Relapse of hepatitis C virus–associated mixed cryoglobulinemia vasculitis in patients with sustained viral response

Authors

  • Dan-Avi Landau,

    1. Université Pierre et Marie Curie-Paris 6, CNRS, UMR 7087, Paris, and Assistance Publique Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Paris, France
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  • David Saadoun,

    1. Université Pierre et Marie Curie-Paris 6, CNRS, UMR 7087, Paris, and Assistance Publique Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Paris, France
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  • Philippe Halfon,

    1. Laboratoire Alphabio, Marseilles, France
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  • Michelle Martinot-Peignoux,

    1. INSERM U773/CRB3, Hôpital Beaujon, Clichy, France
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  • Patrick Marcellin,

    1. INSERM U773/CRB3, Hôpital Beaujon, Clichy, France
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    • Dr. Marcellin has received speaking fees (less than $10,000 each) from Roche, Schering-Plough, Gilead Sciences, Bristol-Myers Squibb, GlaxoSmithKline, and Indenix/Novartis, has served as an investigator and expert witness for Roche, Schering-Plough, Gilead Sciences, Bristol-Myers Squibb, GlaxoSmithKline, Vertex, Idenix/Novartis, Valeant Pharmaceuticals, Human Genome Sciences, Cytheris, InterMune, and Wyeth, and has served as an expert witness for Coley Pharma. Dr. Cacoub has received consulting fees, speaking fees, and/or honoraria (less than $10,000 each) from Servier, Schering-Plough, Sanofi, and Bristol-Myers Squibb.

  • Elena Fois,

    1. Hôpital Cochin, Paris, France
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  • Patrice Cacoub

    Corresponding author
    1. Université Pierre et Marie Curie-Paris 6, CNRS, UMR 7087, Paris, and Assistance Publique Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Paris, France
    • Service de Médecine Interne, Groupe Hospitalier La Pitié-Salpêtrière, 47-83 Boulevard de l'Hôpital, 75013 Paris, France
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    • Dr. Marcellin has received speaking fees (less than $10,000 each) from Roche, Schering-Plough, Gilead Sciences, Bristol-Myers Squibb, GlaxoSmithKline, and Indenix/Novartis, has served as an investigator and expert witness for Roche, Schering-Plough, Gilead Sciences, Bristol-Myers Squibb, GlaxoSmithKline, Vertex, Idenix/Novartis, Valeant Pharmaceuticals, Human Genome Sciences, Cytheris, InterMune, and Wyeth, and has served as an expert witness for Coley Pharma. Dr. Cacoub has received consulting fees, speaking fees, and/or honoraria (less than $10,000 each) from Servier, Schering-Plough, Sanofi, and Bristol-Myers Squibb.


Abstract

Objective

To investigate the clinical characteristics, outcomes, and results of hepatitis C virus (HCV) RNA analyses in a group of patients with HCV-associated mixed cryoglobulinemia (MC) vasculitis who experienced a relapse of vasculitis despite achieving a sustained viral response to treatment with antiviral agents.

Methods

HCV RNA testing was performed by the transcription-mediated amplification (TMA) method in sera and cryoprecipitates (detection limit 2.5 IU/ml). HCV replication was assessed in peripheral blood mononuclear cells (PBMCs) by a modified real-time polymerase chain reaction assay (detection limit 15 IU/106 cells).

Results

We identified 8 patients with relapse of HCV-MC vasculitis despite their having achieved a sustained viral response to treatment. Relapse appeared early after the end of treatment (mean ± SD 2.5 ± 3.5 months) and included mainly purpura (n = 7) and arthralgia (n = 5). Relapse was associated with an increase in serum cryoglobulin levels as compared with end-of-treatment levels (mean ± SD 0.3 ± 0.09 gm/liter and 0.08 ± 0.04 gm/liter, respectively; P < 0.01) and a decrease in C4 levels. In most patients, the relapse was brief, and the MC vasculitis manifestations subsided. A search for HCV RNA by TMA was negative in all patients tested (7 of 8 patients), both in sera and in cryoprecipitates. HCV replication was not found in PBMCs from any of the patients tested (6 of 8 patients). In 3 patients, the MC vasculitis symptoms persisted and were associated with elevated cryoglobulin levels. B cell lymphoma was diagnosed in 2 of these 3 patients.

