Luisa Cavalletto

Serum and liver HCV RNA levels in patients with chronic hepatitis C: correlation with clinical and histological features

Background—Liver disease in chronic hepatitis C virus (HCV) infection ranges from minimal lesions to liver cirrhosis, eventually evolving to hepatocellular carcinoma. Whether and how HCV determines the different clinical and histological manifestations of the disease is not fully understood. Aims—To verify whether the amount of virus in individual patients could be related to the severity of liver injury. Patients and methods—Levels of HCV RNA were measured in serum in 96 consecutive patients with chronic hepatitis type C using a signal amplification assay. The relation between viraemic values and the corresponding viral load in the liver was assessed in a subgroup of 21 patients in whom HCV RNA was measured in serum samples and liver specimens obtained at the same time. Results—A positive correlation was observed between the amount of viral nucleic acid in the two compartments, indicating that levels of viraemia reflect the amount of virus present in the liver. Viral load did not correlate with aminotransferase activities nor with histological diagnosis, and serum and liver levels of HCV RNA were not significantly different in patients infected by the various HCV genotypes. Conclusions—Measurement of HCV replication in serum is a mirror of viral replication in the liver. The extent of replicative activity of HCV does not seem to play a role in the modulation of the associated hepatic disease.

Persistent Hepatitis C Viremia Predicts Late Relapse after Sustained Response to Interferon-α in Chronic Hepatitis C

