With the discovery that the hepatitis C virus (HCV) is the main etiologic agent of mixed cryoglobulinaemia (cryovas), new opportunities and problems in developing therapy for HCV-cryovas have emerged.1–3 The cornerstone of HCV therapy has been and continues to be interferon-α, which has the potential to exacerbate autoimmune disease states.4 ,5 Advances using a triple combination with pegylated interferon-α (Peg-IFN-α), ribavirin and a protease inhibitor in patients infected by the genotype 1 virus have shown promising results. More recently, interferon-free strategies have been developed, again with promising results.6 In more severe cases, combination therapy with rituximab and optimal HCV treatment appears logical, as it may target both mixed cryoglobulin (MC) producing B cells and the viral trigger. This review will focus on advances in our understanding of HCV-cryovas treatment (table 1).

Antiviral agents

Over the last two decades, the treatment of HCV infection (ie, in the absence of HCV-cryovas) has dramatically increased sustained virological response (SVR) rates. This treatment has included interferon (IFN) monotherapy, IFN plus ribavirin4–9 and, more, recently Peg-IFN-α plus ribavirin, leading to an SVR in nearly 50% of patients. Antiviral treatment has also shown improved efficacy in cryovas patients.

Immunosuppressive agents

Before the HCV era, a combination of corticosteroids and immunosuppressants, such as cyclophosphamide and azathioprine, had been used for the control of severe cryovas lesions. In a retrospective study of 105 patients with renal disease associated with cryovas, 80% of patients received corticosteroids and/or cytotoxic agents, while 67% underwent plasmapheresis.13 Despite this aggressive approach, long-lasting remission of the renal disease was achieved in only 14% of cases, and the 10-year survival rate was only 49%.

Corticosteroids, used alone or in addition to IFN-α, did not favourably affect the response of HCV-cryovas manifestations in two controlled studies.14 ,15 In one randomised trial,14 methylprednisolone given alone for 1 year was associated with a clinical response in 16.7% of patients compared with 53.3% and 52.9% in patients receiving IFN-α or IFN-α plus methylprednisolone, respectively. Low-dose corticosteroids may help to control minor intermittent inflammatory signs such arthralgia but they do not succeed in cases of major organ involvement eg, neurologic, renal, cardiac) or in the long-term control of vasculitis.

Plasmapheresis offers the theoretical advantage of removing the pathogenic cryoglobulins from the circulation of patients with HCV-cryovas. Immunosuppressive therapy is usually needed in addition to plasma exchange in order to avoid the rebound increase in cryoglobulin serum levels seen after discontinuation of apheresis. When used in combination with HCV treatment, plasmapheresis did not modify the virologic response when IFN-α was given after each plasma exchange session.16

Rituximab monotherapy

Over the last decade, several groups have reported the efficacy of anti-CD20 monoclonal antibody (rituximab) in patients with HCV-cryovas.17–19 Rituximab is an interesting therapy in cryovas patients, as it targets B cells, which are responsible for cryoglobulin production, immune complex deposition and finally vasculitis lesions.

The results of a literature review20 showed that the main indication for rituximab therapy was non-response to other previous treatments (ie, mostly IFN-α and/or steroids) (n=50), intolerance to previous treatments (n=3) or associated lymphoma (n=2), or as first-line therapy for cryovas (n=2). Most patients received four consecutive weekly intravenous infusions of 375 mg/m² of rituximab. Rituximab infusions proved effective on the main cryovas signs, with a complete clinical response in 24 out of 33 (73%) patients for skin involvement, 16 out of 30 (53%) for arthralgia, 9 out of 25 (36%) for neuropathy and 9 out of 13 (70%) for glomerulonephritis. cryovas relapse was noted in 13 out of 36 (36.1%) patients within a few days (probably related to a reaction mediated by the immune complex21) to 19 months (mean 6.7 months) after the last rituximab infusion. Eight out of 13 relapsers showed complete remission after a second course of rituximab infusion. B cell depletion was achieved in most patients and did not influence the clinical outcome. Two deaths were reported: one from renal failure 12 months after the rituximab infusion, and the other 2  months after the rituximab infusion in a renal transplanted HCV-negative patient due to Candida neoformans meningoencephalitis.