Conclusion

Relapse of MC vasculitis does occur in a few patients with HCV infection, despite achieving a sustained viral response, and this relapse is not related to persistence of virus. Relapse is short-lived and may be induced by the withdrawal of interferon alfa therapy. However, in patients with persistent MC vasculitis symptoms, a different underlying condition should be considered, especially B cell lymphoma.

Chronic infection with hepatitis C virus (HCV) is the principal cause of mixed cryoglobulinemia (MC) vasculitis, a crippling and potentially life-threatening systemic vasculitis that may involve the skin, musculoskeletal system, kidneys, and nervous system (1). The most common type of cryoglobulinemia in patients with HCV is type II MC, which is characterized by the presence of monoclonal immunoglobulins.

Prior to the association of MC with HCV, treatment generally consisted of high-dose glucocorticoids, cytotoxic agents, and plasmapheresis. While effective, this therapy is associated with significant adverse effects and is often only transiently beneficial (2, 3). Antiviral treatment with interferon alfa and ribavirin has offered a new strategy for HCV-associated cryoglobulinemia and has been demonstrated to be successful at achieving remission for HCV-related MC vasculitis (4–6). We recently demonstrated that treatment with PEGylated interferon plus ribavirin is associated with a higher rate of complete clinical response (67.5% versus 56.2%) and viral response (62.5% versus 53.1%) compared with standard interferon alfa plus ribavirin therapy (7).

In most reports of the successful treatment of HCV-associated cryoglobulinemia vasculitis, viral response and clinical remission of vasculitis are closely related (4, 7, 8). Thus, patients who either do not achieve an antiviral effect or experience a viral relapse will often have an incomplete or transient remission of vasculitis symptoms (7, 9–11). We recently observed a subset of patients who had a relapse of HCV-associated MC vasculitis manifestations despite a sustained viral response and repeatedly negative test results for HCV by polymerase chain reaction (PCR) analysis. The aim of this study was to describe the clinical characteristics and outcomes of these patients as well as the results of HCV RNA testing of sera and cryoprecipitates using an ultrasensitive method (12), transcription-mediated amplification (TMA), and of peripheral blood mononuclear cells (PBMCs) using a modified reverse transcription–PCR (RT-PCR) assay.

PATIENTS AND METHODS

Patient population.

Data for this observational study were collected retrospectively on patients with HCV-associated MC vasculitis who were evaluated at the Department of Internal Medicine, Hôpital La Pitié-Salpêtrière, between 1999 and 2006. The patients had pretreatment serum cryoglobulin levels >0.05 gm/liter on at least 2 occasions, which was associated with purpura, arthralgia (13), and sometimes, with renal or neurologic involvement. All patients were positive for HCV RNA, and all had histologically proven chronic active liver disease. In addition to these baseline characteristics, inclusion criteria for the study were as follows: 1) previous treatment with PEG–interferon alfa-2b plus ribavirin or interferon alfa-2b plus ribavirin for a minimum of 6 months; 2) at least 2 negative serum HCV RNA results 6 months after the end of antiviral therapy; and 3) reappearance of signs of MC vasculitis after the end of antiviral therapy. Exclusion criteria were the presence of either hepatitis B surface antigen or anti–human immunodeficiency virus antibodies.

Clinical and laboratory assessments.

The clinical evaluation included age, sex, recent weight loss, neurologic (peripheral and/or central nervous system) involvement (impaired cognitive function and abnormal findings on magnetic resonance imaging of the brain), cutaneous involvement (Raynaud's phenomenon, purpura, ulcers of the distal extremities), arthralgia, myalgia, sicca syndrome, gastrointestinal tract involvement (mesenteric microaneurysms and/or histologically confirmed vasculitis), renal involvement (proteinuria and/or a glomerular filtration rate <70 ml/minute), and clinical signs of hepatic involvement.

