Interferon-α treatment of hepatitis C virus-associated mixed cryoglobulinemia

Journal ofHepatology 1997; 21: 63-l 1 Printed in Denmark . All rights reserved Munksgaard Copenhagen

Copyright 0 European Association for !he Study of the Liver 1997

Journalof Hepatology ISSN 0168-8278

Interferon-a treatment of hepatitis C virus-associated mixed cryoglobulinemia Frank Polzien, Peter Schott, Sabine Mihm, Giuliano

Ramadori

and Heinz Hartmann

Department of Medicine. Division of Gastroenterology and Endocrinology, Georg-August-Universitiit. Gdttingen. Germany

Background/Aims: Chronic hepatitis C virus infection is frequently associated with mixed cryoglobulinemia. The efficacy of interferon-a treatment in the presence of cryoglobulinemia, particularly the rate of sustained responders, has not yet been well defined. Met/zods: Fifty-nine consecutive patients with chronic HCV infection were studied prospectively with regard to the presence of cryoglobulinemia and their biochemical and virological response to interferon-a2, therapy. Results: Cryoglobulins were detected in sera of 23 patients. For this latter group of patients, significant differences were found compared to the 36 patients without cryoglobulinemia, i.e. the prevalence of female sex was higher, the duration of liver disease was longer and distinctive laboratory abnormalities, e.g. higher rheumatoid factor activity, were noted as well as a higher prevalence of cirrhosis. The distribution of HCV genotypes and serum HCV RNA titers was similar in the two groups. Interferon-a treatment regi-

mens were not different regarding mean cumulative dose and mean duration of therapy. The response to therapy was almost identical, i.e. 35% of patients with cryoglobulinemia showed a sustained response compared to 22% of patients without cryoglobulinemia. The percentages of patients showing a relapse or breakthrough were similar in both groups. Pre-treatment viremia levels were higher in non-responders compared to sustained responders. Non-responders appeared to be more frequent among patients infected with genotypes la and lb, especially among male patients without cryoglobulinemia. Conclusions: The presence of cryoglobulinemia per se in chronic HCV-infected patients does not adversely affect the outcome of interferon-a therapy, including the rate of sustained response.

I

precipitates (7), indicating the direct participation of viral particles in precipitate formation. In addition, cryoglobulinemia was observed in HCV-infected patients within a few months after liver transplantation when recurrent disease of the liver allograft was histologically mild (14). An etiological role of HCV was further supported by observations that antiviral therapy directed against HCV, e.g. with interferon-a, resulted in an improvement of vasculitic manifestations and a decrease in the amount of serum cryoglobulins in those HCV-infected patients showing a biochemical and virological response to interferon-a (12,13,15). Interestingly, in patients showing a relapse of HCV infection when interferon-a was discontinued, a recurrence of cryoglobulinemia was reported (15,16). In fact, when HCV-infected patients with symptomatic cryoglobulinemia, e.g. with cutaneous vasculitis, were treated with interferon-a, a sustained response including a continued absence of serum HCV RNA during

abnormalities have been noted in patients with chronic hepatitis C virus (HCV) infection (l-3). In addition, it has been recognized that mixed cryoglobulinemia, i.e. type II as well as type III cryoglobulinemia according to the classification of Brouet et al. (4), is frequently associated with HCV infection (5-9). Extrahepatic disease, e.g. cutaneous purpura, arthralgia, glomerulonephritis and peripheral neuropathy, has also been observed in cryoglobulinemic HCV-infected patients, attributed to systemic vasculitis (9-13). An etiological role of HCV in the pathogenesis of mixed cryoglobulinemia was initially suggested by the demonstration of enriched HCV RNA in serum cryoMMUNOLOGICAL

Received 30 August 1996; revised 9 January; accepted 30 January 1997

Correspondence: Heinz Hartmann, Medizinische Universitatsklinik, Robert-Koch-Strasse 40, D-37075 Giittingen, Germany. Tel: 49 551 396326. Fax: 49 551 398596.

Key words: Hepatitis C genotypes; Hepatitis C virus; Interferon-a; Mixed cryoglobulinemia.