Despite a dramatic reduction in the number of circulating B cells and deletion of B cell clones, Sansonno et al18 observed the appearance of different clones in cryovas patients who responderd to rituximab, which demonstrated that selected antigens may be recognised as part of a limited host response to a virus capable of undergoing spontaneous long-term mutations. B cell depletion (<1% of the total peripheral blood lymphocytes) was achieved in 67% of the cryovas patients regardless of the clinical response.22 Recovery of the B cell count began from 6–9 months. Complete responders to rituximab exhibited an expansion of regulatory T cells accompanied by a decrease in CD8 T-cell activation, and decreased production of interleukin 12 and IFN-γ. Clonal VH1-69⁺ B cells dramatically decreased following treatment (32±6% vs 8±2%, p=0.01).23

De Vita et al24 recently reported the results of a randomised controlled trial in 57 cryovas patients (including 53 HCV-positive patients), which compared conventional treatment (ie, glucocorticoids, azathioprine or cyclophosphamide, or plasmapheresis) and rituximab. None of the HCV-positive patients received concomitant antiviral therapy, either because it was contraindicated (n=28) or had been ineffective or poorly tolerated (n=25). The proportion of patients who continued taking their initial therapy 12 months after treatment was statistically higher in the rituximab group (64.3% vs 3.5%). Sneller et al25 reported the results of an open-label, randomised controlled trial of rituximab compared with immunosuppressive therapy (glucocorticoids, cyclophosphamide, plasma exchanges or methotrexate) for HCV-cryovas patients in whom antiviral therapy had failed to induce remission. A total of 24 patients were enrolled (12 in each treatment group). Remission at 6 months was statistically higher in the rituximab group (83% vs 8%).

A pilot study reported the efficacy of a lower dosage of rituximab (two infusions of 250 mg/m² instead of four infusions of 375 mg/m² per week). All six consecutive cryovas patients had severe or life-threatening disease manifestations. Four of five evaluable patients (excluding one early death) had a >80% decrease of cryocrit and a remission of cryovas at the end of 22–55 weeks (median 40) of follow-up. More recently, the same authors designed a phase II single-arm two-stage study to evaluate the efficacy of a lower dosage of rituximab (250 mg/m² given twice) for refractory cryovas.26 The overall response rate in the first 24 evaluable patients was 79% and the mean time to relapse was 6.5 months (similar to the 6.7 months reported in studies with high-dose rituximab).

In a prospective study, 19 HCV-cryovas patients who had been excluded from antiviral therapy and presented with liver cirrhosis in 15/19 cases were treated with rituximab.27 Consistent improvement in cryovas symptoms was evident at the end of follow-up. Improvement in liver protidosynthetic activity and the degree of ascites was observed at the end of follow-up, especially in more advanced cases.

Rituximab tolerance has been shown to be good in most studies. One potential concern suggested in initial studies was its suspected propensity for worsening HCV viraemia.17 ,18 More recent studies in larger cohorts of HCV-cryovas patients did not confirm these results.28 Some patients may experience systemic drug reactions after a rituximab infusion, particularly those with high MC levels and low C4 levels who received the 1000 mg high-dose rituximab protocol.21 In vitro immunochemical assays showed that rituximab forms a complex with the cryoprecipitating IgM κ, which has rheumatoid factor activity. The in vitro addition of rituximab to serum containing a rheumatoid factor-positive IgM κ type II MC was associated with accelerated cryoprecipitation. Therefore rituximab should be administered cautiously in cryovas patients, with use of the 375 mg/m² protocol combined with plasma exchanges prior to a rituximab infusion in patients with high baseline cryoglobulin levels.

Rituximab plus Peg-IFN-α and ribavirin

Based on the limitations of each therapy (antiviral and rituximab) and the fact that at least 30% of cryovas patients continue to have active disease while receiving rituximab or antiviral therapy, the combination of rituximab with Peg-IFN-α-ribavirin appeared logical. Such combination therapy may target cryoglobulin-producing B cells and the viral trigger.