Laboratory evaluations included a complete blood cell count with differential cell count, serum chemistry profile, rheumatoid factor analysis, levels of the C4 fraction of complement, and cryoglobulin levels. A 24-hour urine collection was also obtained in order to quantify daily levels of protein excretion. Serum HCV RNA was measured by RT-PCR assay (detection level <12 IU/ml; Abbott Laboratory, Rungis, France). A sustained viral response was defined as the absence of serum HCV RNA 6 months after stopping treatment with antiviral agents. HCV genotyping was performed using a second-generation line probe assay (LiPA; Innogenetics, Brussels, Belgium). Liver biopsy specimens were evaluated according to the previously validated Metavir scoring system (14). Cryoglobulins were measured as previously described (15). The diagnosis of non-Hodgkin's lymphoma (NHL) was based on World Health Organization (WHO) criteria (16).

TMA analysis of HCV RNA in sera and cryoprecipitates.

Thawed serum and cryoprecipitate samples from 7 of the 8 patients were tested with the Bayer Versant HCV RNA Qualitative (TMA) Assay (Bayer Diagnostics, Berkeley, CA) according to the manufacturer's instructions for serum samples. An aliquot of 100 μl of cryoprecipitate was diluted in 400 μl of negative serum sample before testing, according to the manufacturer's instructions, without normalization of the cryoglobulin concentrations to this volume between samples. The TMA assay is confirmed by the amplification of an internal control present for each specimen tested. In addition, a positive control, consisting of a cryoprecipitate obtained from a patient with cryoglobulinemia and detectable serum HCV RNA, was included in each run and was tested under the same conditions (100 μl of cryoprecipitate plus 400 μl of negative serum). All stages of testing by this assay (sample preparation, target amplification, and amplicon detection) were performed within a single tube.

Briefly, the capture probe was hybridized to the 5′-untranslated region of the HCV genome, and the complex was captured onto a magnetic microparticle. TMA was performed using Moloney leukemia virus reverse transcriptase and T7 RNA polymerase under isothermal conditions. Hybridization of the amplicons to 2 differentially modified acridinium ester molecules attached to different probes allowed for the simultaneous detection of internal control and HCV RNA targets in the same tube. Chemiluminescence (in relative light units) was measured after oxidation and hydrolysis. Each test result was considered valid if the internal control result was reactive for that sample. The detection level of this assay is considered to be 10 IU/ml. In our laboratory, the sensitivity of the assay assessed with the WHO standard is 2.5 IU/ml. Our in-house experience with this method has shown increased sensitivity. In 81 fresh serum samples obtained from patients during treatment, TMA positivity rates were highest, followed by those for the Cobas TaqMan assay (Roche Molecular Systems, Branchburg, NJ) and those for the RT-PCR assay (Abbott), with positive results in 26%, 11%, and 4% of the patients, respectively. These findings are also supported by published data (17, 18).

Detection of HCV RNA in PBMCs.

HCV replication was assessed in PBMCs using a modified Cobas TaqMan HCV assay with a detection limit of 15 IU/106 cells. The RNA extraction step was performed using silica beads (NucliSens; Organon Teknika, Fresnes, France). One million PBMCs prepared in a BD Vacutainer CPT cell preparation tube (BD Diagnostics, Le Pont de Claix, France) and 4.1 μl of the internal control from the Cobas TaqMan HCV assay were mixed in a lysis tube (9 ml). After centrifugation at 1,500g for 2 minutes, 50 μl of silica was added. After incubation for 10 minutes at room temperature, tubes were centrifuged at 1,500g for 2 minutes. The supernatant was eliminated, and 5 washing steps were performed: 2 with 1 ml of washing buffer, 2 with 1 ml of 70% ethanol, and the last with 1 ml of acetone. Cells were centrifuged at 10,000g for 30 seconds during the washing steps. After acetone elimination, the pellet was dried for 10 minutes at 56°C.