Chronic infection with the hepatitis C virus (HCV) is a major cause of cirrhosis and hepatocellular carcinoma worldwide. In many countries, interferon- therapy is the only licensed method for treating this condition. It has been licensed on the basis of randomized, controlled trials that have shown its effectiveness in reducing disease activity and virus replication [1, 2]. However, few treated patients achieve a complete response that is maintained after therapy, and many others have a transient response or no response at all [3, 4]. Serum alanine aminotransferase activity is the conventional marker used to monitor response to therapy, and sustained biochemical response is defined as normalization that lasts for 6 to 12 months after cessation of interferon- therapy. More recently, serum HCV RNA testing has been recognized as an important component of assessing response to treatment; patients who respond at the biochemical level and maintain normal alanine aminotransferase levels after therapy may remain viremic, indicating that interferon- has suppressed liver disease activity but has not eradicated the virus [5]. There is increasing agreement that patients with biochemical response should be tested for serum HCV RNA to more accurately define the effect and efficacy of treatment. However, little is known of the clinical significance and long-term outcome of persistent viremia with normal alanine aminotransferase activity after interferon- therapy. It is still unclear whether this profile reflects delayed virus clearance or persistence of a noncytopathic type or load of HCV or whether it should instead be regarded as a predictor of reactivation of liver damage and progression of disease. To clarify these issues, we investigated the long-term outcome of a large series of consecutive patients with chronic hepatitis C who had persistently maintained normal alanine aminotransferase activity for at least 1 year after cessation of interferon- therapy. Methods Patients Between January 1990 and November 1993, 440 consecutive patients with chronic hepatitis C were treated with interferon- therapy in three consecutive randomized trials [6, 7] using different schedules of treatment. Inclusion and exclusion criteria were identical for the three studies, and the clinical characteristics of patient subgroups were similar [8]. Enrolled patients were between 18 and 65 years of age and had a persistent (lasting more than 6 months) elevation of alanine aminotransferase levels of more than twice the upper limit of normal positive result on testing for antibody to HCV, and histologic evidence of chronic hepatitis with or without Child grade A cirrhosis. Patients who were also infected with hepatitis B virus or human immunodeficiency virus who had autoimmunity or comorbid conditions were not included. The total interferon- dose ranged from 234 MU to 740 MU, administered for 6 or 12 months. Alanine aminotransferase levels were tested monthly during therapy. Complete biochemical response was defined as normalization of liver enzyme levels during therapy, maintained until withdrawal of therapy. After cessation of interferon- therapy, patients who had responded were monitored for serum alanine aminotransferase at 3-month intervals; sustained biochemical response was defined as enzyme levels that were normal for as long as 12 months after therapy. Of 440 treated patients, 259 (59%) showed complete biochemical response during treatment, but 141 (54%) of the 259 had relapse within 12 months after therapy was discontinued. Of the 118 patients with alanine aminotransferase levels that were normal for as long as 12 months after therapy, 107 could be tested for serum HCV RNA and followed prospectively for an additional 6 to 36 months (mean follow-up after therapy, 35 18 months). In the remaining 11 patients who had responded to interferon-, serum samples were not available for HCV RNA testing 1 year after therapy. Testing Serum HCV RNA was tested using nested polymerase chain reaction [9]; the sensitivity limit of the assay was between 103 and 104 copies/mL. The virus genotype was identified using spot hybridization with genotype-specific oligonucleotide probes [9] and was classified according to the system of Simmonds and colleagues [10]. Liver biopsies were done during the 6 months before therapy. Sixty-three of the 107 patients with persistently normal alanine aminotransferase levels consented to have liver biopsy, done 8 to 12 months after cessation of interferon therapy. The histologic evaluation was made by a pathologist who was blinded to clinical data and to the sequence in which biopsy specimens had been obtained. Continuous variables were compared by using the t-test, and proportions were compared by using the chi-square test. The probability of maintaining a sustained response to therapy in different groups was analyzed by using the Kaplan-Meier method and was compared by using the log-rank test. Results One year after therapy, serum HCV RNA was detectable in 27 (25%) of the 107 patients with sustained biochemical response; 80 patients were negative for HCV RNA. Retrospective analysis of stored serum samples showed that 13 of 27 (48%) patients positive for HCV RNA were viremic at completion of therapy. In the remaining 14 patients (52%), HCV RNA had reappeared either during the first 6 months (6 patients) or the second 6 months (8 patients) after therapy. Patients who were positive for HCV RNA differed substantially from those who were negative for HCV RNA; the former were older and were treated with a smaller total dose of interferon-. Furthermore, a statistically significant difference was seen in the distribution of virus genotypes; HCV RNA-positive patients had a higher prevalence of HCV genotype 2 and a lower prevalence of HCV genotype 3 than did HCV RNA-negative patients. After treatment, a liver biopsy could be done while alanine aminotransferase levels were normal in 14 HCV RNA-positive patients and 49 nonviremic patients. Chronic hepatitis with piecemeal necrosis was seen in 8 of 14 (57%) HCV RNA-positive patients; the remaining patients had chronic hepatitis with portal inflammation but no periportal necrosis (4 patients) or minimal changes (2 patients). In contrast, active histologic findings were seen in only 6 of 49 (12%) HCV RNA-negative patients (P = 0.01); the remaining patients had chronic hepatitis with portal inflammation but no piecemeal necrosis (25 patients) or either normal liver histologic findings or minimal changes (18 patients). When the 107 patients were followed prospectively beyond the first year after therapy had been discontinued, the outcomes in viremic and nonviremic patients clearly differed. All HCV RNA-negative patients maintained normal alanine aminotransferase levels and remained negative for HCV RNA, whereas a biochemical relapse was seen in 8 of the 27 (30%) viremic patients. The estimated probability of a return of elevated alanine aminotransferase levels 4 years after cessation of treatment was 53% in viremic patients and 0% in the negative patients (P < 0.001) (Figure 1). No characteristics could be used to distinguish patients with early (within 12 months) or late (beyond 12 months) relapse after therapy. However, when all patients who had relapse were compared with all patients who had sustained biochemical and virologic long-term responses, the former were older (P < 0.01), had a longer duration of disease (P < 0.01), and had a different distribution of HCV genotype (P < 0.05) with increased prevalence of genotype 1 and decreased prevalences of genotypes 2 and 3. Figure 1. Probability of maintaining normal serum alanine aminotransferase levels at long-term follow-up (Kaplan-Meier curves). P Discussion Our study shows that serum HCV RNA testing is clinically useful when done 1 year after interferon- therapy in patients with chronic hepatitis C who have responded to therapy with persistently normal aminotransferase levels. If the test result is negative 1 year after therapy, the response is permanent; in patients who are still viremic 1 year after therapy, liver disease is often still histologically active and the risk for a late reactivation of hepatitis is substantial (53% of patients in our study had probability of reactivation by 4 years after therapy). Our results are consistent with previous reports on the discrepancy between biochemical and virologic responses to interferon- in chronic hepatitis C and on the possibility of late relapse after a prolonged biochemical response [11, 12]. Only one HCV RNA test was done 1 year after therapy, and we cannot exclude the possibility of some false-negative results caused by intermittent viremia or HCV-RNA levels below the sensitivity of our assay. Furthermore, viremia may not accurately reflect the presence or absence of the virus in the liver. Despite these limitations and the fact that not all of our patients who responded to interferon could have a liver biopsy done after treatment, our results clearly indicate that serum HCV RNA should be tested in all patients who have developed a sustained biochemical response to interferon- to establish whether the response is complete and permanent. Patients who are negative for HCV RNA may be considered cured. Viremic patients with normal alanine amino-transferase levels should be followed carefully well beyond the first year after cessation of therapy, because many will eventually relapse. Studies must be done to determine whether treatment with larger interferon- doses could reduce the prevalence of residual viremia after therapy and whether patients who remain positive for HCV RNA after interferon- therapy could benefit from early retreatment. Dr. Belussi: Divisione Malattie Infettive, O. Le Grazie, 5501 San Marco, 30124 Venezia, Italy. Dr. Martinelli: Divisione Medica, O.C. Cittadella (PD), via Circonvallazione, 35013 Cittadella (PD), Italy.

