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Background Essential mixed cryoglobulinemia is frequently associated with hepatitis C virus (HCV) infection. A beneficial effect of interferon alfa therapy has been reported, but we do not know whether the antiviral activity of the drug affects the clinical and biochemical manifestations of disease.
Methods In a prospective randomized, controlled trial, we studied 53 patients with HCV-associated type II cryoglobulinemia. A group of 27 patients received recombinant interferon alfa-2a thrice weekly at a dose of 1.5 million units for a week and then 3 million units thrice weekly for the following 23 weeks. The 26 control patients did not receive anything apart from previously prescribed treatments. All patients were then followed for an additional 24 to 48 weeks.
Results Interferon was usually well tolerated, but it was permanently discontinued in two patients because of atrial fibrillation and depression. Two of the 26 patients in the control group were lost to follow-up. After the treatment period, serum HCV RNA was undetectable in 15 of the remaining 25 patients who received interferon alfa-2a, but in none of the controls. In comparison with the control group, the 15 patients with undetectable levels of HCV RNA in serum had significant improvement in cutaneous vasculitis (P = 0.04) and significant decreases in serum levels of anti-HCV-antibody activity (P = 0.007), cryoglobulins (P = 0.002), IgM (P = 0.002), rheumatoid factor (P = 0.001), and creatinine (P = 0.006). After treatment with interferon alfa-2a was discontinued, viremia and cryoglobulinemia recurred in all 15 HCV RNA-negative patients. On resumption of treatment, three of four patients had a virologic, clinical, and biochemical response.
Conclusions The therapeutic efficacy of interferon alfa-2a in HCV-associated cryoglobulinemia is closely related to its antiviral activity, thus supporting the idea that HCV infection may be a cause of this disease.
Methods
Patients
We evaluated 58 patients with a history of cutaneous vasculitis, arthralgia, and weakness; IgG and IgM cryoglobulins in the serum; and evidence of HCV infection in the form of a positive test for anti-HCV antibodies or the demonstration of HCV RNA by the polymerase chain reaction (PCR), or both. Patients were excluded if any of the following were present: conditions that might be associated with secondary cryoglobulinemia, such as hematologic tumors, autoimmune disorders, and acute or chronic infectious disease not related to HCV; serious medical illness other than liver disease that might preclude completion of the study; hepatic failure, characterized by a history of ascites, bleeding esophageal varices, endogenous hepatic encephalopathy, a serum bilirubin level above 4 mg per deciliter (68 µmol per liter), a serum albumin level below 3.0 g per deciliter, and a prothrombin time that was more than 3 seconds longer than normal; and cytopenia, as indicated by a platelet count below 60,000 per cubic millimeter, a leukocyte count below 3000 per cubic millimeter, or a hemoglobin level below 10 g per deciliter (6.2 mmol per liter). According to these criteria, five patients were excluded. The remaining 53 patients -- all with HCV-associated type II cryoglobulinemia -- were enrolled between January 1991 and February 1992. After written informed consent was obtained, each patient was randomly assigned to treatment with interferon alfa-2a or to the control group, according to a computer-generated table. All procedures during the trial were conducted in accordance with the Helsinki Declaration of 1975, as revised in 1983.
Treatment
The 27 patients in the treatment group received recombinant interferon alfa-2a (Roferon-A, Hoffmann-LaRoche, Basel, Switzerland) at a dose of 1.5 million units thrice weekly for a week and then 3 million units thrice weekly for the following 23 weeks, usually by self-administered subcutaneous injection. The 26 patients in the control group did not receive anything apart from previously prescribed treatments. Previous treatments including low-dose prednisone (0.2 mg per kilogram of body weight per deciliter or less), diuretics, antihypertensive drugs, and medications to treat associated diseases were continued in both groups (Table 1).
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The patients were seen as outpatients after 2 and 4 weeks, and then at 4-week intervals during the 24-week treatment period. All patients were then seen every eight weeks for at least six months. At each visit, the patients rated the clinical manifestations of their disease and the side effects of therapy on a symptom scale. The examiner searched for signs of cutaneous vasculitis, liver dysfunction, and kidney disease. Blood and urine samples were obtained for blood counts, biochemical tests, and virologic studies.