63

F. Pokien

er ul.

follow-up over 6-12 months was not observed by Misiani

after the end of therapy et al. in 15 patients who

had a complete response at the end of therapy (15). Reports on interferon-a efficacy in HCV-infected patients with less symptomatic cryoglobulinemia are scarce at present. In recent studies, a prevalence of cryoglobulinemia of up to 50% was noted in chronic HCV infection (17,lS). A minority of these latter patients was affected by cryoglobulinemia-related clinical manifestations (17,18). When compared to patients without evidence of cryoglobulinemia, a preponderance of female sex, and of clinically advanced liver disease, and a higher prevalence of cirrhosis were reported among cryoglobulinemic HCV-infected patients ( 17,18). Furthermore, in these latter patients increased levels of serum IgM and of rheumatoid factor activity, as well as a decreased C4 complement factor serum concentration were observed. HCV genotype distribution in cryoglobulinemic patients appeared to be similar to that in patients without cryoglobulinemia (19-21). Treatment of chronic hepatitis C virus infection has mainly relied on interferon-a during recent years. Whereas data from early studies (when efficacy of treatment was assessed using serum biochemical and histopathological parameters) demonstrated a response in approximately 50’%,of treated patients, recent studies (when serum HCV RNA was determined in addition) suggested a lower response rate (22). Modifications of interferon dose and of duration of therapy, however, might result in an improved response rate as well as in a decreased relapse rate after discontinuation of interferon-a (23). Among the factors predictive of response to interferon-a therapy (22). the presence of cryoglobulinemia has not been considered. In the present work HCV-infected patients evaluated for interferon-a therapy were prospectively studied for the presence of cryoglobulinemia. The outcome of therapy, including the number of sustained responders, was determined in cryoglobulinemic patients and compared to contemporaneously treated HCV-infected patients without cryoglobulinemia.

Materials and Methods Patients

Fifty-nine consecutive patients (29-61 years of age; 27 females, 32 males) with chronic HCV infection were studied prospectively. They were referred because of apparent liver disease and evaluated by routine diagnostic procedures. All patients gave informed consent for participation in the study. The protocol was ap64

proved

by the local ethical

committee

of the Georg-

August-Universitat Giittingen. Diagnosis of chronic HCV infection was based on standard criteria, e.g. history, physical findings, laboratory abnormalities, serology. and liver histology. In all patients anti-HCV antibodies were detectable by a second-generation enzyme-linked immunosorbent assay (ELISA). The presence of serum HCV RNA prior to therapy was noted in every patient by reverse transcription polymerase chain reaction (RT/PCR). Duration of disease and the likely route of viral transmission were estimated from the individual’s past medical history (e.g. first evidence of liver disease. receipt of blood transfusion, intravenous drug abuse). Patients with active hepatitis B virus or HIV coinfection and those with continued alcohol abuse were excluded. A liver biopsy was performed in 44 patients within the 13 months prior to therapy. Tissue specimens were processed using standard histopathological methods.

Interferom2

treutment ad

fff$nition

of‘the fj’pe qf

response

All patients received recombinant interferon-cxz, (Roferon A, Hoffmann-La Roche, Basel, Switzerland). None of them had been treated with interferon-a before. Generally a dose of 3 million international units thrice weekly was administered for at least 6 months, but interferon therapy was adapted according to patients’ response and tolerance. e.g. the duration was extended in responding patients for up to 14 months (in some the dose was reduced to 2 million units thrice weekly for the last months on treatment). Three different types of response were defined on the basis of virological and biochemical serum parameters. Patients were classified as showing a sustained response (SR) when serum transaminase activities normalized (biochemical response) and serum HCV RNA became undetectable (virological response) during therapy and remained normal for at least 3 consecutive follow-up determinations (performed at about 1 month apart) within 6 months after interferon discontinuation. The continued re-emergence of serum HCV RNA and/or abnormal transaminase activities after initial biochemical and virological response after a treatment period of at least 3 months was defined as breakthrough. A relapse was assumed if serum HCV RNA became detectable or increased transaminase activity was observed subsequent to discontinuation of therapy in patients who had had a biochemical and virological response during treatment periods. Post-treatment follow-up time was at least 6 months. Patients without normalization of transaminase activities and/or con-