To each cell pellet, we added 50 μl of elution buffer and incubated the tubes for 10 minutes at 56°C and then centrifuged at 10,000g for 2 minutes. We then removed 30–35 μl of elute that had been diluted with specimen Cobas TaqMan HCV assay kit diluent to obtain 75 μl of sample and placed it in a new tube. Real-time PCR was then performed by Cobas TaqMan HCV assay according to the instructions of the manufacturer.

The Cobas TaqMan HCV assay is a real-time nucleic acid amplification assay for the quantitative detection of HCV RNA in human serum or plasma. Like the TaqMan HCV analyte-specific reagent (Roche Molecular Systems), this assay was developed for use with the recently introduced Cobas TaqMan 48 Analyzer (CTM 48; Roche Molecular Systems). Amplification and detection were performed according to the manufacturer's instructions for the TaqMan HCV with the CTM 48 with AmliLink software version 3.0.1 (Roche Diagnostics, Meylan, France). The sensitivity of our method was validated by detection of HCV-infected sera diluted in negative PBMCs. HCV genotype 1 serum quantified at 1,500 IU/ml was diluted by a factor of 100, and 1 ml of diluted serum was added to 1 million PBMCs prepared as described above. The mixture of PBMCs and HCV genotype 1 sera was then quantified with the Cobas TaqMan instrument. The detection limit was 15 IU/106 cells.

Statistical analysis.

Quantitative variables were expressed as the mean ± SD. Comparisons of cryoglobulin and C4 levels at different time points were performed with the paired t-test analysis. All tests were 2-sided at the 0.05 significance level. Analyses were performed using GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA).

RESULTS

Baseline characteristics.

During the study period, 8 patients (6 women and 2 men) were identified who had a relapse of MC vasculitis despite having achieved a sustained viral response, according to the inclusion criteria. Their mean ± SD age at study entry was 60.2 ± 13.7 years (range 43–75 years) (Table 1). Before treatment, MC vasculitis symptoms in this group of patients included arthralgia (n = 5), purpura (n = 7), peripheral neuropathy (n = 6), and nephropathy (n = 1). Neuromuscular biopsy specimens from 3 of the patients showed severe axonal degeneration and an inflammatory process involving the nerves. Renal biopsy specimens showed membranoproliferative glomerulonephritis in 1 of the patients. The remaining patients without histologic confirmation of systemic vasculitis presented with typical signs of “essential” MC vasculitis (i.e., arthralgia, asthenia, and purpura of the lower extremities [13]).

Table 1. Pretreatment characteristics of the patients with HCV-associated mixed cryoglobulinemia vasculitis*
Patient/age/sexHCV genotypeCryoglobulinsArthritis/arthralgiaNeuropathyPurpuraNephropathyAntiviral treatment
TypeLevel, gm/literAgentDuration, months
  • *

    HCV = hepatitis C virus; BiPEG = PEGylated interferon alfa plus ribavirin; Bi = interferon alfa plus ribavirin; NA = not available.

A/73/F1III1.43+++BiPEG15
B/65/F5II IgMκ1.63+++BiPEG25
C/43/F1II IgMκ1.14+++BiPEG20
D/62/F2II IgMκ0.32++BiPEG12
E/66/F1II IgGκ0.6++BiPEG14
F/75/M4II IgMκ0.15++Bi24
G/43/M1II IgMκ1.35++BiPEG12
H/73/FNANAPositive++BiPEG12

Patients were treated with interferon in combination with ribavirin (n = 1) or with PEGylated interferon in combination with ribavirin (n = 7) for a period of 12–25 months (mean ± SD 18 ± 7 months). All patients experienced complete remission of MC vasculitis manifestations during treatment with antiviral agents, with repeatedly negative serum HCV RNA results by RT-PCR, as well as a biochemical response manifested by the normalization of liver function test (LFT) results.