Treatment with interferon(s) of community-acquired chronic hepatitis and cirrhosis type C

Two hundred and thirty-four patients with chronic non-A, non-B hepatitis, 86% positive for anti-HCV by ELISA, were treated with recombinant interferon-alpha 2a or with natural (human-leukocytes-derived) interferon-alpha using different dosage and periods of administration. Interim analysis of follow-up data indicate that 65-70% of patients treated initially with 6 MU, thrice weekly, of recombinant interferon-alpha 2a achieved a complete biochemical response (normalization of alanine aminotransferase: ALT) during therapy compared to 56-58% of those treated with 3 MU, thrice weekly, of recombinant or natural interferon-alpha. A 12-month schedule of interferon administration appeared superior to a 6-month schedule in reducing the probability of reactivation of liver disease after therapy withdrawal, although further data are needed to confirm such a conclusion. The probability of response to interferon in terms of maintaining normal ALT after withdrawal did not appear to be influenced by sex, while it was significantly higher in patients aged below 45 years and in those without cirrhosis.

Progressive increase of SCCA-IgM immune complexes in cirrhotic patients is associated with development of hepatocellular carcinoma.

About 3–4% of cirrhotic patients develop primary liver cancer every year. Specific serologic markers have not yet been identified for screening of high risk patients. The serpin squamous cell carcinoma antigen (SCCA) is overexpressed in liver cancer and circulating SCCA‐IgM complexes have been described in patients with hepatocellular carcinoma (HCC). The aim of the present study was to assess the behavior of SCCA‐IgM in relation to HCC development in patients with cirrhosis. A retrospective, longitudinal study was conducted in a cohort of prospectively followed cirrhotic patients. Two groups with similar clinical profile at presentation were studied : group A included 16 patients who developed HCC during a median follow up of 4 years; group B included 17 patients who did not develop HCC during the same time interval. Circulating SCCA‐IgM immune complexes were determined using a recently standardized ELISA assay. At presentation similar levels of SCCA‐IgM complexes [mean ± SD: 267.40 ± 382.25 U/ml vs. 249.10 ± 446.90 U/ml, p = 0.9006] and of alpha‐fetoprotein [AFP; 24.11 ± 59.04 IU/ml vs. 10.91 ± 23.34 IU/ml, p = 0.3995] were detected in group A and in group B. The increase over time (ϕ) of SCCA‐IgM, assessed within at least one year before clinical diagnosis of HCC, was remarkably higher in group A than in group B (mean ± SD = 280.05 ± 606.71 (U/ml)/year vs. −37.92 ± 95.94 (U/ml)/year, p = 0.0408), while AFP increase was not significantly different (11.89 ± 23.27 (IU/ml)/year vs. 3.67 ± 11.46 (IU/ml)/year, p = 0.2179). Receiver operating characteristic (ROC) curves were plotted for the rate of change in the levels of both markers and the diagnostic accuracy measured as AUROC was higher for SCCA‐IgM ϕ (0.821) than for AFP ϕ (0.654). In conclusion, the progressive increase of SCCA‐IgM over time was associated with liver tumor development, suggesting that monitoring the behavior of SCCA‐IgM might become useful to identify cirrhotic patients at higher risk of HCC development.

