Assessment of liver stiffness in patients with HCV and mixed cryoglobulinemia undergoing rituximab treatment.

INTRODUCTION: Mixed cryoglobulinemia (MC) is a HCV-related lymphoproliferative disorder generally associated with advanced liver disease. Liver stiffness has been significantly correlated with histopathological stage of fibrosis. Moreover, it was influenced by necroinflammatory activity. Rituximab (RTX) is a chimeric anti-CD20 monoclonal antibody inducing transient B lymphocytes depletion that was shown to be useful and safe in the majority of HCV MC patients, leading also to improvement of cirrhotic syndrome. Aim of this study was to evaluate the modifications of liver stiffness following RTX treatment in HCV-related MC patients. MATERIALS AND METHODS: Fourteen consecutive patients (10 F, 4 M; mean age 60.43 ± 43) with HCV-related chronic hepatitis (n = 10) or cirrhosis (n = 4) and MC, eligible for RTX treatment, were prospectively enrolled. Intravenous injection of 1 g of RTX was performed at day 0 and at day 15. Assessment of stiffness was carried out by Fibroscan(®) (Echosens, Paris-France) at baseline, 15 days after the first infusion, and at month 1, 3 and 6 after therapy. RESULTS: MC symptoms significantly improved during the study, especially during the first 3 months. Liver stiffness observed 3 months after treatment was significantly reduced when compared with pre-treatment values (p = 0.01). This difference disappeared after 6 months of follow-up. Cytofluorimetric analysis showed a decrease of CD19+ peripheral blood cells, with the nadir at month 3 after therapy and B cell compartment reconstitution after 6 months. CONCLUSION: This study, for the first time showed that RTX-treatment in HCV-related MC induces a reduction of liver stiffness that is strictly associated with the B-cell depletion

Role of MicroRNA Profile Modifications in Hepatitis C Virus-Related Mixed Cryoglobulinemia

Hepatitis C virus infection is closely related to lymphoproliferative disorders (LPDs), including mixed cryoglobulinemia (MC) and some lymphomas. Modification of the expression of specific microRNAs (miRNAs) has been associated with different autoimmune diseases and/or LPDs. No data exist about the modifications in miRNA expression in HCV-associated LPDs. The aim of this study was to analyze the expression levels of a panel of miRNAs previously associated with autoimmune/LPDs in a large population of HCV patients with and without MC or non-Hodgkin’s lymphoma (NHL), to identify potential markers of evolution of HCV infection. PBMC expression of miR-Let-7d, miR-16, miR-21, miR-26b, miR-146a and miR-155 was evaluated by real-time PCR in 167 HCV patients (75 with MC [MC-HCV], 11 with HCV-associated NHL [NHL-HCV], 81 without LPD [HCV]) and in 35 healthy subjects (HS). A significant increase in miR-21 (p<0.001), miR-16 (p<0.01) and miR-155 (p<0.01) expression was detected in PBMCs from only NHL patients whereas a significant decrease in miR-26b was detected in both MC and NHL subjects (p<0.01) when compared to HS and HCV groups. A restoration of miR-26b levels was observed in the post-treatment PBMCs of 35 HCV-MC patients experiencing complete virological and clinical response following antiviral therapy. This study, for the first time, shows that specific microRNAs in PBMC from HCV patients who developed MC and/or NHL are modulated differently. The specific, reversible downregulation of miR-26b strongly suggests the key role it plays in the pathogenesis of HCV-related LPDs and its usefulness as a biomarker of the evolution of HCV infection to these disorders

HCV-related lymphatic and hepatic malignancies are associated with different microRNA expression patterns in PBMCs

HCV infection leads to chronic liver damage often evolving to Hepatocellular Carcinoma (HCC), but also promotes lymphoproliferative disorders (LPDs) such as mixed cryoglobulinemia (MC) and Non Hodgkin Lymphomas (NHL). Modified expression levels of specific microRNAs have been associated with different autoimmune/lymphoproliferative or neoplastic disorders. We previously showed a downregulation of miR-26b in HCV-related MC and NHL. In this study we analyzed the expression of a panel of microRNAs in peripheral blood mononuclear cells (PBMCs) from a large population of HCV patients with or without LPDs or HCC, in order to identify non-invasive markers of such evolutions of HCV infection. PBMCs levels of miR-Let7d, miR-16, miR-21, miR-26b, miR-146a and miR-155 were evaluated by Real Time PCR in 187 HCV patients (75 with MC [HCV-MC], 11 with HCV-related NHL [HCV-NHL], 20 with HCV-related HCC [HCV-HCC], 81 without LPD or HCC [HCV]) and in 35 healthy blood donors (HS). A significant increase of miR-21 (p&lt; 0.001) expression was detected in PBMCs from only NHL patients, while miR-155 and miR-16 were upregulated both in NHL (p&lt; 0.05) and HCC subjects (p&lt;0.001), when compared to the other groups. A similar level for miR-146a was observed in all groups, except for the HCV-HCC patients, where miR-146a resulted upregulated (p&lt;0.001). A significant decrease of miR-26b was observed in both MC and NHL subjects (p&lt;0.05) when compared to HS and HCV groups. The upregulation of miR-16 and miR-155 is consistent with their reported role as “oncogenic microRNAs” in both HCC and lymphomas, while the upregulation of miR-21 only in the HCV-NHL group confirms its specific role in hematological malignancy. Interestingly, the downregulation of miR-26b in HCV-related LPDs, but not in HCC, suggests its pathogenetic relevance and its potential use as biomarker for evolution into LPDs. Furthermore, the upregulation of miR-146a in PBMC was shown as peculiar profile of HCC patients, suggesting that this could identify an evolution to a hepatic malignancy. In conclusion, this study shows that microRNAs are differently modulated in PBMCs from HCV patients who developed LPDs, NHL or HCC and thus, they could represent clinically relevant tools, useful to discriminate between the two major HCV-related malignancies