Relapse characteristics.

Relapse of MC vasculitis manifestations occurred in most patients shortly after antiviral treatment was discontinued (mean ± SD 2.5 ± 3.5 months) (Table 2). The most common manifestation was purpura (n = 6), followed by arthritis and myalgia/asthenia. Neuropathy was found in 2 patients and nephropathy in 1. MC vasculitis relapse was associated with elevated cryoglobulin levels as compared with those at the end of treatment (mean ± SD 0.3 ± 0.09 gm/liter and 0.08 ± 0.04 gm/liter, respectively; P < 0.01), and a decrease in C4 levels (mean ± SD 0.09 ± 0.02 gm/liter and 0.12 ± 0.04 gm/liter, respectively; P not significant) (Figure 1). In contrast, the biochemical response (normalization of findings on LFTs) persisted throughout the relapse period.

Table 2. Relapse and outcome characteristics of the study patients*
PatientRelapse after antiviral treatment1 year after end of antiviral treatment
Arthritis/arthralgiaPurpuraNeuropathyNephropathyNHLTreatment for relapseSymptomsCryoglobulins
  • *

    NHL = non-Hodgkin's lymphoma; HCQ = hydroxychloroquine; NSAIDs = nonsteroidal antiinflammatory drugs; CYC = cyclophosphamide.

A++Low-dose steroidsPersistent mild symptomsLow levels
B++NoneComplete remissionNone
C++ColchicinePersistent symptomsPersistent
D+HCQ, NSAIDsMild arthralgiaNone
E+++Low-dose steroids, HCQMild arthralgiaNone
F+++Fludarabine, CYCComplete remissionNone
G+NoneComplete remissionNone
H+++Chlorambucil, steroidsDied of lymphoma complicationsPersistent
Figure 1.

Levels of A, cryoglobulins and B, C4 before treatment with antiviral agents, at the end of treatment, at the time of relapse, and 12 months after the end of treatment in 8 patients with hepatitis C virus–associated mixed cryoglobulinemia vasculitis. Values are the mean ± SD. P values were determined by paired t-test (2-sided).

Initially, relapse of MC vasculitis symptoms was presumed to stem from relapse of HCV infection. Therefore, serum tests for HCV RNA by RT-PCR analysis were repeatedly performed (mean of 5.7 tests [range 4–8] over a mean followup period of 2.4 years [range 1.5–5 years]), and the results were found to be negative in all patients at all time points tested.

Faced with the repeated negativity of HCV-RNA by RT-PCR analysis, a search for other causes of vasculitis was conducted. A clinical and laboratory evaluation for connective tissue disorders (including tests for antinuclear antibodies [ANAs], anti–double-stranded DNA antibodies, antinuclear cytoplasmic antibodies, and anti–cyclic citrullinated protein antibodies) was performed, and results were negative in all but 1 patient, who had isolated low titers of ANAs. We also searched for B cell lymphoproliferation as a possible cause of the persistent cryoglobulinemia. B cell lymphoproliferation was identified in 2 patients who had bone marrow involvement. In these 2 patients, a newly identified B cell lymphoproliferation (both with lymphoplasmacytic lymphoma) was the presumed cause of the MC vasculitis relapse.

HCV RNA negativity was confirmed in both serum and cryoprecipitate samples from 7 of the 8 patients with an ultrasensitive test (i.e., TMA). The absence of HCV RNA replication in PBMCs from 6 of the 8 patients was confirmed with the modified RT-PCR.

Long-term followup.

In most patients, the relapse of MC vasculitis manifestations was brief (2 of the 8 patients) or was of weaker intensity. Cryoglobulin levels decreased during followup, and the MC vasculitis manifestations subsided within several months. The mean ± SD cryoglobulin level decreased from 0.3 ± 0.09 gm/liter at the time of relapse to 0.06 ± 0.09 gm/liter at 12 months after treatment withdrawal (P < 0.05), accompanied by a 2-fold increase in the mean ± SD C4 level (0.08 ± 0.07 gm/liter and 0.16 ± 0.1 gm/liter, respectively; P not significant). Treatment in most cases included low-dose antiinflammatory agents (4 of 8 patients), whereas in 2 patients, no treatment was needed.

