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Rituximab combined with Peg-interferon-ribavirin in refractory hepatitis C virus-associated cryoglobulinaemia vasculitis
  1. D Saadoun1,
  2. M Resche-Rigon2,
  3. D Sene1,
  4. L Perard3,
  5. A Karras4,
  6. P Cacoub1
  1. 1
    Pierre et Marie Curie-Paris 6 University I, CNRS UMR 7087, and Department of Internal Medicine, Hôpital Pitié-Salpétrière, Paris, France
  2. 2
    Department of Biostatistics and Medical Data Processing, INSERM U717, Hôpital Saint-Louis, Paris, France
  3. 3
    Department of Internal Medicine, Hôpital Edouard Herriot, Lyon, France
  4. 4
    Department of Nephrology, Höpital European Georges Pompidou, Paris, France
  1. Professor Patrice Cacoub, Université Pierre et Marie Curie-Paris 6, CNRS, UMR 7087, Paris, F-75013 France; AP-HP, Hôpital Pitié-Salpêtrière, Service de Médecine Interne, Paris, F-75013 France; patrice.cacoub{at}psl.aphp.fr

Abstract

Objectives: To report the results of a pilot study using rituximab combined with Peg-interferon (IFN) α2b-ribavirin in severe refractory hepatitis C virus (HCV) related mixed cryoglobulinaemia (MC) vasculitis.

Methods: Sixteen consecutive patients with severe HCV-MC vasculitis that were resistant (n = 11) or relapser (n = 5) to a previous combination treatment with standard (n = 10) or Peg-IFNα2b (n = 6) plus ribavirin were included. They were treated with rituximab (375 mg/m2 intravenously weekly for 4 weeks) combined with Peg-IFNα2b (1.5 μg/kg per week subcutaneously) plus ribavirin (600–1200 mg/day orally) for 12 months.

Results: Fifteen patients (93.7%) showed clinical improvement, 10 of whom (62.5%) were clinical complete responders (CR). HCV RNA and serum cryoglobulin became undetectable in all the clinical CR. Peripheral blood B cell depletion was achieved in all patients (CD19+ cells, 111 (SD 32)/mm3 at baseline versus 2(2)/mm3 after the fourth infusion of rituximab) with reconstitution starting at the end of antiviral treatment. Compared with clinical CR, the partial or non-responders had a 3.6 times longer duration of vasculitis prior to treatment and a lower rate of early virological response. Treatment was well tolerated with no infectious complications. After a mean follow-up of 19.4 (SD 3.6) months, two patients experienced clinical relapse associated with a simultaneous reappearance of HCV RNA and cryoglobulin and an increase in the number of B cells.

Conclusions: Rituximab combined with Peg-IFNα2b-ribavirin represents a safe and effective treatment option in severe refractory HCV-MC vasculitis.

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Mixed cryoglobulinaemia (MC) is a systemic vasculitis that mainly affects the small vessels and, less frequently, medium-sized vessels.1 MC reflects the expansion of B cells producing a pathogenic IgM with rheumatoid factor (RF) activity.2 3 MC leads to clinical manifestations ranging from the so-called MC syndrome (purpura, arthralgia and asthenia) to more serious lesions with neurological and renal involvement.4 Shortly after the discovery of hepatitis C virus (HCV) in 1989, there was evidence that more than 80% of cryoglobulinaemia vasculitis cases were associated with HCV infection.57

Treatment of HCV-MC with severe organ involvement remains difficult and may target either the viral trigger (HCV) or the downstream B cell arm of autoimmunity.8 9 Inducing a sustained virological and clinical response and minimising the use of immunosuppressive drugs are the main goals in the treatment of patients with HCV-MC vasculitis. Antiviral treatment has been shown to reverse bone marrow monoclonal B cell expansion in patients with HCV-MC.10 Patients treated with Peg-IFNα/ribavirin have achieved sustained clinical and virological responses in up to 60% of cases.11 Cryoglobulin clearance was also observed in half of patients. Although this approach affords a satisfactory response rate, additional treatment may be needed in patients with MC with severe organ involvement and/or without an early virological response.11 12

Corticosteroids in combination with cytotoxic agents may be useful initially for the control of life-threatening organ involvement while awaiting the generally slow response to antiviral treatments. More recently, data on the efficacy of anti-CD20 monoclonal antibody (rituximab) have been reported in patients with HCV-MC vasculitis.1320 It appears that rituximab is very efficacious against cryoglobulin production and its clinical consequences, ie, inflammatory vascular lesions. A complete clinical response is achieved in 60–70% of cases,1320 with cryoglobulin clearance in one-third of patients. The absence of efficacy on HCV viral clearance and, furthermore, the potential increase in HCV viral load stress the need for combined antiviral treatment to block the HCV infection trigger. Indeed, up to 30% of patients treated with rituximab experience vasculitis relapse during B cell recovery.1321