Triple antiviral therapy in hepatitis C virus infection with or without mixed cryoglobulinaemia: A prospective, controlled pilot study

Mixed cryoglobulinaemia is strongly related to hepatitis C virus infection. Treatment with peg-interferon and ribavirin has been indicated as first-line therapy for mild/moderate hepatitis C virus-related mixed cryoglobulinaemia. AIM: To evaluate the safety and efficacy of triple boceprevir-based antiviral therapy in patients with or without mixed cryoglobulinaemia previously treated with peg-interferon and ribavirin, and with advanced liver disease. METHODS: Thirty-five hepatitis C virus-positive patients (17 with asymptomatic mixed cryoglobulinaemia, 5 with symptomatic mixed cryoglobulinaemia, and 11 without mixed cryoglobulinaemia) were treated with triple boceprevir-based antiviral therapy. RESULTS: In 19/22 cryoglobulinaemic subjects (86%), the addition of boceprevir induced cryocrit disappearance. Cryocrit behaviour was related to virological response, with improvement of symptoms upon undetectable viraemia and reappearance after virological breakthrough. The rate of sustained virological response was lower in cryoglobulinaemic patients than in patients without mixed cryoglobulinaemia (23.8% vs 70% respectively, p=0.01). CONCLUSION: Boceprevir-based therapy was safe and effective in cryoglobulinaemic patients. The correlation between direct inhibition of hepatitis C virus replication and clinical improvement in mixed cryoglobulinaemic patients is definitive proof of the key pathogenetic role played by viral replication. Further studies are needed to confirm and clarify the reduced virological response in patients with mixed cryoglobulinaemia

Could the presence of Mixed Cryoglobulinemia influence the outcome of standard peg-IFN plus ribavirin anti-HCV therapy?

Background. HCV infection is closely related to the development of mixed cryoglobulinemia (MC). Although MC is present in the majority of HCV patients, only a minority, ranging from 5% to 30% in different studied manifests a MC syndrome (MCS). The possible influence of MC/MCS on the results of anti-HCV treatment is still matter of debate. Being MC/MCS an elusive condition (i.e., the diagnosis of MCS is not well standardized and the MC impossible to determine in stored samples), the possibility that retrospective studies may include MC/MCS patients in the MC-negative control group cannot be ruled out. The aim of this study was to prospectively analyze whether the presence of MC influences the response to the standard peg-IFN+ribavirin therapy (SoC) in HCV+ patients. Patients. 425 HCV-positive patients were consecutively treated with SoC PegIFN and ribavirin therapy and were evaluated for the enrollment. These included: 121 (28.5%) patients with MCS (HCV+MCS), 132 (31.1%) with MC without MCS (HCV+MC) and 159 (37.3%) without MCS nor MC or other autoimmune/lymphoproliferative disorders (HCV). 13 patients (3.1%) resulted of uncertain classification and were excluded from the study. MC/MCS data and virological response were regularly assessed during treatment and in a 6-month post-treatment follow-up. The IL28B genotype was detected by allele-specific real-time PCR. Results. The univariate statistical analysis showed that HCV+MCS group was associated with older age (p=0.0151), more severe liver disease (p=0.02), female sex (p&lt;0.00005) and re-treatment (p=0.0168). No differences were observed in viral genotype distribution, pre-treatment viral load, IL28B allele distribution. During treatment, no significant differences were observed for both RVR and EVR. However, when SVR was considered, a statistically significant difference was observed between HCV+MCS vs. HCV patients (p=0.012) and also HCV+MC vs HCV (p=0.045). The logistic regression analysis showed that HCV+MCS was an independent risk factor of non-response (O.R. 2.48; CI 1.24-6.3; p=0.013). Conclusions. In the present study, for the first time, the virological response of a large number of HCV+ patients with and without MC/MCS was prospectively studied. Results strongly suggest that MCS represents a negative prognostic factors of response to anti-HCV SoC. This does not appear related to a significantly modified early viral kinetics. It may be hypothesized that the higher prevalence of persisting infection in lymphatic reservoirs, previously shown in MCS+ vs. MCS- patients, plays a role

Value of IL28B genotyping in patients with HCV-related mixed cryoglobulinemia: results of a large, prospective study