In 3 patients, severe MC vasculitis manifestations persisted, with elevated cryoglobulin levels; 2 of these patients had lymphoplasmacytic lymphomas and 1 patient (patient c in Table 2) had Sjögren's syndrome (Figure 2). Of the 2 patients with lymphoplasmacytic lymphomas, 1 experienced a relapse 2 months after withdrawal of antiviral treatment. He had severe nephropathy associated with high levels of cryoglobulins and was diagnosed as having bone marrow lymphoma infiltration. He was given 3 courses of combination treatment with fludarabine and cyclophosphamide. Following treatment, he experienced complete remission, with disappearance of the cryoglobulinemia and continuous improvement in renal function. Three years after treatment with cytotoxic agents, the patient's disease is still in complete remission, with no signs of either MC vasculitis or the lymphoproliferative disorder. The second patient with lymphoplasmacytic lymphoma experienced a relapse of MC vasculitis 2 months after withdrawal of antiviral treatment, with purpura and peripheral neuropathy. The type II cryoglobulinemia reappeared, and a lymphocytic B cell lymphoma (medullary and splenic) was diagnosed. She experienced a remission of the lymphoma and vasculitis during treatment with corticosteroids and chlorambucil for ∼2 years. At the end of this period, the patient experienced a relapse of her lymphoma, manifested by severe cytopenia and generalized symptoms. She underwent a splenectomy but died of cardiac failure postoperatively.

Figure 2.

Levels of A, cryoglobulins and B, C4 before treatment with antiviral agents, at the end of treatment, at the time of relapse, and 12 months after the end of treatment in 8 patients with hepatitis C virus–associated mixed cryoglobulinemia vasculitis. Data are shown separately for patients whose relapse was brief and patients whose relapse was persistent. Values are the mean ± SD.

DISCUSSION

We describe herein a subset of HCV patients who experienced MC vasculitis relapse despite successful elimination of the virus from the serum with effective antiviral treatment. In this group of 8 patients, most relapses were short-lived and were less severe than the initial MC vasculitis disease experienced before treatment with an antiviral agent. In 5 of the 8 patients, the cryoglobulin levels continued to decrease, and they experienced a complete or nearly complete remission with minimal treatment within several months. However, in 3 patients, persistent symptoms remained, and more importantly, in 2 of them, an underlying B cell lymphoproliferative process was identified.

This study joins a previous study by Levine et al (19), who described 4 patients with HCV-induced MC vasculitis who experienced complete remission of vasculitis symptoms accompanied by negative findings on serum HCV PCR testing after treatment with antiviral agents. Those patients experienced a relapse of symptoms during the first year after withdrawal of antiviral agents, accompanied by rising cryoglobulin levels and decreasing C4 levels. An exhaustive search for lymphoproliferative disorders was conducted in 3 of the 4 patients, with negative results. In addition, HCV PCR analysis was also performed on cryoprecipitates from 3 of the 4 patients, and the results were negative. Two of the 3 patients who continued followup were treated with prednisone and anti-CD20.

B cell lymphoproliferative disorders have been reported to occur in association with chronic HCV infection, mostly in patients with MC (20, 21). In a meta-analysis published in 2003, Gisbert et al (22) showed that the prevalence of HCV infection in patients with B cell NHL was ∼15%. The estimated odds ratio for NHL in HCV-seropositive persons relative to HCV-seronegative persons is 5.7 (95% confidence interval 4.09–7.96) (23). The overall risk of NHL in patients with HCV-MC is even higher, estimated to be 35 times higher than that in the general population (24).

