75.8) 93.6 (35.8) Median HCV RNA, IU/ml (range) ( ) (2, ) ( ) (5, ) (4, ) Ishak staging score 0 2, n (%) 233 (49) 83 (45) 80 (51) 57 (62) 13 (35) 3 4, n (%) 132 (28) 54 (29) 45 (29) 20 (22) 13 (35) 5 6, n (%) 106 (23) 48 (26) 32 (20) 15 (16) 11 (30) ALT, alanine aminotranferase; BMI, body mass index; HCV, hepatitis C virus; HCV-1, HCV genotype 1; HCV-2, HCV genotype 2; HCV-3, HCV genotype 3; HCV-4, HCV genotype 4; IVDA, intravenous drug abuse. Liver biopsies were performed with a 16-gauge Tru- Cut needle (Uro-Cut 16 G; TSK, Tokyo, Japan) and read by a single pathologist (MFD). Liver biopsies were considered to be adequate for fibrosis assessment if they were longer than 15 mm or had at least 12 portal tracts. The severity of hepatic inflammation was evaluated by the Ishak score in separate reports for grading and staging [15]. The maximum score for grading was 18, ranged from 0 to 4 for piecemeal necrosis, focal necrosis and portal inflammation, and from 0 to 6 for confluent necrosis. The score for staging ranged from 0 (representing no fibrosis) to 5 (incomplete cirrhosis) and 6 (cirrhosis). Disease duration was calculated by considering the date of blood transfusion received prior to 1992 or the period of drug injection as the onset of infection. In patients with an unknown source of infection, the date of the first abnormal ALT test was arbitrarily taken as the start of infection. Definition of response Clearance of serum HCV RNA, determined using in-house nested RT-PCR, was assessed at week 4 (rapid virological response [RVR]), at week 12 (complete early virological response [cevr]), at week 24 and at week 48 of treatment (end-of-treatment response [ETR]). A sustained virological response (SVR) was undetectable HCV RNA by RT- PCR at week 24 of post-treatment. Patients with an ETR who were tested as HCV-RNA-positive during follow-up were classified as relapsers. Patients who had any other virological response were considered as non-responders. Statistical analyses Data were analysed by intention-to-treat analysis. Patients missing HCV RNA determinations during follow-up and those who prematurely suspended treatment were considered as treatment failures and were categorized as non-responders. The descriptive analysis included absolute and relative frequencies for grouped data and mean (±sd) for continuous scaled data. Statistical comparison of SVR rates, stratified by the degree of fibrosis, were performed by the χ 2 test. The level of significance was 0.05 (two-sided) for all statistical tests. All confidence intervals (CIs) provided were at 95%. The odds ratios (ORs) for treatment failure and corresponding 95% CIs were estimated by unconditional multiple logistic regression equations using a stepwise method. Results Patient demographics and clinical characteristics are shown in Table 1. All patients had a liver biopsy specimen that was considered as adequate for histological staging by the pathologist (MFD). The 106 patients with histological diagnosis of cirrhosis were more often male (66% versus 55%; P=0.05), of older age (57 years versus 49 years; P<0.0001) and had a higher body weight (72.5 kg versus 68.5 kg; P=0.003) than the 365 non-cirrhotic patients (Table 2). All cirrhotic patients were in class A5 of the Child-Pugh score. Overall, 22 patients (5%; 5 cirrhotic and 17 non cirrhotic Antiviral Therapy

4 A Aghemo et al. Table 2. Demographic and clinical features of the 471 patients enrolled, stratified by degree of fibrosis Feature Ishak staging score 0 4 (n=365) Ishak staging score 5 6 (n=106) P-value Male gender, n (%) 199 (55) 70 (66) 0.05 a Mean age, years (±sd) 49 (12.5) 57 (9.3) < b Mean body weight, kg (±sd) 68.5 (12.6) 72.5 (11.8) b Mean BMI, kg/m 2 (±sd) 23.8 (3.3) 24.4 (3.4) 0.12 b BMI>27 kg/m 2, n (%) 60 (16) 21 (20) 0.51 a Source of infection Parenteral, n (%) 69 (19) 15 (14) 0.36 a IVDA, n (%) 63 (17) 13 (12) 0.26 a Sporadic, n (%) 233 (64) 78 (74) 0.08 a Median disease duration, months (range) 120 (10 720) 120 (7 360) 0.87 b Mean ALT, IU/l (±sd) (114.7) (105.5) 0.14 b Median HCV RNA, IU/ml (range) ( ) (1, ) 0.57 b HCV genotype 1 and 4, n (%) 163 (47) 59 (56) 0.06 a 2, n (%) 125 (33) 32 (30) 0.5 a 3, n (%) 77 (20) 15 (14) 0.14 a a χ 2 Test. b Student s t-test. ALT, alanine aminotranferase; BMI, body mass index; HCV, hepatitis C virus; IVDA, intravenous drug abuse. patients) prematurely discontinued treatment because of severe asthaenia (n=8), neutropaenia (n=5), anaemia (n=3), development of bacterial infections (n=3), severe hypersensitivity to PEG-IFN-α2b (n=2) and ALT flare (n=1). Overall, 449 (95%) patients completed the treatment and the programmed post-treatment follow-up, and 73% adhered to the predetermined treatment schedule, with comparable rates between cirrhotic and non- cirrhotic patients (70% versus 74%; P=0.1). Dose reductions for PEG-IFN-α2b and RBV were deemed necessary in 6 (1%) and 126 (27%) patients, respectively, with similar rates among cirrhotic and non-cirrhotic patients (PEG- IFN-α2b 2% versus 1% and RBV 30% versus 26%). Overall, 261 (55%) achieved a RVR, 336 (71%) achieved an ETR and 261 (55%) achieved a SVR. By univariate analysis, age >60 years (P=0.02), male gender (P=0.02), presence of cirrhosis (P<0.0001), and HCV-1 and HCV-4 (P<0.0001) emerged as predictors of treatment failure. By multiple logistic regression analysis, cirrhosis (OR 3.00, 95% CI ), and HCV-1 and HCV-4 (OR 7.44, 95% CI ) were the only independent predictors of treatment failure. Although RVR rates were not modified by the degree of fibrosis (58% in non-cirrhotic versus 50% in cirrhotic patients; P=0.15), cevr rates were significantly lower in cirrhotic patients (64% versus 76%; P=0.01). When stratified by HCV genotype, RVR rates were not affected by the presence of cirrhosis across all HCV genotypes (Table 3). By contrast, EVR rates were significantly lower among HCV-1 and HCV-4 patients with cirrhosis when compared with the rates attained in non-cirrhotic patients (42% versus 59%; P=0.04; Table 3). Both ETR and SVR rates were significantly lower in patients with cirrhosis than those who were non-cirrhotic (ETR 74% non-cirrhotic versus 62% cirrhotic patients; P=0.02 and SVR 61% non-cirrhotic versus 35% cirrhotic patients; P=0.0001). Compared with non-cirrhotic patients, cirrhotic patients showed reduced SVR rates across all genotypes. ETR rates were also affected by cirrhosis in HCV-1 and HCV-4 patients, but not in HCV-2- and HCV-3-infected patients (Table 3). HCV-2 and HCV-3 patients with cirrhosis showed higher rates of post-treatment relapse (29% and 61%, respectively) than non-cirrhotic patients (10% and 12%, respectively; Table 4). HCV-1 and HCV-4 patients showed higher relapse rates, although not reaching statistical significance, between cirrhotic and non- cirrhotic patients (55% versus 31%; Table 4). By multiple logistic regression analysis, HCV-1 and HCV-4 emerged as the only independent predictors of primary non-response (OR 11.64, 95% CI ), whereas cirrhosis (OR 4.95, 95% CI ) and HCV-1 and HCV-4 (OR 2.96, 95% CI ) predicted post-treatment relapse. Discussion The present study confirms that both HCV genotype and cirrhosis strongly influence PEG-IFN-α2b and RBV treatment outcome and unequivocally suggest the existence of an interaction or an addictive effect of these two moderators of anti-hcv treatment outcome. Indeed, although we found that cirrhosis did not influence RVR, it did affect later end-points of response and treatment failures resulting from a primary nonresponse to therapy in HCV-1 and HCV-4 cirrhotic patients, whereas post-treatment relapse accounted for International Medical Press

5 HCV genotype and cirrhosis Table 3. RVR, cevr, ETR and SVR stratified by degree of fibrosis and HCV genotype Overall Patients with Ishak staging Patients with Ishak staging Response (n=471) score 0 4 (n=365) score 5 6 (n=106) P-value a HCV-1 and HCV-4 RVR, n (%) b 75/222 (34) 58/163 (36) 17/59 (29) 0.43 cevr, n (%) b 121/222 (56) 96/163 (59) 25/59 (42) 0.04 ETR, n (%) 108/222 (49) 86/163 (53) 22/59 (37) 0.06 SVR, n (%) 69/222 (31) 59/163 (36) 10/59 (17) 0.01 HCV-2 RVR, n (%) b 124/157 (79) 102/125 (82) 25/32 (78) 0.84 cevr, n (%) b 144/157 (92) 114/125 (91) 30/32 (94) 0.91 ETR, n (%) 146/157 (93) 115/125 (92) 31/32 (97) 0.5 SVR, n (%) 126/157 (80) 104/125 (83) 22/32 (69) 0.09 HCV-3 RVR, n (%) b 62/92 (67) 53/77 (69) 11/15 (73) 0.96 cevr, n (%) b 79/92 (86) 67/77 (87) 13/15 (87) 0.97 ETR, n (%) 82/92 (89) 69/77 (90) 13/15 (87) 0.7 SVR, n (%) 66/92 (72) 61/77 (79) 5/15 (33) a χ 2 Test. b Tested with in-house nested PCR (limit of detection 25 IU/ml). cevr, complete early virological response; ETR, end-of-treatment response; HCV, hepatitis C virus; HCV-1, HCV genotype 1; HCV-2, HCV genotype 2; HCV-3, HCV genotype 3; HCV-4, HCV genotype 4; RVR, rapid virological response; SVR, sustained virological response. Table 4. HCV RNA post-treatment relapse rates stratified by degree of fibrosis and HCV genotype Degree of liver fibrosis Genotype Ishak staging score 0 4 Ishak staging score 5 6 P-value a HCV-1 and HCV-4, n (%) 27/86 (31) 12/22 (55) 0.07 HCV-2, n (%) 11/115 (10) 9/31 (29) 0.01 HCV-3, n (%) 8/69 (12) 8/13 (61) a χ 2 