Based on the limitations of each treatment (ie, antiviral and rituximab), and the 30% of patients with MC that continue to have active disease while receiving anti-CD20 monoclonal antibody or antiviral treatment, the combination of rituximab with Peg-IFNα-ribavirin appear logical. We present here the results of a pilot study using rituximab combined with Peg-IFNα2b-ribavirin in 16 patients with HCV-MC who were consecutive, unselected and who were resistant to a previous antiviral combination treatment with standard or Peg-IFNα2b plus ribavirin.

PATIENTS AND METHODS

Patients

Sixteen consecutive, unselected, patients with HCV-MC vasculitis who were resistant (n = 11) or relapser (n = 5) to a previous combination antiviral treatment with standard (n = 10) or Peg-IFNα2b (n = 6) plus ribavirin seen between 2003 and 2006 were included. Overall, in our MC cohort, 30–35% of patients are resistant to combination treatment, 5–10% are relapsers and less than 5% are intolerant to antiviral treatment. Informed consent was obtained for each patient and the study conformed to the ethical guidelines of the Declaration of Helsinki (1975). Patients had a serum cryoglobulin >0.05 g/l on at least two occasions, which was associated with purpura, and arthralgia with renal and/or neurological involvement. All patients were positive for HCV RNA.

Inclusion criteria for the study were as follows: (a) chronic active HCV infection; (b) severe organ involvement of MC vasculitis (ie, renal and/or neurological involvement); (c) non-response (n = 11) or relapse (n = 5) to a previous combination antiviral treatment with standard (n = 10) or Peg-IFNα2b (n = 6) plus ribavirin for a minimum of 6 months; (d) a minimum of 6 months of follow-up after stopping anti-HCV treatment.

Exclusion criteria were the presence of hepatitis B surface antigen or anti-human immunodeficiency virus (HIV) antibodies. All 16 patients had histologically confirmed systemic vasculitis (nerve (n = 8), kidney (n = 6) and skin (n = 2)). None of the patients had received previous treatment with either cyclophosphamide or rituximab.

Previous combination antiviral treatment included standard IFNα-ribavirin (n = 10) and Peg-IFNα-ribavirin (n = 6). The treatment schedule consisted of the weekly administration of four intravenous infusions of rituximab at 375 mg/m2 (on days +1, +8, +15, +22) over a period of 1 month; this was followed 1 month later by an antiviral combination with Peg-IFNα2b (1.5 μg/kg per week subcutaneously) plus ribavirin (600–1200 mg/day orally) for 12 months. Premedication with methylprednisolone 40 mg intravenously was given before each infusion of rituximab. Rituximab and antiviral treatment were not started at the same time to avoid cumulative side effects. For each patient, clinical and biological data were recorded at the time of the initial evaluation, at 3 months, 6 months, 9 months, 12 months and at the end of follow-up. The diagnosis of non-Hodgkin’s lymphoma was based on World Health Organization (WHO) criteria.22

Immunological and virological markers

Cryoglobulins were measured and classified as previously described.23 24 Glomerular filtration rate was determined as previously described by Cockcroft and Gault.25 A 24 h urine collection was also performed in order to quantify daily protein excretion.

Flow cytometry analysis of the CD19+ B cell marker was evaluated in peripheral blood mononuclear cells at baseline, and then monthly in all the patients during a 12-month period.

Serum HCV RNA was measured by a reverse transcription–polymerase chain reaction assay with a threshold of detection of 2.7 log copies/ml. Liver biopsy specimens were evaluated according to the previously validated Metavir scoring system.26

Treatment efficacy

The response to treatment was defined as previously described11 by comparing clinical, immunological and virological parameters at the initial evaluation, at 3 months, 6 months, 9 months, 12 months, and at the end of follow-up. Clinical response was defined by analysing the progression of the following main clinical signs: skin involvement (absence of purpura and/or leg ulcer), peripheral neuropathy (clinical and electrophysiological improvement on two successive examinations), renal involvement (normalisation of serum creatinine level and disappearance of proteinuria and/or haematuria), and the absence of arthralgia.

A complete clinical response of MC vasculitis (CR) was defined by an improvement in all baseline clinical manifestations. A partial response (PR) was defined by an improvement of at least half of the baseline clinical manifestations. All other patients were classified as non-responders (NR).

A sustained virological response was defined by the absence of detectable serum HCV RNA 6 months after the end of antiviral treatment; the remaining patients were classified as virological non-responders.