Interferon-cr for HCV

tinued presence of serum HCV RNA while receiving therapy were classified as non-responders. Laboratory testing of sera Activities of serum transaminases, y-GT and AP as well as concentrations of serum bilirubin, total protein, albumin, IgG, IgA, IgM, complement factors (C~C, C4) and rheumatoid factor activity were measured before, during and after discontinuation of therapy by routine methods, including nephelometry (Behring Nephelometric Analyzer, Marburg, Germany). Serum samples from all patients were systematically analyzed for the presence of cryoglobulins. Serum cryoglobulins were quantified by determining the cryocrit using the method described by Schubothe et al. (24). Anti-HCV antibodies (ELISA 2nd, Ortho Diagnostic Systems, Raritan, N. J., USA) and serum HCV RNA were repeatedly measured at intervals of about 4-6 weeks. Detection and classiJication of cryoglobulins Venous blood samples were collected in prewarmed syringes and allowed to clot at 37°C for 2 h. Thereafter, sera were prepared by centrifugation at 37°C 3000 rpm for 10 min (Hettich centrifuge, Tuttlingen, Germany), transferred into calibrated Nissel tubes (Hecht, Sontheim, Germany) and kept for 96 h at 4°C. After centrifugation at 4°C for 10 min at 1200 rpm (Rotanta Centrifuge, Hettich, Tuttlingen, Germany), visible precipitations were quantified as percent of volume. For further analysis, cryoprecipitates were washed four times in a small volume (0.5 ml) of ice-cold phosphatebuffered saline (PBS, pH 7.2). Samples were centrifuged at 10000 g for 2 min at 4°C between each washing. Finally, precipitates were dissolved in 100 ~1 PBS and stored at -20°C for further analysis. Cryoglobulins were classified into type I, II or III according to Brouet et al. (4) by immunofixation. After dilution of samples in PBS, 0.5% (w/v) CHAPS (Sigma, Deisenhofen, Germany) to an immunoglobulin concentration of 1 g/l, a commercially available test system (Minifix, Binding Site, Heidelberg, Germany) was used according to the instructions of the manufacturer. Detection of HCV RNA by nested RT/PCR Serum samples (150 ~1) were taken up in a freshly prepared and prewarmed mixture of 800 yl lysis buffer containing 0.05 M Tris pH 8.0, 1 mM EDTA, 0.1 M sodium chloride, 0.5% (w/v) SDS, 20 ~1 proteinase K (25 mg/ml, Boehringer Mannheim, Germany) and incubated at 37” C for 1 h with slight agitation. After phenol/chloroform extraction the material was precipi-

cryoglobulinemia

tated in ethanol overnight at -20°C. The precipitate was taken up in 70% ethanol for 1 h at -80°C and for another hour at -20°C. After centrifugation (30 min, 20000 rpm, 4”C), RNA was dissolved in 30 ~1 HZ0 containing 40 U of the ribonuclease inhibitor RNasin (Serva, Heidelberg, Germany). The reverse transcriptase reaction was carried out in 0.1 mol/l Tris-(Hydroxymethyl)-aminomethane pH 8.3, 10 mmol/l MgC12, 10 mmol/l dithiotreitol, 1 mmol/l of desoxynucleotides (dATP, dCTP, dGTP, dTTP, Boehringer, Mannheim, Germany) each, 200 U of moloney murine leukemia virus reverse transcriptase (M-MLVRT, Gibco BRL, Eggenstein, Germany) and 16 U RNasin in a total volume of 25 ~1 for 1 h at 40°C using isolated, denaturated RNA corresponding to 50 ~1 of serum and an antisense oligonucleotide primer (SRl 5’-TGC ACG GTC TAC GGA GAC CT3’, nt -2 to -20). Amplification of cDNA was carried out as a two-step reaction with two pairs of nested primer (outer primer pair: SF1 5’-GCC ATG GCG TTA GTA TGA GT3’, nt -259 to -240, and SRl; inner primer pair: SF2 5’-GTG CAG CCT CCA GGA CCC CC3’, nt -236 to -217 and SR2 5’-GGG CAC TCG CAA GCA CCC TAT3’, nt -25 to -45) derived from highly conserved regions within the 5’noncoding region of HCV Five microliters of the first PCR reaction were transferred into the second PCR reaction. Amplification was carried out with denaturation temperature at 94°C for 1 min, annealing temperature at 60°C for 55 s and extension temperature at 72°C for 1 min. Amplification products (212 bp) were separated on a 1.5% agarose gel and visualized by ultraviolet light after ethidium bromide staining. QuantiJication of HCV viremia Determination of the HCV viremia was done using a commercially available competitive RT/PCR assay (Amplicor HCV Monitor, Hoffmann-La Roche, Grenzach-Wyhlen, Germany). Test procedures were performed according to the manufacturer’s manual. Genotyping of HCV Determination of HCV genotypes was performed using RT/PCR with type-specific primers of the HCV core gene according to Okamoto et al. (25,26), except that RNA extractions from sera and the reverse transcription were carried out as described above. Individual genotypes were confirmed by a restriction enzyme analysis. In brief, the amplification product of HCV type I was subjected to digestion by Xhol(28+21), that of type II by Xho (no digestion) and AvaI (66+24+54 or 66+78), that of type III by EcoRV (94+78) and AvaI (154+20), that of type IV by Sty1 (99+24) and 6.5