To assess the severity of cutaneous vasculitis, a scoring system was used in which a score of 0 indicated the absence of active skin lesions; a score of 1, rare (fewer than 10) purpuric spots on the lower limbs (mild acral purpura); a score of 2, 10 or more purpuric spots on the lower limbs (severe acral purpura); a score of 3, extension of acral purpura to the trunk, upper limbs (widespread purpura), or both; and a score of 4, the presence of skin ulcers, gangrene, or both.
Serum alanine aminotransferase levels were determined with an AutoAnalyzer. Serum cryoglobulins were measured as the protein concentration of isolated cryoprecipitate, as previously described15. The serum concentration of immunoglobulins, rheumatoid factor, and C3 and C4 complement components were measured by routine nephelometric assays.
Serum and urinary creatinine and proteinuria were measured by standard methods. The protein-creatinine index in spot urine samples (the protein concentration, in milligrams per liter, divided by the creatinine concentration, in milligrams per liter) was measured instead of 24-hour urinary protein excretion. The latter determination is frequently affected by collection and measurement errors.
Anti-HCV antibodies were measured with a second-generation enzyme-linked immunosorbent assay that used c22-c200 antigens (Abbott Laboratories, North Chicago, Ill.). Post-treatment changes in serum anti-HCV antibodies were measured as the activity of anti-HCV antibodies (IgG anti-HCV antibodies divided by total serum IgG), as previously reported7. This ratio was calculated as the optical density multiplied by 1000 divided by the serum concentration of IgG (in milligrams per deciliter). The pattern of different antibodies to HCV was tested semiquantitatively with a dot blot immunoassay (Abbott Matrix) that includes spots of recombinant core (c22-3), NS3 (33c), and NS4 (c100-3) antigens of HCV in the nitrocellulose membrane.
Serum HCV RNA was detected by reverse transcription and nested PCR with primers derived from the highly conserved 5' untranslated region of the viral genome, as previously described16. Each test was performed in duplicate. Only reproducible results obtained in at least two independent experiments were considered in the study.
For the purposes of this trial, we defined a response as the disappearance of HCV RNA from serum by the end of treatment with interferon alfa-2a.
Statistical Analysis
To assess the similarity of the two groups at entry into the study, we compared base-line values using a two-sample Wilcoxon test17. To compare the changes in the severity of cutaneous vasculitis, the patients' post-treatment scores were subtracted from their pretreatment scores. The differences were then ranked, and the scores in the groups were compared with a two-sample Wilcoxon test. The changes in the results of biochemical tests and in the activity of anti-HCV antibodies, as measured at the end of the treatment period, were calculated as the percent change from the pretreatment base-line value for each patient. The values were used for comparisons between the groups with a two-sample Wilcoxon test. All P values are two-tailed; the 0.05 level was used to determine statistical significance.
Results
Characteristics of the Patients
At entry, the two groups had similar demographic, clinical, biochemical, and virologic features (Table 1). About three quarters of the patients had renal involvement, manifested by microscopic hematuria, proteinuria, hypertension, or mild-to-moderate renal failure with serum creatinine levels of up to 3.5 mg per deciliter (310 µmol per liter). Two of the 27 patients assigned to the interferon alfa-2a group did not complete the treatment because of side effects of the medication. Two of the 26 patients in the control group were lost to follow-up. Complete results were therefore available for 25 patients in the interferon alfa-2a group and 24 patients in the control group. Data on these patients were analyzed.
Response to Interferon Alfa-2a
At the start of the trial, all patients in the interferon alfa-2a group and 23 of the 24 patients in the control group had HCV RNA in their serum. After 24 weeks of therapy, HCV RNA became undetectable in 15 of the 25 patients receiving interferon alfa-2a (60 percent). In contrast, at the end of the treatment period, all 24 patients in the control group were HCV RNA-positive, including the single patient who was initially HCV RNA-negative.