A complete immunological response was defined by the absence of serum cryoglobulin, and a partial immunological response by a >50% decrease in the baseline cryoglobulin level.

Clinical relapse was defined as the reappearance of clinical signs of vasculitis, virological relapse as the reappearance of detectable HCV RNA and immunological relapse as the reappearance of serum cryoglobulin. The response of lymphoma was defined by physical examinations (presence of lymphadenopathy and/or splenomegaly) and by analysing peripheral blood and computed tomography scans. Complete remission of lymphoma was defined as the total disappearance of the lymphomatous mass, and a partial remission as a 75% reduction.

Statistical analysis

Data are expressed as the mean (SD) and median (ranges) for comparison between CR and PR/NR. Comparisons between baseline and end of follow-up values were tested using MacNemar’s test or Wilcoxon’s paired test. Associations between variables and clinical response were tested using Fisher’s exact test or Wilcoxon’s exact test as applicable. All tests were two-sided at a 0.05 significance level. Analyses were performed using the R statistical package (online at http://www.R-project.org).

RESULTS

Characteristics of the patients with hepatitis C virus-associated cryoglobulinaemia vasculitis

Patient characteristics are detailed in table 1. Sixteen HCV-MC patients (three men and 13 women) with a mean age of 58 (10.5; range 40–78 years) were included. The mean duration of HCV-MC vasculitis was 50.2 (57.6; range 10–192 months). Clinical features included purpura and polyneuropathy in 13 patients (81.2%), renal involvement in seven (43.7%), arthralgia in six (37.5%) and leg ulcers in two (12.5%). One patient (6.2%) had haemolytic autoimmune anaemia, and three had B cell non-Hodgkin’s lymphoma (B-NHL) (lymphocytic (n = 2) and marginal zone lymphoma (n = 1)).

Table 1 Characteristics of the 16 HCV-MC vasculitis patients at baseline and at the end of follow-up*

All patients had MC, with a mean cryoglobulin level of 1.1 g/l (range 0.1–2.9). Fourteen (87.5%) patients had a type II cryoglobulin with monoclonal IgMκ in all cases, and two (12.5%) patients had type III. C4 and CH50 serum levels were low in 87.5% of patients. RF activity was observed in 11 (68.7%) patients.

The distribution of HCV genotypes was genotype 1 (n = 10), genotype 2 (n = 2), genotype 3 (n = 3) and genotype 5 (n = 1). Eleven patients (68.7%) had elevated serum alanine aminotransferase (ALT) levels and the mean ALT concentration was 1.8-fold the upper limit of normal value. The mean HCV RNA level was 5.8 (0.6) log (range 4.3–6.7). Four patients (25%) had cirrhosis.

Treatment-related data

The main treatment-related data are summarised in tables 1 and fig 14.

Figure 1 Complete response rate of hepatitis C virus-associated cryoglobulinaemia vasculitis with rituximab combined with Peg-interferon-α2b-ribavirin. Arrows indicate the number of weekly rituximab infusions and the duration of antiviral treatment.
Figure 2 Dynamics of CD19+ B cell depletion and recovery during treatment with rituximab combined with Peg-interferon-α2b-ribavirin in patients with hepatitis C virus-associated cryoglobulinaemia vasculitis. (A) In patients with complete clinical response (n = 10) (circles), and (B) in the partial and non-responders (n = 6) (squares).
Figure 3 Course of hepatitis C virus RNA viral load during treatment with rituximab combined with Peg-interferon-α2b-ribavirin in patients with hepatitis C virus-associated cryoglobulinaemia vasculitis. (A) In patients with complete clinical response (n = 10) (circles), and (B) in the partial and non-responders (n = 6) (squares). Dashed line indicates the threshold of detection of hepatitis C virus RNA (2.7 log copies/ml). Arrows indicate the number of weekly rituximab infusions and the duration of antiviral treatment. EOF, end of follow-up.
Figure 4 Immunological parameters in patients with hepatitis C virus-associated cryoglobulinaemia vasculitis during treatment with rituximab combined with Peg-interferon-α2b-ribavirin. RF, rheumatoid factor; C4, C4 complement; EOF, end of follow-up.