F. Polzien TABLE

et rrl

1

Demographic, treatment

biochemical,

and histopathologic

data

of 59 patients

with chronic

Patients with cryoglobulincmia (11=23) lhnogruphic

Lmhorator~ duto Cryocrit (vol ‘%I) ALT (U/I) (g/l)

C4 (mg/dl) RF (W/l) HistologJx’ CPH ((j/o) CAH (‘Xl) Cirrhosis (‘Xl) ‘I X2-test. bUnpaired otherwise indicated.

C virus infection

before

Patients without cryoglobulinemia (n=36)

initiation

of interferon-n

J’

data

Age (years) Sex (m/f) Presumed duration of infection (years) History of blood transfusion (I%)

IgM

hepatitis

47.7213.3 7i16 14.7Z8.9’ 39

41.11-12.4 25/l I 8.2?6.0” 33

1.6’5.8 84.1243.2 3.410.5 22.729.8 353.81822

0 75.lir70.5

appropriate f-test and

tests, i.e. unchi-square-test,

1,s

IIS

23.6k6.8 17.113.45

/KO.O’h ns p
80.6 9.7 9.7

‘KO.05~ ns p
two-tailed t-test. “Available in 44 patients. +Obtained in 17 patients. CPH: chronic persistent hepatitis; CAH: chronic active hepatitis.

Statistical analysis Data were analyzed using paired two-tailed Student’s as indicated.

p<0.008b

1.71-0.3

56.5 13.0 30.5

EcoRV (94+22), and that of type V by Mae111 (68+20) and AvaI (no digestion). Numbers in parentheses indicate the length of the expected digestion products in basepairs. All restriction endonucleases were purchased from Boehringer (Mannheim, Germany). HCV genotypes I to V according to Okamoto et al. correspond to genotypes la, lb, 2a, 2b and 3a as classified by Simmonds et al. on the basis of sequence variations within the 5’-noncoding region of the genome (27).

ns

p-am’

“Obtained

in 26 patients.

Data are mean

?SD

unless

1; ~~0.02) and rheumatoid factor activity (353 vs. 17 IU/ml; ~~0.05) were increased (Table 1). HCV infection was most likely acquired by the receipt of blood transfusions in 39% and 33% of patients, respectively (Table 1). In both groups few patients gave a history of intravenous drug usage. No significant differences were noted when comparing patients with to those without cryoglobulinemia regarding presumed mode of HCV transmission. In 12 patients cryoprecipitate samples were available for immunochemical classification. Among these, the majority had type III cryoglobulinemia (X/12), while one third (4/12) had type II cryoglobulinemia. Clinical signs of systemic vasculitis characteristic of mixed cry-

Results Dernogruphic, clinical und histopathological dutu Cryoglobulinemia was detected in 23 out of 59 patients (prevalence 39%). The mean cryocrit level was 1.6% (vol/vol) in cryoglobulinemic patients. When comparing the group of patients with cryoglobulinemia to those without, no significant differences were found between groups for age, ALT activity, and C4 concentration before initiation of therapy. Females were more frequently encountered in the group with cryoglobulinemia (16 females vs. 7 males; ~~0.02). In this latter group liver disease was present for longer (14.7 vs. 8.2 years in patients without cryoglobulinemia; p
66