Performance and utility of transient elastography and noninvasive markers of liver fibrosis in primary biliary cirrhosis.

BACKGROUND The performance of transient elastography in primary biliary cirrhosis has yet to be fully established. AIM To assess: (1) the performance of transient elastography in identifying significant fibrosis in primary biliary cirrhosis by comparison with surrogate markers (AST platelet ratio index (APRI), FIB-4, Fibroindex, Forns, aspartate aminotransferase/alanine aminotransferase ratio); (2) the correlation between liver stiffness and Mayo score prognostic index. METHODS One hundred and twenty patients with primary biliary cirrhosis were consecutively enrolled. The performance of each marker and of liver stiffness was compared with histological staging and METAVIR at time of liver biopsy. RESULTS The area under receiver operating characteristic (ROC) of liver stiffness were 0.87, 0.88, 0.99 for histological stage ≥II, ≥III and =IV and 0.89, 0.92, 0.99 for METAVIR ≥2, ≥3 and =4. Transient elastography alone proved better able in identifying any grade of fibrosis or cirrhosis than noninvasive markers. Combining each surrogate marker with transient elastography did not improve the area under ROC. Transient elastography correlated positively with the Mayo score (P<0.001). Logistic regression analysis showed that transient elastography was associated with an advanced fibrosis (P<0.001). CONCLUSIONS Transient elastography proved a simple, reliable and useful method for assessing liver fibrosis in primary biliary cirrhosis, whereas noninvasive surrogate markers proved unsatisfactory in predicting significant fibrosis.

Effects of PEG-interferon alpha plus ribavirin on tryptophan metabolism in patients with chronic hepatitis C.

The currently recommended therapy for chronic hepatitis C (HCV) is a combination of pegylated interferon-alpha (PEG-IFN alpha) and ribavirin. Psychiatric disorders, including depression, are frequent in HCV patients under therapy. We investigated the effect of the antiviral treatment on tryptophan (Trp) metabolism along both serotonin pathway (via 5-hydroxytryptophan, 5-HTP) and kynurenine (Kyn) pathway and on the onset of depressive symptoms in patients with HCV. The key enzyme of the Kyn pathway is indoleamine 2,3-dioxygenase (IDO), an intracellular haem protein enzyme expressed in several tissues. It was also investigated the influence of the therapy with PEG-IFN-alpha-2a or PEG-IFN-alpha-2b plus oral ribavirin and possible differences between genders. Free and total Trp, 5-hydroxytryptophan (5-HTP) and Kyn serum concentrations and the presence of depressive symptoms [Beck Depression Inventory (BDI) scores] were evaluated in 45 patients with HCV infection treated with PEG-IFN alpha-2a or -2b at four different times: baseline (before treatment), 1 and 6 months during therapy, and 3 months after the end of therapy. The concentration of serum total TRP (free+protein bound) as well as that of 5-HTP significantly decreased after 1 and 6 months of therapy, and then returned to baseline values 3 months after the end of therapy, while the levels of free TRP did not vary significantly during and after the therapy. On the contrary, the time course of Kyn markedly arose during treatment, paralleled by a significant increase of [Kyn/Trp]×10(3) ratio, an index used to measure IDO activity. No significant difference was detected between males and females neither between PEG-IFN-alpha-2a or -2b treatment. The BDI scores significantly increased during therapy, and returned to baseline values 3 months after the end of therapy. Our results support the hypothesis that the increased IDO-mediated tryptophan metabolism along the Kyn pathway, leading to plasma Trp depletion and a decline of serotonin pathway, concurs to the development of depressive symptoms observed in HCV patients undergoing IFN-alpha therapy.