Ten patients (62.5%) were complete clinical responders (CR) (fig 1), five (31.2%) were partial responders (PR) and one (6.2%) was a non-responder (NR). Clinical improvement was observed after a mean time of 6 (4.1) months. Leg ulcers resolved in 100% of patients, purpura improved in 84.6%, arthralgia in 83.4%, renal involvement in 57.2% and polyneuropathy in 38.4% (table 1). Haematuria and proteinuria disappeared in seven of seven (100%) and five of seven (71.4%) patients respectively. The glomerular filtration rate improved in four of six (66.6%) patients with renal insufficiency (table 1). The CR of haemolytic autoimmune anaemia was noted. CR was achieved in two patients with a stage III lymphocytic lymphoma and PR was observed in the remainder with a stage IV marginal zone lymphoma. The median time to haematological response was 3 months.

B cell depletion in the peripheral blood was achieved in all patients (fig 2). The mean number of CD19+ cells at baseline (111 (32)/mm3) dropped to 2 (2)/mm3 after the fourth infusion of rituximab. Recovery of the B cell count began after 6 months. There was a delayed B cell reconstitution that actually started at the end of antiviral treatment (35 (9)/mm3 at month +12, and 68 (20)/mm3 at month +15) (fig 2).

A slight non-significant increase in the HCV viral load from 5.8 (0.6) to 6.0 (0.4) log copies/ml was observed after the fourth infusion of rituximab (ie, before the institution of Peg-IFNα2b-ribavirin) (fig 3). The association of Peg-IFNα2b-ribavirin induced a significant decrease in the HCV viral load from 6.0 (0.4) to 3.2 (1.1) log copies/ml at the end of treatment and to 4.2 (1.4) log copies/ml at the end of follow-up, with HCV RNA being undetectable in 11 (68.7%) patients. Alanine aminotransferase levels decreased from 1.8 to 1.3 the upper limit of normal value and normalised in seven of 11 (63.6%) patients (table 1).

Serum cryoglobulin disappeared in 10 (62.5%) patients and decreased by more than 50% from the baseline level in five (31.2%). The cryoglobulin level decreased significantly from 1.1 to 0.2 g/l at the end of follow-up. The C4 complement level increased from 0.06 to 0.12 g/l and normalised in eight of 14 (57.1%) patients. RF and IgM levels decreased from 173.3 to 42.6 IU/l and from 1.8 to 1.2 g/l respectively. A significant reduction in the serum levels of cryoglobulins (p = 0.01) and RF (p = 0.01) was noticed after treatment in conjunction with a significant increase in the serum levels of C4 (p = 0.009) (fig 4). A trend toward a decrease in the IgM level was observed after treatment (p = 0.21).

The mean follow-up was 19.4 (3.6) months with at least 6 months of follow-up after cessation of antiviral treatment in all the patients. One patient with cirrhosis died from liver failure. Two patients with clinical CR experienced a relapse, with the median estimated time to relapse being 2.5 months after discontinuing antiviral treatment. Clinically, relapse was characterised by the reappearance of purpura (n = 2) and peripheral neuropathy (n = 2) and the recurrence of arthralgia (n = 1). The simultaneous reappearance of detectable HCV RNA and the increment of cryoglobulin and B cell number were also demonstrated in these two patients.

Treatment was well tolerated in 14 of 16 patients. In the two remaining patients, side effects included worsening of peripheral neuropathy (n = 1) and flare-up of skin psoriasis (n = 1), both linked to Peg-IFNα treatment. Antiviral treatment interruption was required in these two patients at months +4 and +8 respectively. Minor side effects were noted in 12 (75%) patients and included anaemia (n = 8), fatigue (n = 3), fever (n = 2), mild depression (n = 2) and thrombocytopenia (n = 1).

Factors associated with complete clinical response

Table 2 summarises the comparison between the complete clinical responders (CR) and partial (PR) or non-responders (NR). At the end of treatment, 10 patients (62.5%) were CR and six (37.5%) were PR (n = 5) or NR (n = 1). Clinical CR had a lower vasculitis duration and a lower HCV viral load at month +3 (25.9 (33.9); median (range), 12.5 (10–120)) vs 90.8 (68.8) (102.0 (12–192)) months and 2.9 (0.4); median (range), 2.7 (2.7–3.7)) vs 5.3 (1.6) (5.9 (2.7–7.1)) log copies/ml, respectively, p<0.05) compared with PR or NR. Negative HCV RNA at month +3 was achieved in eight of 10 (80%) CR compared with only two of six (33.3%) PR or NR. No significant differences between CR and PR or NR were found for baseline clinical signs, virological parameters, serum levels of cryoglobulin, C4, RF and IgM or the number of CD19+ B cells.