TABLE

2

Cumulative interferon-a dose, treatment duration. outcome in 59 patients with chronic HCV infection

Interjbron trmtnwni Cumulative dose (mega U) Duration (months)

Rrs,tmsc~ to trcwttm’tir Sustained Break-through/relapse Non-response

and therapeutic

Patients with cryoglobulincmia (11=X)

Patients without cryoglobulinemiu (n-36)

,/j

309.6+ 147.6

291.6597.2

ns

x. 122.7

ns

X.624.1 8 5 10

8 3 25

ns ns /1<0.05”

Interferon-a

for HCV

cryoglobulinemia

TABLE 3 Response to interferon-a treatment according to sex and the presence of cryoglobulinemia Response

Sustained Break-through/relapse Non-response

Patients with cryoglobulinemia

Patients without cryoglobulinemia

Male

Female

Male

Female

5 1 1

3 4 9

4 1 20

4 2 5

oglobulinemia (i.e. purpura, arthralgia, proteinuria) were present only in a few patients (4/23). None was affected by severe complications, e.g. renal failure. Response

to interferon-a

ns ns p‘CO.02”

vs. lo/23 patients; p
treatment

The mean duration of treatment and the mean cumulative interferon-a dose were comparable in the two groups (Table 2). The percentages of patients showing a sustained response to treatment were statistically not different (35% in the presence of cryoglobulinemia compared to 22% in the absence), as were the percentages for those with a relapse or breakthrough (21 vs. 8%). Non-responders appeared to be more frequent in the group of patients without cryoglobulinemia (25/36

180

p

1

Response

-a-

to interferon-a

treatment

160

-+SR

160 -

of cryoglobulinemia

Cryocrit levels were measured before, during and after treatment. The mean cryocrit before treatment was 1.6k5.8 and decreased to 0.75k2.56 at the end of therapy. A decrease of cryocrit was observed in about 60% of patients, while cryoglobulinemia disappeared in 40%. Cryoglobulinemia-related clinical symptoms improved in each of the four affected patients.

Relapse

B

A

tNR

I

140 120 2 5

3

Z’OO .t6 0 80-

100 & .@ .$ 80

interferon treatment

z 1

2

60

60 40

20

I

I

+1

+2

r

+3

observation period (months)

*. ::: Rk

O-m*

observation period (months)

Fig.1 A and B. Time course of ALT levels in 36 patients without (A) and in 23 patients with cryoglobulinemia (B) before, during and after interferon-a therapy. SR=sustained responders (n=8 in A and n=8 in B); Relapse=relapsing patients (n= 2 in A and n=4 in B); NR=non-responders (n=25 in A and n=lO in B). Data are depicted as mean values, representative standard deviations (SD) are given at some time points for illustration. Normal range for ALT was <22 U/l for men and ~17 U/l for women. Mean duration of treatment was 8.6 and 8.1 months, respectively. Data are given for time points before (-I), during (1,2,3,4), at the end (0) and during follow-up (+1,+2,+3) of treatment (months). 67