Monitoring SCCA-IgM complexes in serum predicts liver disease progression in patients with chronic hepatitis.

Summary.  About 30% of the patients with chronic hepatitis develop a progressive liver disease and one of the most intriguing issues is the detection of noninvasive markers for fibrosis stage and disease progression. High levels of squamous cell carcinoma antigen (SCCA)‐immunoglobulin M (IgM) are detectable in hepatocellular carcinoma and their increase in cirrhotic patients can predict tumour development. As SCCA‐IgM can also be detectable at low percentages in patients with chronic hepatitis, the aim of this study was to assess SCCA‐IgM complexes in relation to disease outcome in this group of patients. An ELISA assay was used to determine the presence of SCCA‐IgM in 188 patients with chronic hepatitis and in 100 controls. An additional serum sample was available after a median period of 6 years in 57 untreated patients: these patients were subdivided in group A, including eight patients with a fibrosis score increase ≥2 in a second liver biopsy and group B, including 49 patients without fibrosis progression during a similar follow up. SCCA‐IgM complexes were detectable in 63 of 188 (33%) patients but in none of the controls. A significant increase of SCCA‐IgM levels over time was observed in patients with fibrosis progression (mean ± SD: 117 ± 200 U/mL/year), but not in those without histologic deterioration (mean ± SD: –8.8 ± 31 U/mL/year, P < 0.0001). In conclusion, monitoring SCCA‐IgM levels over time appears a useful approach to identify patients with chronic hepatitis at higher risk for cirrhosis development.

Evidence for sequence selection within the non‐structural 5A gene of hepatitis C virus type 1b during unsuccessful treatment with interferon‐α

Resistance of the hepatitis C virus (HCV) to interferon‐α (IFN‐α) therapy in patients with hepatitis C may be genetically controlled by an IFN sensitivity‐determining region (ISDR) within the non‐structural 5A (NS5A) gene. To assess whether HCV 1b strains carrying a ‘resistant’ type of ISDR are selected during unsuccessful IFN therapy, we analysed the evolution of the NS5A quasispecies, as detected by the clonal frequency analysis technique, and of the ISDR sequence by nucleotide sequence determination, in 11 patients showing no virological response during two consecutive cycles of IFN‐α therapy. IFN‐resistant patients had a homogeneous ISDR quasispecies with sequences identical to those described as ‘resistant‐’ or ‘intermediate‐’ type ISDR. After retreatment with IFN, further selection towards a homogeneous viral population was observed and 10 out of 11 patients had only one variant of HCV with no or just one single amino acid mutation within the ISDR sequence. Treatment and retreatment with IFN was associated in our non‐responder patients with evolution of the ISDR quasispecies towards a rather homogeneous viral population carrying a conserved or minimally mutated ISDR motif, supporting the idea that this motif may be relevant for IFN resistance in HCV 1b‐infected individuals.

The pattern of response to interferon alpha (α-IFN) predicts sustained response to a 6-month α-IFN and ribavirin retreatment for chronic hepatitis C