HCV-related mixed cryoglobulinemia (MC) is characterized by clonal expansion of B-cells producing a polyreactive natural antibody (rheumatoid factor) and interferon (IFN)-based therapy is the first therapeutic option in mild-moderate MC. Single nucleotide polymorphisms (SNPs) proximal to genes involved in the innate response (IL28B/IFN-λ gene family) are strongly associated with spontaneous and IFN-induced viral clearance in hepatitis C, but no data exist about their role in HCV-positive MC (MC-HCV). A large cohort of MC-HCV patients was studied in order to evaluate the influence of IL28B genotype on the response to treatment and/or the evolution to MC of HCV infection. The rs12979860/rs8099917 IL28B polymorphisms were analyzed in 481 consecutive HCV-positive subjects (250 with MC-HCV and 231 without MC -controls-) using Real-time PCR and direct sequencing. 115 MC-HCV patients received standard anti-HCV therapy and the results were evaluated according to the IL28B SNP distribution. Similar IL28B SNPs allele frequencies were recorded for MC-HCV patients and controls. IL28B major allele homozygosis (for both SNPs tested) was tightly correlated with virological and clinical response (p=0.002). A statistically significant association was limited to “difficult-to-treat” (G1/4) HCV genotypes. The IL28B genotype was a strong independent predictor of response (p=0.007, O.R. 6.06; C.I. 1.65-22.22). The IL28B genotype was confirmed to be a useful predictor of response to IFN-based therapy in also MC-HCV patients. The very close correlation between IL28B SNPs distribution, virological and clinical response definitively confirmed the key role played by HCV in MC. Conversely, the IL28B genotype does not seem to influence the evolution to MC

Main clinical and laboratory data of 35 healthy subjects (HS) and 167 HCV-chronically infected patients with mixed cryoglobulinemia (MC-HCV) or HCV-related non-Hodgkin’s lymphoma (NHL-HCV) or without MC or NHL (HCV).

<p>Results are presented as mean± standard deviation;</p><p>ns, not significant;</p>∧<p>ALT, alanine aminotransferase;</p><p>ULN, upper limit of normal.</p>#<p>Complement C3, normal values: 83 to 177 mg/dL;</p>‡<p>Complement C4, normal values: 20 to 150 mg/dL;</p>†<p>Rheumatoid Factor, normal values: <25 IU/mL.</p>*<p>HS vs MC-HCV; HS vs NHL-HCV; HCV vs MC-HCV.</p>**<p>HS vs MC-HCV; HS vs NHL-HCV; HCV vs MC-HCV; HCV vs NHL-HCV.</p>***<p>HCV vs MC-HCV.</p><p>°HS or HCV vs MC-HCV or NHL-HCV.</p

Mixed cryoglobulinemia syndrome is a negative prognostic factor in the standard of care (Peg-IFN+ribavirin) anti-HCV therapy

HCV leads to chronic hepatitis (CHC) and mixed cryoglobulinemia (MC). MC is observed in the majority of HCV pts, but is clinically evident (MC syndrome, MCS) in a minority of cases. Antiviral therapy is the first therapeutic option for both CHC and MCS. In previous reports, generally based on retrospective analyses and/or limited populations, no difference was observed in MC/MCS virological response. This is an elusive condition (the diagnosis is not well standardized and the MC impossible to determine in stored samples), and the possibility that some MC/MCS pts were included in the negative control group cannot be ruled out. In this study, we prospectively analyzed MC/MCS data and virological response of a large population of HCV pts treated with anti-HCV SoC (Peg-IFN+RBV). Pts were consecutively recruited from July 2003 to July 2010. Results: 424 pts were enrolled. These included: 121 pts with MCS (HCV+MCS), 132 with MC without MCS (HCV+MC) and 158 without MCS nor MC (HCV). 13 patients (3.1%) resulted of uncertain classification. HCV+MCS group was associated with older age (p=0.0151), more severe liver disease (p=0.02), female sex (p&lt;0.00005) and re-treatment (p=0.0168) in the univariate analysis. No differences were observed in viral genotype distribution, pre-treatment viral load, IL28B allele distribution. No significant differences were observed for both RVR and EVR. However, when SVR was considered, a statistically significant difference was observed between HCV+MCS vs. HCV patients (p=0.012) and also HCV+MC vs HCV (p=0.045). The logistic regression analysis showed that HCV+MCS was an independent risk factor of non-response (O.R. 2.48; CI 1.24-6.3; p=0.013). Conclusions: This study strongly suggests that MCS represents a negative prognostic factor of response to anti-HCV SoC. This does not appear related to a modified early viral kinetics. It may be hypothesized that the higher prevalence of persisting infection in lymphatic reservoirs, previously shown in MCS+ vs. MCS- patients, plays a role

Principal mixed cryoglobulinemia (MC) manifestations present in the 75 HCV patients with MC (MC-HCV).

<p>Principal mixed cryoglobulinemia (MC) manifestations present in the 75 HCV patients with MC (MC-HCV).</p