Sixteen consecutive HCV-cryovas patients were first reported as having received rituximab combined with Peg-IFN-α2b plus ribavirin for 12 months.23 Fifteen patients (93.7%) showed clinical improvement, 10 of whom were complete responders. Clinical improvement was observed after a mean time of 6±4.1 months. HCV RNA and serum cryoglobulin became undetectable in 11 (68.7%) and 10 (62.5%) patients, respectively. Compared with clinical complete responders, the partial or non-responders had a duration of vasculitis that was 3.6 times longer prior to therapy and a lower rate of early virologic response. After a mean follow-up of 19.4±3.6 months, one death occurred due to liver failure. Two patients (12.5%) experienced clinical relapse associated with the simultaneous reappearance of HCV-RNA and cryoglobulin, and an increase in the number of B cells.

Two recent controlled clinical trials compared the efficacy and safety profile of Peg-IFN-α/ribavirin versus rituximab plus Peg-IFN-α/ribavirin in cryovas patients. In both studies, patients treated with rituximab plus Peg-IFN-α/ribavirin had a shorter time to clinical remission, better renal response rates, and higher rates of cryoglobulin clearance than those who had received Peg-IFN-α/ribavirin.29 ,30 Treatment was well tolerated, with no worsening of viraemia on rituximab and 11% discontinuation due to antiviral therapy.

Immunomodulation: low-dose interleukin 2

Patients with HCV-cryovas have been shown to have a reversible quantitative defect of the CD4⁺CD25⁺⁺FoxP3⁺ regulatory T cells (Tregs) after resolution of HCV infection and vasculitis cure. Interleukin 2 (IL-2), a cytokine that promotes Treg survival and function, could be beneficial for patients who are resistant to HCV therapy. The safety and immunological effects of low-dose IL-2 were recently reported in a prospective open-label phase I/IIa study.31 Ten patients with HCV-cryovas that was refractory to conventional antiviral and/or rituximab therapy received one IL-2 course of 1.5 million IU/day for 5 days, followed by three 5-day courses of 3 million IU/day at weeks 3, 6 and 9. There were no drug-related adverse events greater than grade 1. The treatment did not induce effector T cell activation, vasculitis flare or increased viraemia. Improvement of the vasculitis symptoms was found in 8 out of 10 patients. Administration of low-dose IL-2 was followed by an increase in the percentage of CD4⁺CD25^hiCD127^–Foxp3⁺ Tregs, with potent suppressive activity in all subjects and a concomitantly decreased proportion of marginal zone B cells. Transcriptome studies of peripheral blood mononuclear cells revealed that IL-2 induced global attenuation of inflammatory/oxidative stress mediators.

Treatment of HCV-related vasculitis relapse

Recurrent cryovas symptoms after withdrawal of antiviral therapy, together with virological relapse, can be successfully treated with another course of combination antiviral therapy. Careful monitoring for adverse effects is mandatory, since some manifestations of HCV-cryovas, such as peripheral neuropathy or skin ulcers, may worsen with IFN-based therapy. In most cases, however, IFN-based treatment can be reinitiated without further problems. Ribavirin is also reasonably well tolerated, with the exception of haemolytic anaemia, which requires dosage reduction in some patients and dose adjustment in case of renal failure.

The occurrence rate of B cell non-Hodgkin's lymphoma (B-NHL) is higher in cryovas patients compared with the general population, even in patients who cleared HCV RNA following antiviral therapy. The authors in one study reported on eight patients who presented with cryovas due to chronic active HCV infection with no evidence of underlying malignant disease.32 After successful treatment of their HCV infection, the patients persistently remained HCV RNA-negative. cryovas-related symptoms reappeared later, with no HCV infection relapse, but malignant B-NHL was found in two cases.

The safety and efficacy of a combination treatment with fludarabine (40 mg/m² orally on days 2–4), cyclophosphamide (250 mg/m² orally on days 2–4) and rituximab (375 mg/m² on day 1), taken every 4 weeks for 3–6 cycles has been recently reported in patients with severe and refractory cryovas associated with B-NHL. All patients had a clinical response, with three (42.9%) patients achieving a complete remission and four (57.1%) patients reporting a partial remission.33

Prognosis of HCV-related vasculitis

Cryovas is associated with significant morbidity and mortality. The worst prognostic factors in previous studies were age older than 60 years and renal involvement, with renal failure reported as the main cause of death.13 ,34–36 The cumulative 10-years probability of survival for patients with cryovas-related glomerulonephritis was 49%.13 A fourfold increased risk of developing B-NHL has been reported.37 A serum cryoglobulin level higher than 0.6 g/L (OR 1.44) and the presence of cryovas (OR 4.3) and hypogammaglobulinaemia (OR 6.7) were independently associated with B-NHL.