Abstract Background/Aims: In chronic hepatitis C, interferon-alpha (α-IFN) and ribavirin combination therapy improves sustained response compared to α-IFN monotherapy, both in naive patients and in previous α-IFN relapsers, but the efficacy of such therapy remains limited in non-responder cases. The aim of this study was to assess whether the pattern of response to α-IFN alone may predict sustained response to combination therapy during retreatment. Methods: Fifty previous α-IFN relapsers and 50 previous α-IFN non-responders were retreated with a high α-IFN dose (6 MU/thrice weekly for 2 months; induction phase) and then randomised to continue with α-IFN alone (3 MU/thrice weekly) or to receive combination therapy (3 MU/thrice weekly of α-IFN and 1000–1200 mg/daily of ribavirin) for an additional 6 months according to the biochemical response to α-IFN shown after the induction phase. All patients were also evaluated for virological and histological response. Results: Eleven of 25 (44%) relapsers treated with combination therapy and 4/25 (16%) treated with α-IFN alone achieved a sustained response. The corresponding figures among non-responders were 1/25 (4%) and 0/25, respectively. Among 26 patients with a complete ALT and HCV-RNA response after 2 months of α-IFN, sustained response was seen in 11/14 (79%) treated with combination therapy and in 4/12 (33%) treated with α-IFN alone ( p =0.05). On the other hand, of 74 cases still HCV-RNA positive after 2 months of α-IFN alone, biochemical and virological end of therapy response was better with combination therapy (11/36; 30.5%) compared to α-IFN alone (4/38; 10.5%), but only one patient developed a sustained response (1/36; 3%). Conclusions: The retreatment with a 6-month combination therapy was associated with a high rate of sustained response only in patients showing a complete biochemical and virological response to α-IFN alone. Longer retreatment with combination therapy may be needed to achieve a sustained response in patients without a prompt virological response to α-IFN.

Incidence of DAA failure and the clinical impact of retreatment in real-life patients treated in the advanced stage of liver disease: Interim evaluations from the PITER network

Background Few data are available on the virological and clinical outcomes of advanced liver disease patients retreated after first-line DAA failure. Aim To evaluate DAA failure incidence and the retreatment clinical impact in patients treated in the advanced liver disease stage. Methods Data on HCV genotype, liver disease severity, and first and second line DAA regimens were prospectively collected in consecutive patients who reached the 12-week post-treatment and retreatment evaluations from January 2015 to December 2016 in 23 of the PITER network centers. Results Among 3,830 patients with advanced fibrosis (F3) or cirrhosis, 139 (3.6%) failed to achieve SVR. Genotype 3, bilirubin levels >1.5mg/dl, platelet count <120,000/mm3 and the sofosbuvir+ribavirin regimen were independent predictors of failure by logistic regression analysis. The failure rate was 7.6% for patients treated with regimens that are no longer recommended or considered suboptimal (sofosbuvir+ribavirin or simeprevir+sofosbuvir±ribavirin), whereas 1.4% for regimens containing sofosbuvir combined with daclatasvir or ledipasvir or other DAAs. Of the patients who failed to achieve SVR, 72 (51.8%) were retreated with a second DAA regimen, specifically 38 (52.7%) with sofosbuvir+daclatasvir, 27 (37.5%) with sofosbuvir+ledipasvir, and 7 (9.7%) with other DAAs ±ribavirin. Among these, 69 (96%) patients achieved SVR12 and 3 (4%) failed. During a median time of 6 months (range: 5–14 months) between failure and the second DAA therapy, the Child-Pugh class worsened in 12 (16.7%) patients: from A to B in 10 patients (19.6%) and from B to C in 2 patients (10.5%), whereas it did not change in the remaining 60 patients. Following the retreatment SVR12 (median time of 6 months; range: 3–12 months), the Child-Pugh class improved in 17 (23.6%) patients: from B to A in 14 (19.4%) patients, from C to A in 1 patient (1.4%) and from C to B in 2 (2.9%) patients; it remained unchanged in 53 patients (73.6%) and worsened in 2 (2.8%) patients. Of patients who were retreated, 3 (4%) had undergone OLT before retreatment (all reached SVR12 following retreatment) and 2 (2.8%) underwent OLT after having achieved retreatment SVR12. Two (70%) of the 3 patients who failed to achieve SVR12 after retreatment, and 2 (2.8%) of the 69 patients who achieved retreatment SVR12 died from liver failure (Child-Pugh class deteriorated from B to C) or HCC complications. Conclusions Failure rate following the first DAA regimen in patients with advanced disease is similar to or lower than that reported in clinical trials, although the majority of patients were treated with suboptimal regimens. Interim findings showed that worsening of liver function after failure, in terms of Child Pugh class deterioration, was improved by successful retreatment in about one third of retreated patients within a short follow-up period; however, in some advanced liver disease patients, clinical outcomes (Child Pugh class, HCC development, liver failure and death) were independent of viral eradication.