Effective antiviral treatment has been demonstrated to induce hematologic remissions in HCV-associated NHL. A recent systematic review (25) found that complete remission was achieved in 75% (95% confidence interval 64–84%) of 65 patients with HCV infections. In uncontrolled studies, interferon treatment, either alone or in combination with ribavirin, induced complete remission in patients with nodal and splenic marginal-zone lymphoma (26, 27) and immunocytoma (28). Recent controlled trials in HCV patients with splenic lymphoma with villous lymphocytes have shown that reduction of the HCV viral load leads to regression of the tumor burden (29, 30), with a complete hematologic response observed in 89% of patients. It is important to note, that as is the case with antiviral treatment in HCV-MC, regression of lymphoproliferative disease is closely associated with the viral response (26, 30) and will recur if HCV replication reappears (29).

Previous studies, however, suggest that B cell proliferation may eventually reach an autonomous phase in which it may become HCV-independent, as demonstrated by the finding that monoclonal immunoglobulin gene rearrangement was still detectable in the blood even though a complete hematologic response had been achieved (29). This is perhaps because B cell clones may become fully transformed and require the presence of HCV for their proliferation but not for their survival. The high rate of B cell proliferative disorders in our current study emphasizes the importance of a thorough evaluation of B cell lymphoproliferation in patients with relapse of MC vasculitis in whom a sustained viral response has been achieved. Interestingly, the most severe MC vasculitis manifestations were observed in patients who developed concomitant B cell lymphoproliferation with higher levels of serum cryoglobulins.

Two recent reports serve as an important reminder that patients with a sustained viral response may not be necessarily cured. Radkowski et al (31) demonstrated the persistence of small quantities of HCV RNA in PBMCs as well as in the liver of patients with a sustained viral response. In a small number of patients, replicative forms of HCV RNA were also detected in lymphocytes or macrophages (31). Pham et al (32) demonstrated the presence of traces of replicative forms of HCV RNA in PBMCs cultured with mitogens. In the present study, however, we used the most sensitive method available to date for the detection of HCV RNA, the TMA method, and did not identify any viral RNA in the serum or cryoprecipitate of patients who tested negative by conventional RT-PCR analysis. The results of our search for HCV RNA replication in PBMCs using a modified RT-PCR technique were also negative. These results significantly reduce the likelihood that active viral replication was the cause of the relapse of MC vasculitis.

Relapse in most of our study patients occurred shortly after discontinuation of antiviral agents, which may suggest a direct immunomodulatory role of interferon treatment in suppressing the production of cryoglobulins. Interferon alfa is a potent inhibitor of interleukin-7–dependent growth of early B cell lineage progenitors, effectively aborting further B cell lineage differentiation at the pro–B cell stage (33, 34). Interferon alfa has been demonstrated to prevent B cell outgrowth due to Epstein-Barr virus (35). Peters et al (36, 37) identified a direct effect of interferon alfa on B cells, in which high concentrations of interferon suppressed mitogen-induced antibody production. This direct effect of interferon may partly explain the relapse of MC that appears shortly after discontinuation of treatment in patients with a sustained viral response.

In conclusion, although a relapse of MC vasculitis occurred in only a few patients with HCV-related MC vasculitis who had achieved a sustained viral response, it is important to consider that such a relapse may occur despite successful treatment with antiviral agents. The results of the TMA analysis in sera and cryoprecipitates as well as of RT-PCR analysis in PBMCs reported herein effectively rule out persistence of HCV RNA as a probable cause of the vasculitis relapse. In such patients, different underlying condition should be considered, with a special emphasis on B cell lymphoproliferative disorders.

AUTHOR CONTRIBUTIONS

Dr. Cacoub had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Saadoun, Halfon, Cacoub.

Acquisition of data. Landau, Saadoun, Martinot-Peignoux, Fois, Cacoub.

Analysis and interpretation of data. Landau, Saadoun, Martinot-Peignoux, Marcellin, Cacoub.

Manuscript preparation. Landau, Saadoun, Halfon, Cacoub.

Statistical analysis. Landau, Cacoub.

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