Table 2 Comparison between characteristics of clinical responders and partial or non-responders*

DISCUSSION

In this pilot study, we reported on the tolerance and efficacy of rituximab combined with Peg-IFNα2b-ribavirin in 16 consecutive patients with HCV-MC who were resistant (n = 11) or relapser (n = 5) to a previous combination antiviral treatment with standard (n = 10) or Peg-IFNα2b (n = 6) plus ribavirin. The treatment schedule was borrowed from earlier open studies in patients with HCV-MC in which rituximab was infused weekly13 27 and Peg-IFNα2b plus ribavirin were given for 12 months.11

The present study is the first to show the results of rituximab combined with Peg-IFN-ribavirin in a large series of patients with HCV-MC. Fifteen patients (93.7%) showed clinical improvement, 10 (62.5%) of whom were complete responders. HCV RNA and cryoglobulin became undetectable in all clinical CR. This combination treatment was well tolerated in 14 of 16 patients, with only minor side effects. In the two remaining patients, the flare-up of skin psoriasis and worsening of polyneuropathy were attributable to Peg-IFNα2b. Taken together these data suggest that rituximab combined with Peg-IFNα-ribavirin may act synergistically and represents a good treatment strategy in patients with HCV-MC with severe organ involvement.

Rituximab is an interesting treatment in patients with HCV-MC as it targets B cells that are responsible for the cryoglobulin production, immune complex deposition and, finally, MC vasculitis lesions. With the use of rituximab as monotherapy, a clinical response was achieved in two-thirds of patients, but relapse developed in nearly 40% of cases after a mean delay of 7 months.1321 However, most of the reported patients were previously treated with IFNα monotherapy and/or corticosteroids and as such should not be considered as refractory HCV-MC. In addition, as shown in our study, one potential concern regarding the use of such treatment is its propensity to worsen HCV viraemia.13 28 29 In this setting, rituximab cannot be seen as a curative treatment as long as the viral starter antigen of the vasculitis (ie, HCV) remains. Indeed, despite dramatic reduction in the number of circulating B cells and deletion of B cell clones, Sansonno et al observed the appearance of different clones in patients with MC who were responders to rituximab; this demonstrated that selected antigens may be recognised as part of a limited host response to a virus capable of undergoing spontaneous long-term mutations.13 In the present study, although an initial increase in HCV viral load was evidenced after rituximab infusions, a significant number of patients achieved eradication of HCV RNA and normalisation of serum transaminases after completion of Peg-IFNα-ribavirin. These results are consistent with the case report by Lamprecht et al14 showing that rituximab initiated remission in refractory HCV-MC vasculitis and underlying lymphoproliferative disease; HCV RNA was subsequently eliminated by antiviral treatment.

The present combination of rituximab and Peg-IFNα2b-ribavirin proved to be very effective on skin vasculitis manifestations (purpura and leg ulcers) and articular involvement. Peripheral neuropathy and nephropathy remain the most challenging clinical complications to treat in patients with HCV-MC and were present in 13 and seven cases, respectively. Indeed, complete remission of peripheral neuropathy and nephropathy were achieved in 38.4% and 57.2% of cases, respectively. The B cell clones producing MC were effectively targeted by the present combination treatment. A significant decrease in mean serum cryoglobulin and RF and IgM levels was observed. The disappearance of cryoglobulin and the normalisation of the C4 complement level were achieved in up to 50% of cases. Peripheral blood B cell depletion was rapidly achieved and persisted for up to 6 months in all patients. In contrast with previous studies using rituximab as the sole treatment,13 18 we observed a delayed B cell reconstitution that actually started at the end of antiviral treatment (at month +12), without infectious complications. A relapse occurred 2 months after stopping antiviral treatment in two patients and was associated with the simultaneous reappearance of HCV RNA and cryoglobulin and an increase in the number of peripheral blood B cells.

The comparison between clinical complete responders and partial or non-responders revealed that the MC disease duration was a significant predictive factor. The duration of MC vasculitis before treatment in the partial or non-responders was 3.6 times longer than the complete clinical responders. This suggests that this treatment schedule should be considered as the initial treatment for patients with HCV-MC, at least in those with severe organ involvement. Consistent with a previous report in patients with HCV-MC treated with antiviral treatment,11 an early virological response (ie, 3 months) to Peg-IFNα2b-ribavirin was another factor associated with a complete clinical response. This result underscores the trigger role of HCV in MC vasculitis and the need for an antiviral treatment in such patients.

In conclusion, rituximab combined with Peg-IFNα2b-ribavirin is well tolerated, and affords a satisfactory response rate in severe and refractory HCV-MC vasculitis. A randomised trial with rituximab plus Peg-IFNα-ribavirin versus Peg-IFNα-ribavirin in patients with HCV-MC is warranted.

Acknowledgments

We wish to thank P. Ghillani-Dalbin for help with the immunological data.

REFERENCES

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Footnotes

  • Competing interests: None.

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