All but one of the patients in the interferon alfa-2a group and all the patients in the control group were initially positive for anti-HCV antibodies. These results did not change during the treatment period. When anti-HCV antibodies in serum were measured in terms of activity, however, the effect of interferon alfa-2a therapy became apparent. The activity of anti-HCV antibodies decreased by an average of 19 percent in the patients with a response to treatment with interferon alfa-2a (indicated by the disappearance of HCV RNA from serum) and increased by 23 percent in the control patients -- a significant difference (P = 0.007). In contrast, antibody activity was essentially unchanged in patients who had no response to interferon alfa-2a. The decrease in the activity of anti-HCV antibodies was confirmed by the dot blot immunoassay in 10 of 12 patients with a response. This assay also showed that the titers of different antibodies to HCV generally changed in parallel.
Clinical and Biochemical Changes
Treatment with interferon alfa-2a improved the signs and symptoms of cryoglobulinemia in the 15 patients in whom HCV RNA disappeared from serum. In particular, among the seven patients with cutaneous vasculitis, purpuric lesions disappeared in six and improved in the seventh (P = 0.04 as compared with the control group). In contrast, during the treatment period, the clinical condition of many patients in the control group, and to a lesser extent the patients who had no response to interferon alfa-2a, appeared to deteriorate progressively.
Table 2 shows the pretreatment and post-treatment values of all biochemical variables measured in the patients with a response to interferon alfa-2a and in the control group. By the end of the treatment period, serum cryoglobulin levels had decreased markedly in the patients who responded to therapy, whereas a small increase was observed in the control group (P = 0.002). A comparison of the patients who responded to interferon alfa-2a with those who did not also suggested that the decrease in serum cryoglobulin levels was related to the antiviral activity of the drug. A statistically significant and progressively increasing difference between these groups was observed from the 16th week of treatment onward (Figure 1). Similarly, the patients with a response had significant decreases in serum IgM (P = 0.002) and rheumatoid factor (P = 0.001) in comparison with the patients in the control group (Table 2). In addition, a small but consistent reduction in the serum creatinine concentration was noted in the patients with a response, but not in the control group (P = 0.006).
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Follow-up
After the treatment period, 25 patients in the interferon group and 24 patients in the control group were seen every 8 weeks for 24 to 48 additional weeks (median, 32). Among the 15 patients in whom HCV RNA could not be detected at the end of interferon alfa-2a therapy, viremia recurred within six months in 13 and after one year in the other 2. The reappearance of HCV RNA in the serum was accompanied or followed by worsening clinical manifestations of cryoglobulinemia.
Four of the 15 patients who had a flare-up of their disease after stopping treatment received interferon alfa-2a for an additional six months. In three of these patients the disappearance of serum HCV RNA on resumption of treatment was associated with clinical and biochemical improvement in cryoglobulinemia. The response of one of these patients is illustrated in Figure 2.
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Side Effects
Interferon alfa-2a therapy was well tolerated by most patients. It was permanently discontinued in two patients because of atrial fibrillation and depression. Thrombocytopenia developed in two patients, requiring a temporary reduction in the dose. Nearly all patients had a short influenza-like illness around the time of the first few injections. Many patients had a decrease in white-cell and platelet counts of 10 to 20 percent; 13 patients lost their appetites and had a slight weight loss. Alopecia (five patients), insomnia (two patients), and mouth ulcers (one patient) were less common side effects.
Discussion
The clinical syndrome of essential mixed cryoglobulinemia is usually thought to result from a widespread vasculitic process induced by cryoprecipitable circulating immune complexes. Conventional treatment includes corticosteroids, cytotoxic agents, and plasmapheresis. The goal is to control the formation, tissue deposition, and inflammatory effects of cryoglobulins18,19,20,21,22. A small preliminary clinical study9 and a more recent larger trial conducted by the same authors10 reported a beneficial effect of interferon alfa-2a in most patients with essential mixed cryoglobulinemia. The rationale for the use of interferon alfa-2a was related to its antiproliferative and immunomodulating effects in a disease thought to be the expression of a low-grade lymphomatous disorder. This interpretation has been challenged by the demonstration in large studies5,6,7,8 that most patients with essential mixed cryoglobulinemia are infected with HCV. Interferon has been reported to be efficacious for the treatment of HCV-associated cryoglobulinemia11,12,13,14. Yet, controlled studies with large numbers of patients and appropriate virologic tests are necessary to establish whether the improvement in clinical features is related to antiviral or other effects of interferon.