A recent study described the prognosis of patients with HCV-cryovas and identified prognostic survival factors.38 The 1-year, 3-year, 5-year and 10-year survival rates were 96%, 86%, 75%, and 63%, respectively. Deaths were mainly related to serious infections and end-stage liver disease. Baseline factors associated with a poor prognosis were the presence of severe liver fibrosis (HR 5.31), central nervous system involvement (HR 2.74), kidney involvement (HR 1.91) and heart involvement (HR 4.2). Treatment with the combination of Peg-IFN-α plus ribavirin was associated with a good prognosis (HR 0.34), whereas treatment with immunosuppressive agents was associated with a poor outcome, even after adjustment for vasculitis severity (HR 4.05).

Interestingly, a recent study analysed the baseline factors associated with prognosis in patients with HCV-negative cryovas.39 It found results that were quite similar to those in HCV-positive cryovas, as the 1-year, 2-year, 5-year and 10-year overall survival rates were 91%, 89%, 79% and 65%, respectively (figure 1). Deaths were related to serious infections in half the cases and to vasculitis flare in 20%.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

Kaplan–Meier estimate of survival rates in patients with cryoglobulinaemia vasculitis according to the type of cryoglobulin and hepatitis C virus (HCV) status (from Terrier and Cacoub 39).

Therapeutic guidelines

Aggressive optimal antiviral therapy with Peg-IFN-α and ribavirin (plus a protease inhibitor in the case of HCV genotype 1 infection) should be considered as induction therapy for HCV-cryovas patients with mild to moderate disease severity and activity (ie, without rapidly progressive nephritis, motor neuropathy or other life-threatening complications) (figure 2). The duration of therapy has not yet been rigorously determined, but the current treatment duration in HCV-cryovas patients is 48 weeks for all HCV genotypes.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

Therapeutic options in patients with hepatitis C virus (HCV)-induced vasculitis. If failure or contraindication to Peg-INF/ribavirin, rituximab may be used alone. In case of HCV genotype 1 infection, the exact role of the new antiviral combination of Peg-IFN/ribavirin/protease inhibitor still needs to be defined. HCV, hepatitis C virus; Peg-IFN, pegylated interferon-alpha; CNS, central nervous system. Access the article online to view this figure in colour.

In patients presenting with more severe HCV-cryovas disease (ie, worsening of renal function, mononeuritis multiplex, extensive skin disease including ulcers and distal necrosis), an immunosuppression induction phase is often necessary while awaiting the generally slow response to antiviral treatments. The following therapeutic schedule is recommended: (1) weekly administration of four intravenous infusions of rituximab at 375 mg/m² (on days 1, 8, 15 and 22) over a 1-month period and (2) antiviral combination starting after the last rituximab infusion for 12 months.

For patients presenting with the fulminant forms (‘catastrophic HCV-cryovas disease’, including peripheral necrosis of the extremities; rapidly progressive nephritis; digestive, cardiac, pulmonary and/or central nervous system involvement; and/or signs and symptoms of hyperviscosity), apheresis can have immediate beneficial effects but must be combined with immunosuppression. The combination of rituximab, fludarabine and cyclophosphamide appeared to be an effective salvage treatment for refractory cryovas associated with lymphoma.

In patients who failed to respond or have a contraindication to optimal antiviral therapy, rituximab may be used alone. The following therapeutic schedule is therefore recommended: (1) weekly administration of four intravenous infusions of rituximab at 375 mg/m² (on days 1, 8, 15 and 22) over a 1-month period and (2) administration of one infusion of rituximab, every 6–9 months at a dose of 200–500 mg.

Biological treatment with B cell-directed therapy is promising in the treatment of HCV-cryovas, but many questions remain regarding the appropriate role of this strategy in treatment. The duration of effect appears finite, with the response duration typically lasting 6–12 months, and it is necessary to combine it with antiviral drugs. The safety of repeated therapy in HCV-cryovas requires further investigation.