Test. HCV, hepatitis C virus; HCV-1, HCV genotype 1; HCV-2, HCV genotype 2; HCV-3, HCV genotype 3; HCV-4, HCV genotype 4. most therapeutic failures in HCV-2 and HCV-3 patients with cirrhosis. These findings, which exclusively apply to PEG-IFN-α2b therapy, stem from treatment of consecutively enrolled patients over a period span where the standards for patient selection and treatment had remained unchanged and adherent to the NIH consensus conference guidelines [12]. Interestingly, the peculiar interplay between HCV genotype and cirrhosis in anti-hcv therapy with PEG- IFN-α2b and RBV went undetected both in the registration trial and in subsequent studies, possibly because too few patients with cirrhosis were enrolled and treatment outcome was jointly analysed in patients with HCV-2 and HCV-3 infection [3,16]. The latter was also responsible for the late discovery of the greater sensitivity to IFN-based therapies of HCV-2-infected patients compared with HCV-3-infected patients with or without cirrhosis [16 18]. As literature on HCV therapy cumulated, discrepant patterns of treatment failure among patients with different HCV genotypes were more easily revealed by real world studies such as ours [19,20]. Indeed, these studies have included more patients with advanced hepatitis and comorbidities, which ultimately test the therapeutic effectiveness of IFN-based therapies better than registration trials enrolling highly selected patients [21]. Unsurprisingly, therefore, recent real world studies [10,22] reported lower than expected SVR rates to PEG- IFN-α2b and RBV in HCV-1 patients (36% and 34%, respectively). This was a finding similar to ours (31%) in patients chronically infected with HCV-1 (and HCV-4), possibly because of the number of patients with cirrhosis included (27%), who ultimately responded less satisfactorily to PEG-IFN-α2b and RBV than non-cirrhotic patients (SVR 17% versus 36%). Similar disappointing figures of response to PEG-IFN-α2b and RBV in HCV-1 patients with cirrhosis were also reported by a multicentre study in Italy and in a small study in Australia, which also included patients with HCV-4 infection [23,24]. In our study, the influence of cirrhosis as a moderator of treatment outcome extends beyond HCV-1- and HCV-4-infected patients. Our findings of 33% SVR rates in HCV-3 cirrhotic patients compared with the 79% rates for those with minimal or moderate hepatic fibrosis, being attributable to a high rate of post- treatment Antiviral Therapy

6 A Aghemo et al. relapse, confirm previous observations [18,19]. The high rates of relapsers we observed in HCV-3 cirrhotic patients cannot be attributed to differences in rates of adherence or RBV dose reductions compared with noncirrhotic patients (93% versus 88% and 29% versus 27%, respectively). Moreover, social or psychiatric difficulties were not the culprits as only 50% of patients had a past history of intravenous drug abuse and all patients were abstinent from concurrent alcohol abuse; however, because of the relatively small sample size, we cannot completely rule out a statistical error. Cirrhosis also had a moderate influence on SVR rates to PEG- IFN-α2b and RBV in patients infected by HCV-2 (69% compared with 83% for non-cirrhotic patients) as we previously reported in patients receiving standard IFN plus RBV therapy [20]. Although molecular analysis studies have provided some insights into the putative mechanisms of IFN responsiveness of HCV-1 patients, leading to the recognition of molecular signatures predictive of a response to IFN therapy [25 27], the mechanisms by which cirrhosis selectively attenuates PEG-IFN-α2b and RBV efficacy in hepatitis C patients of any genotype remain elusive. Although patients with cirrhosis in the present study were skewed toward male gender and older age compared with non-cirrhotic patients, we could not ascertain differences in HCV genotype distribution with respect to the degree of fibrosis. The finding of similar baseline HCV RNA levels in cirrhotic and non-cirrhotic patients rules out that viraemia, otherwise an important predictor of SVR rates in HCV patients [3 5], could have accounted for the discrepant response rates we found between cirrhotic and non-cirrhotic patients. Finally, our finding of similar rates of adherence to therapy between cirrhotic and non-cirrhotic patients (70% versus 74%), indicate that reduced tolerance to therapy was not a determinant of treatment outcome in cirrhotic patients. It is noteworthy that only a minority (5%) of all patients discontinued treatment because of the onset of severe side effects