Our study indicates that the beneficial effect of interferon therapy is limited to patients in whom HCV RNA disappears from the serum. All clinical improvements, such as a decrease in cutaneous vasculitis, were only observed in patients with a response to interferon alfa-2a, whereas the condition of patients who had no response and of the patients in the control group progressively worsened. Some markers of immunologic activity (cryoglobulins, rheumatoid factor, and serum IgM), HCV infection (activity of anti-HCV antibodies), and renal function (serum creatinine) decreased in patients in whom HCV RNA disappeared from the serum, but increased or remained unchanged in patients with no response and in control patients. Treatment with interferon alfa-2a was associated with a slight but insignificant decrease in alanine aminotransferase levels in patients with a response. A likely explanation is that in most patients with cryoglobulinemia, alanine aminotransferase levels are normal before treatment. Thus, any effect of interferon alfa-2a on this marker of liver inflammation may not be seen.
In all patients who were previously negative for HCV RNA, signs and symptoms of cryoglobulinemia and viremia recurred after interferon alfa-2a therapy was stopped. This finding and the virologic, clinical, and biochemical response following the resumption of treatment in three of these patients reinforce the evidence that the therapeutic efficacy of interferon alfa-2a in HCV-associated cryoglobulinemia is closely related to its antiviral activity. This conclusion is consistent with the finding in other studies that the clinical and serum biochemical response to interferon alfa in patients with chronic HCV infection is associated with a loss of detectable HCV genome from serum and that long-term remission of the disease is accompanied by the sustained absence of viremia23,24,25,26.
Although 60 percent of our patients had a virologic, clinical, and biochemical response to interferon alfa-2a therapy, none became completely free of the abnormalities that characterize mixed cryoglobulinemia. All these patients relapsed after interferon alfa-2a was discontinued. A recent uncontrolled trial of 21 patients10 found that the initial rate of clinical and biochemical response to interferon alfa was 77 percent and that the long-term rate of remission in the absence of treatment was 24 percent. A higher dose of interferon and a longer treatment period were used in that study.
Our results suggest that eradicating HCV infection potentially cures HCV-associated cryoglobulinemia. To accomplish this task, higher doses of interferon alfa-2a, a longer treatment period, and the use of additional antiviral drugs may be helpful. A large comparative trial may be needed to establish the relative efficacy of different therapeutic regimens. Our current policy is to use, if well tolerated, doses of 4.5 to 6 million units of interferon alfa-2a thrice weekly for six months. In patients who respond to interferon alfa-2a, this treatment is continued at a dose of 3 million units thrice weekly for six additional months. Then, the dose of interferon alfa-2a is tapered over the next six-month period, and the treatment is discontinued. Throughout the treatment and follow-up periods, testing for serum HCV RNA is conducted every six months to monitor the response to therapy and to predict disease relapse.
Supported by a grant (C-33432) from the Italian Ministry of Health.
Source Information
From the Division of Nephrology and Dialysis (R.M., D.M.), the Department of Immunohematology and the Blood Transfusion Center (P.B., O.V.), the Laboratory of Clinical Chemistry (P.Z., A.V.), and the Division of Medicine I (P.L.S.), Ospedali Riuniti di Bergamo, Bergamo; the Laboratory of Clinical Chemistry (D.F.) and the Dialysis Unit (M.M., G.V.), Ospedale di Treviglio e Caravaggio, Treviglio; and the Institute of Virology, University of Milan, School of Medicine, Milan (E.T., A.Z.) -- all in Italy.
Address reprint requests to Dr. Bellavita at the Department of Immunohematology and the Blood Transfusion Center, Ospedali Riuniti di Bergamo, 24128 Bergamo, Italy.
References
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Related Letters:
Interferon Alfa-2a for Cryoglobulinemia Associated with Hepatitis C Virus
Agnello V., Knight G., Abel G., Ganger D. R., Jensen D. M., Misiani R., Bellavita P., Vicari O.
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Full Text
N Engl J Med 1994;
331:400-401, Aug 11, 1994.
Correspondence
This article has been cited by other articles:
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