F. Polzien et ul. Relation oj’patient sex, cryoglobulinemia, HCV genotypes, serum HCV RNA level and response

100

to

interferon-u therapy Cryoglobulinemia was more frequently observed in female patients (Table 3). Among those cryoglobulinemic patients showing no response to interferon-a, only one male patient was noted, and nine were female. In this latter group, 71% (5/7) of male patients showed a sustained response. Among patients without cryoglobulinemia, however, non-responders were predominantly male (80% males vs. 2O”X females). Statistical analysis for sex proportion according to the rate of non-response showed significant differences between male and female patients (~K0.02, x:-test, Table 3). In patients with and in those without cryoglobulinemia (15 in each group were analyzed), no significant difference in the prevalence of individual HCV genotypes was noted. Regarding response to therapy (irrespective of the presence of cryoglobulinemia), patients with a sustained response (n= 11) were infected by genotypes la (n=2; 18.2X), lb (n=4; 36.4 ‘%), 2b (n= 1; 9%) and 3a (n=4; 36.4%). In non-responders (n=19) the genotypes la (n=6; 31.6%). lb (n=8; 42.1’S), 2a (n=l; 5.2X), 2b (n= 1; 5.2%) and 3a (n=3; 15.9%) were found. A significantly increased prevalence of genotypes la (6 vs. 2; pcO.02) and lb (8 vs. 4; ~~0.02) was observed in non-responders compared to sustained responders. In the group of non-cryoglobulinemic non-responding male patient a high prevalence of genotypes la and lb was noted as well. Among these latter patients (n=20) genotypes were determined in ten patients, five of whom were infected by genotype la and four by genotype lb, respectively. The level of serum HCV RNA was determined in sera obtained before interferon therapy in 23 patients, of whom 11 had cryoglobulinemia. The mean serum HCV RNA titer in cryoglobulinemic patients (301.212’-+474.017 copies/ml; n= 11) was not different compared to patients without cryoglobulinemia (382.0622362.143 copies/ml; n= 12). In contrast. pretreatment titers in sustained responders (irrespective of the presence of cryoglobulinemia) were significantly lower when compared to non-responders (154.108+179.690 vs. 499.9322483.690 copies/ml; pCO.02). Time course of chnnges qf’ ALT activity and cryocrit in relation to response to interferon-a The time course of serum ALT activity changes during therapy (and after discontinuation) was similar in patients with cryoglobulinemia compared to those without in the different groups of treated patients, i.e. in 68

Fig 2. Cryocrit levels before, during and afier interferon-u treatment in three patients shol~?ng u distinct response pattern, e.g. sustuined rrsponsr (SRI, nowresponse / NR) ot relupse. Durution qf’ intcrjkon-a treutment was I I months in SR und NR each and 7 months in the relapsing patient. Cryocrit levels are g&n before i - I 1. dwing Il,3,51 ard after ( •t 3) treatment (man ths).

responding, relapsing and non-responding patients. respectively (Fig. 1). In responding patients normalization of ALT activity was observed during the first 3 months of interferon-a treatment; in relapsing patients ALT activity increased within 6 weeks after discontinuation of therapy (Fig. 1). In the majority of cryoglobulinemic patients cryocrit ranged from 0.5 to 10%; a high cryocrit value of up to 30”/0 was observed in a single patient. During interferon-a therapy, serum cryoglobulin levels appeared to decrease in responding patients. remaining low or becoming undetectable in sustained responders. Changes of cryocrit in three patients (selected according to the type of response to interferon-lx) are demonstrated in Fig. 2.

Interferon-a for HCV cryoglobulinemia

Discussion A prevalence of cryoglobulinemia of up to 50% has previously been reported in patients with chronic HCV infection (17,18). Thus, the prevalence of 39% observed in the present study when prospectively measuring serum cryoglobulins in 59 consecutive patients evaluated for interferon-lx therapy confirms these reports. We also identified distinctive demographic, clinical and laboratory features in cryoglobulinemic HCV-infected patients when compared to patients without cryoglobulinemia, (Table l), similar to previous reports (17,18). Classification of cryoglobulinemia by immunochemical methods demonstrated a high prevalence of type III cryoglobulinemia in the present series. It should be noted that a minority of patients studied was affected by symptomatic disease related to cryoglobulinemia. Thus, the present data are similar to the prevalence rate of 68% for type III cryoglobulins previously reported in a similar cohort of 69 HCV-infected patients with cryoglobulinemia (17). Recently, an even higher proportion, i.e. 19 out of 21 cryoglobulinemic HCVinfected patients, were shown to have type III cryoglobulinemia (28). Interferon-a therapy is effective in a minority of patients with chronic HCV infection (22). Several parameters predictive of a beneficial response to therapy have been identified in recent years, e.g. infection by HCV genotypes other than type lb, as well as clinical characteristics like the absence of cirrhosis of the liver (22). The importance of cryoglobulinemia per se has received less attention, probably because routine searching for serum cryoglobulins is technically difficult, particularly since stored serum samples cannot be utilized. In turn, interferon-a has been used to treat patients presenting with clinical symptoms of vasculitis indicative of cryoglobulinemia and found to be infected with HCV (lo-13,15,16,29-31). A sustained response to interferon-a regarding HCV infection and chronic liver disease has hardly been demonstrated in these latter patients. Interestingly, patients eliminating serum HCV RNA during interferon treatment showed a decrease in serum cryoglobulins and an improvement in vasculitic manifestations, and thus the therapeutic efficacy of interferon-a in cryoglobulinemic vasculitis appeared to be closely related to its antiviral activity. A similar decrease in serum cryocrit was seen in responding patients in the present study as well. It should be noted, however, that determining serum cryocrit levels might only partially reflect cryoglobulinemic disease activity and the response to interferon therapy. A beneficial effect of interferon-a was reported in patients with symptomatic “essential” mixed cryoglobulinemia at a time when the association with chronic