and that this finding, along with satisfactory rates of adherence to the predefined treatment schedules, was the result of the careful and thorough physician-driven care, as reported by others [10]. The main objective of this study was to evaluate whether the interaction between genotype and liver disease severity could result in different patterns of treatment failure. Indeed, treatment failure in HCV-1 and HCV-4 cirrhotic patients was characterized by high rates of primary non-response, whereas HCV-2 and HCV-3 cirrhotic patients with treatment failure showed predominantly high rates of post-treatment relapse. The observation that cirrhosis determines primary non-response in HCV-1 patients is not a complete novel finding. In fact, Everson et al. [28] convincingly demonstrated the negative influence of cirrhosis on virological response at week 20 of treatment, albeit in treatment-experienced patients receiving retreatment with PEG-IFN-α2a. By contrast, the high posttreatment relapse rates in HCV-2 and HCV-3 patients with cirrhosis observed in our study are novel. Unfortunately, because of the retrospective design of the study, the mechanisms by which cirrhosis is associated with post-treatment relapse in these patients remains unknown. We can reasonably exclude RBV underdosing to be the culprit, mainly on the basis of the weightbased dose schedule we utilized, the similar rates of adherence in HCV-2 and HCV-3 cirrhotic patients compared with non-cirrhotic patients (66% versus 78%; P=0.2 and 93% versus 88%; P=0.9) and, ultimately, because dose reductions occurred with similar frequencies among cirrhotic and non-cirrhotic patients (30% versus 26%; P=0.9). Conversely, we cannot completely rule out false ETR caused by residual viraemia or occult replication of HCV in the liver or in peripheral blood mononuclear cells as potential causes of post-treatment relapse in our patients [29,30]. Also, we can not exclude the potential confounding role of other moderators of treatment outcome, such as insulin resistance and liver steatosis [16,31], although the role of these cofactors in attenuating SVR in HCV-2 and HCV-3 patients is debated [19,32,33]. Whatever the mechanisms underlying the different patterns of treatment failure in cirrhotic patients, the recognition of an interaction between HCV genotype and liver fibrosis might help to improve management of patients with hepatitis-c-related cirrhosis. Phase II studies with NS3/NS4 protease inhibitors suggest that triple therapy with PEG-IFN-α and RBV might accelerate HCV clearance in HCV-1-infected patients compared with PEG-IFN-α2a and RBV combination therapy, although the ability of this new regimen to circumvent primary non-response to IFN remains to be demonstrated [34,35]. By contrast, extended treatment with standard of care regimens could be an option for improving SVR rates in patients with high rates of post-treatment relapse, similar to what happened with HCV-1 patients with a slow response to PEG- IFN-α2a and RBV therapy [8 11]. Therefore, studies that prospectively analyse the extension to 48 weeks of treatment in HCV-2 and HCV-3 patients with cirrhosis, regardless of RVR, are needed. In conclusion, we demonstrated an interaction between HCV genotype and cirrhosis, which supports significant differences in the pattern of failure to PEG- IFN-α2b and RBV therapy in patients with chronic hepatitis C. With all the caveats of a retrospective single-centre study, which by definition carries a lower grade of evidence than a multicentre randomized study because it reflects the clinicians experience on patient selection and management of antiviral therapy, International Medical Press

7 HCV genotype and cirrhosis and therefore needs to be prospectively and externally validated, we think that our findings might help to refine our approach to HCV patients with cirrhosis. Acknowledgements The authors thank Caterina M Puricelli for her expert secretarial assistance. The present study was supported by a grant from the University of Milan in Disclosure statement MC has served as a speaker for Schering Plough, Roche and GlaxoSmithKline, is an advisory board member for Schering Plough and Roche, and has received research funding from Schering Plough and Roche. All other authors declare no competing interests. References 1. Seeff LB. Natural history of chronic hepatitis C. Hepatology 2002; 36:S35 S Hoofnagle JH, Seeff LB. Peginterferon and ribavirin for chronic hepatitis C. N Engl J Med 2006; 355: Manns MP, McHutchison JG, Gordon SC, et al. 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