HCV infection had not been identified (32,33). It can be assumed that the majority of these latter patients were infected with HCV (9). A dissociation of the antiviral effects of interferon-a and its effect on cryoglobulinemia has been claimed; however, only three patients have been reported in whom serum HCV RNA persisted despite improvement of vasculitis (34). It should be noted that in these latter patients transaminase activity decreased during therapy and serum HCV RNA was not quantified (34). The presence of serum cryoglobulins did not appear to affect the response to interferon-a therapy in the present series of HCV-infected patients, i.e. 35% of patients with cryoglobulinemia showed a sustained response compared to 22% of patients without cryoglobulinemia treated contemporaneously. Among cryoglobulinemic patients, response to therapy was observed in patients with relatively low cryocrit levels (e.g. 0.53%), as well as patients with higher cryocrit levels. However, because only a small number of individuals studied showed a high cryocrit, the effect of cryoglobulin levels on response to therapy cannot be reliably assessed at present. Interferon-a cumulative dose and duration of treatment were almost identical for both groups of patients. It is noteworthy that negative predictive factors of a response to interferon-a, e.g. infection by HCV genotype lb and the presence of cirrhosis (22) did not appear to be less frequent in cryoglobulinemic patients. Serum HCV RNA level - a genotype-independent predictive factor - was similar in patients with and in those without cryoglobulinemia. However, significantly lower pre-treatment HCV RNA levels were observed in patients showing (irrespective of the presence of cryoglobulinemia) a sustained response compared to nonresponding patients, thus confirming the predictive value described before (22). Furthermore, infection with HCV genotypes la and lb was recognized as a negative predictive factor in the present study as well. In comparison to reports on symptomatic cryoglobulinemic patients, the high proportion of patients with cryoglobulinemia showing a sustained response of HCV infection to interferon-a might be unexpected. However, the patients in the present series obviously received therapy at a different clinical stage of cryoglobulinemia, when severe symptomatic disease (e.g. renal failure) was not present. Asymptomatic cryoglobulinemia or mildly symptomatic disease appears to predominate in HCV-infected patients (35). It is possible that patients presenting with clinically more advanced HCV-associated cryoglobulinemia respond differently to interferon-a. The small number of patients with symptomatic cryoglobulinemic disease in the present 69

F. P&h

et al.

series, means that the response to therapy in this group cannot be judged. In the present study, 54% of the 59 patients were male. Interestingly, a correlation between non-response to interferon-a, absence of cryglobulinemia and male sex was suggested. The presence of cryoglobulinemia in males did not affect the outcome of antiviral therapy adversely (sustained response in 5 out of 7), whereas in females only 19% (3/l 6) of female cryoglobulinemic patients showed a sustained response. HCV genotypes were determined in 10 non-cryoglobulinemic, non-responding males and a high prevalence of genotypes lb (n=4) and la (n=5) was noted. Therefore, in view of the genotype distribution in this latter group of patients, the negative predictive value of male sex in the absence of cryoglobulinemia remains uncertain. Regarding the contribution of other predictive factors, e.g. the presence of cirrhosis, conclusions seem to be impossible because of the small number of patients studied. In summary, the presence of cryoglobulinemia per se does not preclude a sustained response to interferon-a therapy in patients with chronic HCV infection presenting without overt clinical signs of vasculitis.

Acknowledgements The help of Prof. U. Kaboth in the laboratory analysis of cryoglobulins is gratefully acknowledged. We thank Dr. A. Fayyazi, Department of Pathology. for histopathological interpretation of liver biopsies and R. Faber for secretarial assistance.

References 1. Pawlotsky

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3.

4.

5.

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